streptococcus pyogenes endoglycosidase and methods of use

专利摘要:
ENDOGLICOSIDASE OF STREPTOCOCCUS PYOGENES AND METHODS OF USEThe invention provides an endoglycosidase, referred to as EndoS49 and having the amino acid sequence SEQ ID NO: 1. EndoS49 was isolated from strain Streptococcus pyogenes NZ131 and is an EndoS homolog. EndoS49 has specific endoglycosidase activity in native IgG and will join a greater variety of EndoS Fc glycans. A mutant, where the glutamic acid at position 186 of SEQ ID NO: 1 has been replaced, said mutant has been produced without endoglycosidase activity but is capable of binding to IgG. Methods using EndoS49, their deletions and said mutant, especially for assessing IgG glycosylation or for isolating IgG are disclosed.
公开号:BR112014005816A2
申请号:R112014005816-4
申请日:2012-09-12
公开日:2020-12-29
发明作者:Mattias Collin；Maria Allhorn；Jonathan Sjögren
申请人:Genovis Ab；
IPC主号:

专利说明:

[0001] [0001] The present invention relates to a new endoglycosidase, its mutants devoid of glycan hydrolysis activity, and its use in glycoprotein glycan hydrolysis methods. Background of the Invention
[0002] [0002] Endoglycosidase S (EndoS) is secreted by a number of Streptococcus pyogenes serotypes and has a specific activity of native IgG endoglycosidase by hydrolysis of conserved glycans bound to asparagine 297 residue in heavy chains of IgG, Collin and Olsen, The EMBO Journal , 2001, 20, 3046-3055. EndoS is the first known bacterial enzyme with a unique specificity for native IgG. In contrast, the activities of other known endoglycosidases require or are enhanced by denaturation of the glycoprotein substrate.
[0003] [0003] Antibodies such as IgG have many applications in basic research, as well as in the diagnosis and development of drugs. In some of these applications, such as immunohistochemistry, immunoassays, tumor detection, radiation therapy, crystallography studies of antibody binding sites and immunosegmentation, it is more convenient to use Fab fragments of entire IgG molecules. Some of the advantages of using Fab fragments are that they will not be affected by Fc receptors in cells or precipitate the antigen, they exhibit reduced immunogenicity and are less susceptible to phagocytosis, and that Fab fragments are radiolabeled more quickly removed from tissue than the IgG molecules. For other applications, the use of IgG Fc fragments is desirable. In other applications, it may be desirable to use deglycosylated versions of antibodies or other glycoproteins.
[0004] [0004] IgG cleavage into Fab and Fc fragments is most often performed using proteolytic enzymes such as pepsin or papain. These enzymes generally cleave other proteins, so the cleavage reaction usually has to be carried out on a purified IgG fraction. In addition, pepsin and papain will typically pick up IgG in more than one place. This means that the fragments obtained often do not correspond to complete Fab or Fc fragments, and even if cleavage results in Fab and Fc fragments, which are typically susceptible to further cleavage into smaller fragments. Isolation of Fc fragments from Fab fragments is most often performed with protein A or G affinity separation columns, which use the Fc-binding properties of bacterial proteins A and G.
[0005] [0005] Many different glycoproteins are useful in therapeutic applications. Methods for analyzing the glycosylation of these proteins are useful in research and development of proteins as therapeutic agents. It may also be desirable to provide deglycosylated versions of these proteins. Summary of the Invention
[0006] [0006] The inventors have identified a new M49 serotype endoglycosidase, Streptococcus pyogenes, referred to herein as EndoS49. EndoS49 was isolated from strain NZ 131, a nephritogenic and highly transformable strain of serotype M49. Strain NZ131 is a clinical isolate from a case of acute post-streptococcal glomerulonephritis in New Zealand. At the protein level, EndoS49 has less than 40% EndoS identity, and is a 90 kDa protein, compared to 108kDa of EndoS. EndoS49 has deglycosylation activity for a wide range of EndoS proteins.
[0007] [0007] The enzyme is a 90 kDa enzyme, which has a family of 18 catalytic domains glycoside hydrolases. EndoS49 hydrolyzes glycan in human glycoproteins, and in particular, IgG1-4, and alpha-1-
[0008] [0008] The modified EndoS49 polypeptide that lacks endoglycosidase activity can be used in methods to isolate glycosylated and / or functionally active IgG. By using such a modified EndoS49 polypeptide in combination with an additional IgG binding reagent that is capable of binding denatured and / or deglycosylated IgG, the inventors have also identified a method for assessing the state of glycosylation or the functional quality of a sample containing IG G.
[0009] [0009] According to the present invention, there is thus provided a polypeptide comprising (a) the amino acid sequence of SEQ ID NO: 1; (b) a variant of yours with at least 50% identity to the amino acid sequence of SEQ ID NO: 1 and having endoglycosidase activity, or (c) a fragment of any of these that has endoglycosidase activity.
[0010] The invention also provides a polypeptide capable of binding to IgG and which has no endoglycosidase activity comprising (a) the amino acid sequence of SEQ ID NO: 2; (b) a variant of yours with at least 50% identity to the amino acid sequence of SEQ ID NO: 1, in which the equivalent amino acid of glutamic acid at position 186 is substituted, or (c) a fragment of any of these .
[0011] [0011] The invention also provides polynucleotides, expression vectors and host cells that encode or express the polypeptides of the invention. The invention also relates to the use of the polypeptides of the invention in a process of determining or analyzing the glycosylation status of the protein, and in particular an antibody, in particular an IgG antibody.
[0012] [0012] The invention also provides a method for isolating IgG from a sample containing IgG, whose method comprises: (a) contacting said sample containing IgG with a modified EndoS49 polypeptide that lacks endoglycosidase IgG activity (b) separating said EndoS49 of the contacted sample; thus obtaining isolated IgG.
[0013] [0013] In addition, the method of assessing the state of glycosylation or the functional quality of a sample containing IgG is provided, the method of which comprises having a first and a second sub-sample of the sample containing IgG, and in which steps ( a) and (b) according to the method above are applied to the first sub-sample, in which steps (a) and (b) as above apply to the second sub-sample except the EndoS49 polypeptide is replaced with a alternative IgG binding reagent, which is capable of denatured and / or deglycosylated IgG binding, and which further comprises: (c) quantifying the amount of IgG bound to the EndoS49 polypeptide in the first sub-sample, and the amount of IgG bound to the reagent of alternative Igg binding in the second sub-sample; and (d) comparing the bound IgG values determined in (c); and thereby assess the state of glycosylation or the functional quality of a sample containing IgG.
[0014] [0014] The modified enzyme of the present invention can also be used in methods for the isolation of IgG Fab or Fe fragments. The methods of the present invention make use of a highly specific S. pyogenes IgG cleavage enzyme, IdeS (Immunoglobulin G S. pyogenes degrading enzyme), and an EndoS49 polypeptide.
[0015] [0015] In a method of the invention, a sample containing IgG is contacted with Ides and an EndoS49 polypeptide, which is a modified EndoS49 polypeptide that lacks endoglycosidase activity as described above.
[0016] [0016] In the methods of the invention, IgG Ides generally cleaves into Fab and Fc fragments and the polypeptide binds to EndoS49 Fc fragments. The Fe fragments are then separated from the Fab fragments.
[0017] [0017] This method is particularly useful for the isolation of Fab fragments or Fc fragments from samples comprising purified IgG. More specifically, it is useful for the isolation of Fab or Fc fragments from a sample comprising the purified IgG using the modified EndoS49 polypeptide of the invention. However, the method can also be adapted for use with samples containing unpurified IgG, such as serum, cell lysate or cell culture medium.
[0018] [0018] Kits are also provided for carrying out the methods according to the invention. Brief Description of the Figures
[0019] [0019] Figure 1. ClustalW alignment of EndoS49 and EndoS reveals two different proteins. EndoS49 and Endos were aligned using ClustalW in the MacVector software. GH18 catalytic motif (D ** D * D * E) is present in position 179-186 with Glul86 as a catalytic residue.
[0020] [0020] Figure 2. EndoS49 has activity on glycoproteins. A. 1 µg of EndoS49, its catalytic mutant and the truncated versions were incubated with 3 µg of human IgG in PBS overnight at 37 ° C and analyzed on an SDS-PAGE gel and with LCAlectin blot. B. 1
[0021] [0021] Figure 3. EndoS49 binds to IgG. 4, 2 and 1 µg of EndoS49 and its mutants were immobilized on a PVDF membrane and incubated with human IgG and later with HRP-coupled protein G.
[0022] [0022] Figure 4. The genomic context of ndoS49 and NDOS. A comparison of the genes surrounding ndoS49 and ndoSm GAS strains NZ131 (M49) and 5005 (Ml) was performed in MacVector.
[0023] [0023] Figure 5. Phylogenetic analysis of EndoS49 and other bacteria endoglycosidases. Figure 6. Avastin and Erbitux SDS gel page after digestion with EndoS or EndoS49 followed by IdeS digestion. Brief Description of the Strings
[0024] [0024] SEQ ID NO: 1 is an amino acid sequence of an EndoS49 polypeptide isolated from S. pyogenes serotype M49 NZ 131.
[0025] [0025] SEQ ID NO: 2 is an amino acid sequence of a modified EndoS49 polypeptide (E186L) derived from the sequence of SEQ ID NO: 1.
[0026] [0026] SEQ ID NO: 3 is a nucleotide sequence encoding EndoS49 polypeptide.
[0027] [0027] SEQ ID NO: 4 is an IdeS amino acid sequence isolated from S. pyogenes API. Detailed Description of the Invention General characteristics of polypeptides
[0028] [0028] The present invention relates to a new EndoS49 polypeptide. The invention also provides several methods that use bacterial proteins EndoS49 and IdeS, as well as other proteins. The terms protein, peptide and polypeptide are used here interchangeably. It should be understood that certain polypeptides and methods of the invention require a polypeptide having EndoS49 endoglycosidase activity, whereas other polypeptides and methods of the invention require a modified EndoS49 polypeptide lacking said activity.
[0029] [0029] The following section refers to general characteristics of all polypeptides of the invention, and in particular to the variations, alterations, modifications or derivations of amino acid sequence that are included within the polypeptides of the invention. It should be understood that these variations, alterations, modifications or derivations of polypeptides as described herein, are subject to the requirement that the polypeptides retain any other required activity or characteristic that can be specified in subsequent sections of this description.
[0030] [0030] Polypeptide variants of the invention can be defined by particular levels of amino acid identity, which are described in more detail in the following sections of this description. Amino acid identity can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate homology or align sequences (such as identifying equivalent or corresponding sequences (usually in their default configurations), for example, as described in Altschul SF (1993) J Mol Evol 36 : 290 - 300; Altschul, S, F et al (1990) J Mol Biol 215: 403-10 Software for performing BLAST analyzes is available to the public through the National Biotechnology Information Center (http: // www This algorithm involves first identifying high sequence pair (HSP) punctuation, identifying short words of length W in the query string that match or satisfy any positive T threshold score of value when aligned with a same length in a database string. T is referred to as the neighborhood word limit score (Altschul et al, supra). These initial neighborhood words act as finishing seeds to initiate p searches to find HSPs containing them. Word hits are extended in both directions throughout each sequence, as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are interrupted when: the cumulative alignment score falls by the amount X of its maximum value reached, the accumulated score goes to zero or below, due to the accumulation of one or more negative punctuated residue alignments, or the end of any sequence is reached. The parameters of the BLAST algorithm W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses a standard word length (W) of 11, the BLOSUM62 matrix to mark (see Henikoff and Henikoff (1992) Proc Natl Acad Sci USA 89: .... 10,915-10,919) alignments (B) of 50, the expectation (E) of 10, M = 5, N = 4, and a comparison of both chains.
[0031] [0031] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. A measure of the similarity provided by the BLAST algorithm is the least probability of sum (P (N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the probability of the smallest sum when comparing the first sequence with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0 , 01, and more preferably less than about 0.001. Alternatively, the UWGCG package provides the BESTFIT program that can be used to calculate homology (for example used in its standard configurations) (Devereux et al (1984) Nucleic Acids Research 12, 387- 395).
[0032] [0032] It will be understood that the polypeptide variants of the invention also include substitution variants. Substitution variants preferably involve replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid can be replaced by an alternative amino acid that has similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids that can be used to select suitable substituents are as follows: Aliphatic, hydrophobic, neutral Hydrophobic Met, neutral Polar Cys, hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar, hydrophilic, charged (-) Hydrophobic, neutral, polar Glu, hydrophilic, charged (-) Polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic, neutral Polar, hydrophilic, neutral His aromatic, polar, hydrophilic , Thr polar, hydrophilic, neutral charged (+) Ile aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged (+) Trp aromatic, hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic
[0033] [0033] The polypeptides of the invention and for use in the present invention can be in a substantially isolated form. It is to be understood that the polypeptide can be mixed with vehicles or diluents that do not interfere with the intended purpose of the polypeptide and still be considered to be substantially isolated. A polypeptide for use in the invention can also be in a substantially purified form,
[0034] [0034] The amino acid sequence of the polypeptides of the invention and for use in the invention can be modified to include non-naturally occurring or amino acids to increase the stability of the compound. When polypeptides are produced by synthetic means, such amino acids can be introduced during production. Polypeptides can also be modified after the production of synthetic or recombinant.
[0035] [0035] Polypeptides of the invention or for use in the invention can also be produced using D-amino acids. In such cases, the amino acids will be linked in the reverse sequence in the C to N orientation. This is conventional in the art for the production of such polypeptides.
[0036] [0036] A number of modifications of the side chain are known in the art and can be made for the side chains of the polypeptides, the polypeptides subject to retention of any other required activity or as a characteristic can be specified herein.
[0037] [0037] It will also be understood that the polypeptides of the invention and used in the invention can be chemically modified, for example, post-translationally modified. For example, they can be glycosylated, phosphorylated or modified to comprise amino acid residues. They can be modified by adding a signal sequence to promote insertion into the cell membrane.
[0038] [0038] The polypeptides of the invention can also be derived or modified to facilitate their isolation or purification. Thus, in an embodiment of the invention, the polypeptide for use in the invention is derivatized or modified by the addition of a ligand that is capable of binding directly and specifically to a separation medium.
[0039] Alternatively, the polypeptide is derived or modified by adding a member of a binding pair and the separation medium comprises a reagent that is derivatized or modified by adding another member of a binding pair. Any suitable connection pair can be used. In a preferred embodiment, where the polypeptide for use in the invention is derivatized or modified by the addition of a member of a binding pair, the polypeptide is preferably histidine-labeled or biotin-labeled. Typically, the amino acid coding sequence of the histidine marker, or biotin, is included at the gene level and proteins are recombinantly expressed in E. coli. The histidine or biotin marker is normally present at one end of the polypeptide, either at the N-terminus or the C-terminus. The histidine marker typically consists of six histidine residues, but it can be longer than this, typically over 7, 8, 9, 10 or amino acids or less, for example, 5, 4, 3, 2 or 1 amino acids. In addition, the histidine marker may contain one or more amino acid substitutions, preferably conservative substitutions, as defined above.
[0040] [0040] EndoS49s polypeptide having endoglycosidase activity
[0041] [0041] The EndoS49 polypeptide in this case is preferably S. pyogenes EndoS49, or a variant or fragment of S. pyogenes EndoS49 that retains endoglycosidase activity. The variant can be a polypeptide from another EndoS49 Streptococcus equi, Streptococcus zooepidemicus, or, preferably, Streptococcus pyogenes.
[0042] [0042] The polypeptide can comprise EndoS49: (a) the amino acid sequence of SEQ ID NO: 1;
[0043] [0043] Preferably, the polypeptide comprises or consists of, the sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the EndoS49 sequence of S. pyogenes. The EndoS49 polypeptide of the invention may additionally not comprise a signal sequence.
[0044] [0044] The variant polypeptides described in this section are those for which the amino acid sequence varies from SEQ ID NO: 1, but which retain the endoglycosidase activity of EndoS49. Such variants may include allelic variants and the deletion, modification or addition of individual amino acids or groups of amino acids within the protein sequence, provided the peptide maintains IgG endoglycosidase activity.
[0045] [0045] Variant sequences typically differ from at least 1, 2, 3, 5, 10, 20, 30, 50, 100 or more mutations (which can be substitutions, deletions or insertions of amino acids). For example, from 1 to 100, from 2 to 50, 3 to 30 or 5 to 20 amino acid substitutions, deletions or insertions can be made, as long as the modified polypeptide retains activity as an IgG endoglycosidase.
[0046] [0046] The amino acid sequence variants of SEQ ID NO: 1 preferably contain residues 179-186 of SEQ ID NO: 1, and in particular include the motif D ** D * D * E. These amino acids constitute a family of 18 glycoside hydrolases catalytic domain. Glutamic acid at position 186 is essential for enzyme activity. More preferably, therefore, the variant of SEQ ID NO: 1 contains glutamic acid at the position equivalent to position 186 of SEQ ID NO: 1. The variant of SEQ ID NO: 1 can contain residues 179-186 of SEQ ID NO: 1 having one or more conservative substitutions, provided that the variant contains glutamic acid in the position equivalent to position 186 of SEQ ID NO: 1.
[0047] [0047] Typically, polypeptides that exhibit EndoS49 endoglycosidase activity with more than about 50%, 55% or 65% identity, preferably at least 70%, at least 80%, at least 90% and so particularly preferred at least 95%, at least 97% or at least 99%) identity, with the amino acid sequence of SEQ ID NO: 1 are considered variants of the protein The identity of SEQ ID NO: 1 variants can be measured by over a region of at least 100, at least 250, at least 500, at least 800, at least 810, at least 820, at least 930, at least 940 or more contiguous amino acids of the sequence shown in SEQ ID NO: 1, or more preferably more than the full length of SEQ ID NO: 1.
[0048] [0048] The fragment of the EndoS49 polypeptide used in the invention is typically at least 400, 500, 600, 700, 750, 800, or 825 amino acids in length, as long as it retains the EndoS IgG endoglycosidase activity. Preferably, the fragment of the EndoS49 polypeptide used in the invention encompasses residues 179-186 of SEQ ID NO: 1.
[0049] [0049] Polypeptides for use in the present invention can be isolated from any appropriate organism that expresses an EndoS49 polypeptide or a variant of an EndoS49 polypeptide. Typically, the polypeptide is isolated from suitable EndoS49 EndoS49 expressing strains of Streptococcus, preferably strains of S. pyogenes, and in particular those of serotype M49.
[0050] [0050] The isolation and purification of EndoS49 from an expressing S. pyogenes culture, or from cell cultures expressing other EndoS49 is typically based on endoglycosidase activity. Preferably, the purification method involves an ammonium sulfate precipitation step and an ion exchange chromatography step. According to one method, the culture medium is fractionated by adding increasing amounts of ammonium sulfate. Ammonium sulfate amounts can be 10 to 80%.
[0051] [0051] Preferably, the culture medium is fractionated with 50% ammonium sulphate, and the resulting supernatant is further precipitated with 70% ammonium sulphate. Granulated polypeptides can then be subjected to ion exchange chromatography, for example, on a FPLC Mono Q column. Eluted fractions can be tested for endoglycosidase activity and peak activity fractions can be pooled. Fractions can be analyzed by SDS-PAGE. Fractions can be stored at -80 ° C.
[0052] [0052] Polypeptides for use in the present invention can also be prepared as fragments of such isolated polypeptides. In addition, EndoS49 polypeptides can also be made synthetically or by recombinant means. For example, a recombinant EndoS49 polypeptide can be produced by transfecting cultured mammalian cells with an expression vector comprising a nucleotide sequence that encodes the polypeptide operably linked to suitable control sequences, cell culture, extraction and purification of the EndoS49 polypeptide produced by the cells.
[0053] [0053] The polypeptides of the invention EndoS49 described in this endoglycosidase activity section presentation. Preferably, the polypeptide hydrolyzes fragments of IgG or IgG Fc by hydrolysis of bound glycan from a full length of IgG polypeptide heavy chain. Preferably, the EndoS49 polypeptide of the invention also has endoglycosidase activity, and is capable of hydrolysis of alpha-1-microglobulin glycan.
[0054] [0054] Endoglycosidase activity can be determined using an appropriate assay. For example, a test polypeptide can be incubated with glycoprotein, such as IgG or alpha-1-
[0055] [0055] The endoglycosidase activity of the polypeptides can be further characterized by inhibition studies.
[0056] [0056] The EndoS49 polypeptide is capable of hydrolyzing glycoprotein molecules present in a sample taken from a subject. Thus, in which the subject is a human being, the EndoS49 polypeptide is capable of hydrolyzing glycans of an object's glycoproteins, such as for example on the heavy chains of human IgG or alpha-1-microglobulin.
[0057] [0057] EndoS49 is capable of hydrolyzing human IgG from all four subclasses (IgGi_4). In preferred embodiments, the EndoS49 polypeptide has the ability to hydrolyze human IgG and alpha-1-microglobulin. EndoS49s polypeptide that do not have endoglycosidase activity
[0058] [0058] The EndoS49 polypeptide in this example can also be modified from S. pyogenes EndoS49, which was designed for lacking endoglycosidase activity, but which has an IgG binding activity. Such a modified EndoS49 is particularly useful for the methods described here. By IgG-binding activity it will be understood that the modified EndoS49 binds to IgG, or a variant or fragment thereof, in particular, the Fc fragment thereof, which is normally glycosylated. By "normally glycosylated" it will be understood that the IgG molecule, or a fragment thereof, variant, is a glycoprotein comprising at least the IgG heavy chain polypeptide (or fragment variant) coupled to at least one group of carbohydrates as found naturally coupled to occurring IgG molecules. In particular, the at least one carbohydrate group is a glycan attached to the asparagine residue corresponding to residue 297 of a full-length IgG of heavy chain polypeptide.
[0059] [0059] The EndoS49 polypeptide is preferably constructed by site-directed mutagenesis. By IgG binding activity it will be understood that the EndoS49 polypeptides described in this binding section are at least one, preferably two, three or all four IgG subclasses, IgGi_ 4. Preferably, the at least one subclass of IgG is linked with high affinity and / or high specificity.
[0060] [0060] High affinity means that the constant binding affinity (KD) for the interaction of the modified EndoS49 with an IgG subclass is greater than 0.05 μΜ, preferably greater than 0.06 μΜ, 0.07 or 0.08 μΜ μΜ. The binding activity can be determined, and the binding affinity can be assessed by any suitable means. For example, by resonance surface plasmon interaction analysis, dialysis balance analysis, or any standard biochemical methods in conjunction with, for example, Scatchard analysis.
[0061] [0061] The variant can be derived from an EndoS49 polypeptide from another organism, such as another bacterium, as described in the previous section, with the exception that the variant does not have,
[0062] [0062] The variant polypeptides described in this section are those for which the amino acid sequence varies from SEQ ID NO: 2, but which lack endoglycosidase activity and retain Igg binding activity. Such variants may include allelic variants and the deletion, modification or addition of individual amino acids or groups of amino acids within the protein sequence, provided that the peptide maintains the above characteristics.
[0063] [0063] Variant sequences typically differ from at least 1, 2, 3, 5, 10, 20, 30, 50, 100 or more mutations (which may be amino acid substitutions, deletions or insertions). For example, from 1 to 100, from 2 to 50, 3 to 30 or 5 to 20 amino acid substitutions, deletions or insertions can be made, provided that the modified polypeptide lacks endoglycosidase activity and retains IgG binding activity.
[0064] [0064] Typically, polypeptides, which lack endoglycosidase activity and retain IgG-binding activity with more than about 50%, 55% or 65% identity, preferably at least 70%, at least 80%, at least 90% and particularly preferably at least 95%, at least 97% or at least 99% identity, with the amino acid sequence of SEQ ID NO: 2 are considered protein variants The identity of SEQ ID NO variants : 2 can be measured over a region of at least 100, at least 250, at least 500, at least 750, at least 800, at least 820, at least 830 or more contiguous amino acids from the sequence shown in SEQ ID NO : 2, or more, preferably along the total length of SEQ ID NO: 2.
[0065] [0065] The fragment of the EndoS49 polypeptide used in the invention is typically at least 300, 400, 500, 600, 700, 750, 800 or 830 amino acids in length, while it lacks endoglycosidase activity and retains binding activity IG G.
[0066] [0066] In an alternative method, an EndoS49 protein with the desired characteristics can be produced by altering a nucleotide encoding an EndoS49 protein, and then expressing said nucleotide in a suitable system. Suitable methods include mutagenesis directed at the nucleotide site that encodes the protein. This technique has been widely used in the study of protein functions. The technique is generally based on oligonucleotide and involves the following steps: (1) Cloning the DNA encoding the protein of interest into a plasmid vector. (2) Denaturation of plasmid DNA for the production of single strands. (3) The contact of the denatured DNA with a synthetic oligonucleotide (or oligonucleotides) complementary to the target sequence, but which incorporates the desired mutation (s) (point mutation, deletion, or insertion), such that the oligonucleotide synthesis hybridizes with the target region. (4) Extending the strand mutated by a DNA polymerase using the plasmid DNA strand as the template.
[0067] [0067] After propagation, about 50% of the heteroduplexes produced are mutants and the other 50% are of the "wild" type (without mutation). Selection and enrichment methods are used to favor the production of mutants. For example, the parental unmutated strand can be digested with a restriction enzyme that only digests methylated DNA (Dpnl). This allows the removal of the parent strand from the reaction before the transformation of E. coli once the newly synthesized strands are non-methylated, while the parent strand (if purified from the correct E. coli background) is methylated.
[0068] [0068] Alternatives to site-directed mutagenesis include: (1) Polymerase chain reaction (PCR) using mutagenic primers specific methods, or error-prone PCR with subsequent screening for desired mutations or loss / gain of protein based function . (2) Introduction of a plasmid that houses the gene of interest in a mutant E. coli strain (deficient in DNA review systems) and subsequent selection of desired mutations or loss / gain of protein function. (3) Chemical synthesis of partial or integral genes containing the desired mutations and subsequent introduction into an appropriate protein expression system.
[0069] [0069] Alternatively, an EndoS49 protein with the desired characteristics can be produced by independent DNA methods, which include the chemical synthesis of parts of a polypeptide with the desired mutation.
[0070] [0070] Polypeptides for use in the present invention can also be prepared as fragments of such isolated polypeptides. In addition, EndoS49 polypeptides can also be made synthetically or by recombinant means. For example, a recombinant EndoS49 polypeptide can be produced by transfecting mammalian cells in culture with an expression vector comprising a nucleotide sequence that encodes the polypeptide operably linked to suitable control sequences, cell culture, extraction and purification of the EndoS49 polypeptide produced by the cells.
[0071] [0071] The EndoS49 polypeptide is able to bind to IgG molecules present in a sample made from a subject. Thus, where the subject is a human being, the EndoS49 polypeptide is able to bind to human IgG.EndoS49 is able to bind to human IgG from all four subclasses (IgGi_4). Polynucleotides, vectors and host cells
[0072] [0072] The invention also concerns polynucleotides that encode the above polypeptides, and their use in medicine. In particular, the invention relates to polynucleotides comprising or consisting of (a) the coding sequence of SEQ ID NO: 3 or a sequence complementary thereto, (b) the sequence which hybridizes under stringent conditions to the defined sequences in (a), (c) sequence that is degenerated as a result of the genetic code to sequence, as defined in (a) or (b), (d) the sequence has at least 60% identity with the sequences defined in (a ), (b) or (c) and (e) fragments of the above sequences.
[0073] [0073] Typically, the polynucleotide is DNA. However, the invention may comprise RNA polynucleotides. Polynucleotides can be single or double stranded, and can include synthetic or modified nucleotides within them.
[0074] [0074] A polynucleotide of the invention can hybridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 3 at a level significantly above the background. Background hybridization can occur, for example, due to other DNAs present in a DNA library. The level of signal generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 3 is typically at least 10 times, preferably at least 100 times, as intense as the interactions between other polynucleotides and the coding sequence of SEQ ID NO: 3. The intensity of the interaction can be measured, for example, by radio tagging the probe, for example, with 32 P. Selective hybridization can be achieved, typically using high medium conditions rigor. However, such hybridization can be carried out under any suitable conditions known in the art (see Sambrook et al, 1989. For example, suitable conditions if high stringency is necessary to include 0.1 to 0.2 x SSC at 60 ° C until 65 ° C suitable conditions see less rigor it is necessary to include 2 x SSC at 60 ° C.
[0075] [0075] The coding sequence of SEQ ID NO: 3 can be modified through nucleotide substitutions, for example, from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The polynucleotide of SEQ ID NO: 3, may alternatively or additionally be modified by one or more insertions and / or deletions and / or by an extension at one or both ends. Additional strings, such as signal strings can also be included. The modified polynucleotide generally encodes a polypeptide that has endoglycosidase activity. Alternatively, a polynucleotide that encodes an epitope portion of an EndoS49 polypeptide. Degenerate substitutions can be made and / or substitutions can be made, which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the table above.
[0076] [0076] A nucleotide sequence that is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 3 is generally at least 60%, at least 70%,
[0077] Any combination of the above-mentioned degrees of sequence identity and minimum sizes can be used to define the polynucleotides of the invention, with the most stringent combinations (i.e., greater sequence identity over longer lengths) being preferred. Thus, for example, a polynucleotide that has at least 90%> sequence identity over 25, preferably more than 30 nucleotides form an aspect of the invention, as well as a polynucleotide that has at least 95% identity sequence with more than 40 nucleotides.
[0078] [0078] Polynucleotide fragments, such as those that are suitable for use as probes or primers will preferably be at least 10, preferably at least 15 or at least 20, for example, at least 25, at least 30 or at least 40 nucleotides of lenght. They will normally be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. The probes and fragments can be more than 150 nucleotides in length, for example, up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even some nucleotides, such as five or ten nucleotides, short of the sequence coding of SEQ ID NO: 3.
[0079] [0079] The polynucleotides according to the invention can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be cloned by conventional techniques. Polynucleotides are typically provided in isolated and / or purified form.
[0080] [0080] In general, the primers will be produced by synthetic means, involving a sensible step of making the desired nucleotide sequence of nucleotide sequence one at a time. Techniques for accomplishing this using automated techniques are readily available in the art.
[0081] [0081] Longer polynucleotides will generally be produced using recombinant means, for example, using PCR (polymerase chain reaction), cloning techniques. This will involve making a pair of primers (for example, about 15-30 nucleotides) for a region of the ndoS49 gene that you want to clone, putting the primers in contact with the DNA obtained from a bacterial cell, performing a polymerase chain reaction under conditions that cause amplification of the desired region, isolate the amplified fragment (for example, purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. Primers can be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.
[0082] [0082] Although, in general, the techniques mentioned herein are well known in the art, reference can be made in particular to Sambrook et al, Molecular Cloning: A Laboratory Manual, 1989.
[0083] [0083] The polynucleotides according to the invention have utility in the production of polypeptides according to the invention, which can take place in vitro. The polynucleotides of the invention can be used as a primer, for example, a PCR primer, a primer for an alternative amplification reaction, a probe, for example tagged with a developing marker by conventional means using radioactive or non-radioactive markers, or polynucleotides can be cloned into vectors.
[0084] [0084] Polynucleotides or primers of the invention can carry a developer tag. Suitable tags include radioisotopes, such as 32 P or 35 S, enzyme tags, or other protein tags, such as biotin. Such tags can be added to the polynucleotides or primers of the invention and can be detected using techniques known per se.
[0085] [0085] The polynucleotides or primers of the invention or their fragments, labeled or unlabeled, can be used by a person skilled in the art in tests based on nucleic acid for detecting and sequencing ndoS49 in a sample. Such tests to detect generally comprise bringing a sample containing the DNA or RA in contact with a probe comprising a polynucleotide or primer of the invention, under hybridization conditions and detecting any duplex formed between the probe and the nucleic acid in the sample. Such detection can be achieved using techniques such as PCR or by immobilizing the probe on a solid support, removing nucleic acid in the sample that is not hybridized to the probe, and then detecting nucleic acid that has been hybridized to the probe . Alternatively, the sample nucleic acid can be immobilized on a solid support, and the amount of probe attached to such a support can be detected.
[0086] [0086] The invention can conveniently be packaged in the form of a test kit in a suitable container. In such kits the probe can be connected to a solid support where the test format for which the kit was designed requires such a connection. The kit can also contain reagents suitable for treating the sample to be probed, hybridizing the nucleic acid probe to the sample, control reagents, instructions and the like.
[0087] [0087] Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector can be used to replicate the nucleic acid in a compatible host cell. Thus, the polynucleotides of the invention can be made by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell and growing the host cell under conditions that cause the vector to replicate.
[0088] Preferably, the vector is an expression vector comprising a nucleic acid sequence that encodes a polypeptide of the invention. Such expression vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA and appropriate primers, promoters, enhancers and other elements, which may be necessary, and which are positioned with the correct orientation, in order to allow protein expression. Other suitable vectors will be apparent to those skilled in the art. As an additional example, in this respect we refer to Sambrook et al. 1989.
[0089] [0089] The polynucleotides according to the invention can also be inserted into the vectors described above, in an antisense orientation, in order to provide the production of antisense RNA. Antisense RNA or other antisense polynucleotides or interfering RNA, RNAi can also be produced by synthetic means. Such antisense polynucleotides or RNAi can be used as test compounds in the assays of the invention, or can be useful in a method of treating the human or animal body by therapy.
[0090] Preferably, a polynucleotide of the invention or for use in the present invention in a vector is operably linked to a control sequence that is capable of providing expression of the coding sequence by the host cell, that is, the vector is a vector of expression. The term "operatively linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in their intended manner. A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.
[0091] The vectors can be, for example, plasmid, virus or phage supplied with vectors from a source of replication, optionally a promoter for the expression of said polynucleotide and optionally a regulator of the promoter. The vectors can contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector.
[0092] [0092] Promoters and other expression regulation signals can be selected to be compatible with the host cell for which the expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. promoter and ADH promoter. Mammalian promoters include the metallothionein promoter that can be induced in response to heavy metals, such as cadmium. Viral promoters such as those of SV40 large T antigen promoter or adenovirus promoters can also be used. All such promoters are readily available in the art.
[0093] [0093] Mammal promoters, such as β-actin promoters, can be used. Tissue-specific promoters are especially preferred. Viral promoters can also be used, for example, the long-term repeat Moloney murine leukemia virus (MMLV LTR), the Rous sarcoma virus (RSV), the LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV ) IE promoter, adenovirus, HSV promoters (such as HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.
[0094] [0094] Expression vectors can be transformed into a suitable host cell to provide expression of a polypeptide or polypeptide fragment of the invention. The host cell, transformed or transfected with an expression vector as described above, is grown under conditions that allow expression of the polypeptide or fragment, and the expressed polypeptide or fragment is recovered. Isolation and purification can be carried out as described above. The host cells will be chosen to be compatible with the vector and will preferably be of bacterial origin. The host cells can also be cells of a non-human animal, or a plant transformed with a polynucleotide of the invention. IdeS
[0095] [0095] IdeS is an extracellular cysteine protease produced by the human pathogens S. pyogenes and is described in WO 03/051914. Ides was originally isolated from a group of a strain of strain M1 serotype, but the ides gene has now been identified in all groups tested for strep strains. Ides has an extraordinarily high degree of substrate specificity, with its substrate only identified as IgG. Ides catalyzes a single proteolytic cleavage in the lower region of the human IgG hinge. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the death of group A Streptococcus. IDEs also cleave some subclasses of IgG in various animals and efficiently convert IgG into Fc and Fab fragments. The ides gene was cloned and expressed in E. coli as a GST fusion protein.
[0096] [0096] The Ides polypeptide for use in the methods of the invention is preferably S. pyogenes Ides, or a variant or fragment of S. pyogenes Ides that retains the cysteine protease activity. The variant may be a polypeptide Ides from another organism, such as another bacterium. The bacterium is preferably a streptococcus. Streptococcus is preferably a group of a Streptococcus, a Streptococcus of group C or Streptococcus of group G. In particular, the variant may be a polypeptide Ides of a group C Streptococcus, such as S. equii or S. zooepidemicus. Alternatively, the variant may be Pseudomonas putida. The IdeS polypeptide can include (a) the amino acid sequence of SEQ ID NO: 4; (b) a variant with at least 50% identity to the amino acid sequence of SEQ ID NO: 4 and having IgG cysteine protease activity, or (c) a fragment of any of these having protease activity of IgG cysteine.
[0097] Preferably, the IdeS polypeptide comprises or consists of, the sequence of SEQ ID NO: 4. SEQ ID NO: 4 is the sequence of the mature form of Ides, without the signal sequence.
[0098] [0098] Variant polypeptide ides are those for which the amino acid sequence varies from SEQ ID NO: 4, but which exhibit the same IgG cysteine protease activity as Ides. Typically, polypeptides with more than about 50%, 55% or 65%> identity, preferably at least 70%, at least 80%, at least 90% and particularly preferably at least 95%), at least 97% or at least 99% identity, with the amino acid sequence of SEQ ID NO: 4 are considered variants of the protein. Such variants may include allelic variants and the deletion, modification or addition of individual amino acids or groups of amino acids within the protein sequence, provided that the peptide maintains the basic functionality of Ides. The identity of SEQ ID NO: 4 variants can be measured over a region of at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 275, at least 300 or more contiguous amino acids of the sequence shown in SEQ ID NO: 4, or more, preferably along the total length of SEQ ID NO: 4.
[0099] [0099] The amino acid sequence variants of SEQ ID NO: 4 preferably contain residues Lys-55 and / or Cys-65 and / or His-
[00100] [00100] Variant sequences typically differ from at least 1, 2, 5, 10, 20, 30, 50 or more mutations (which can be substitutions, deletions or insertions of amino acids). For example, from 1 to 50, 2 to 30, 3 to 20 or 5 to 10 amino acid substitutions, deletions or insertions can be made. The modified polypeptide retains activity as an IgG-specific cysteine protease. Preferably, the variant polypeptides comprise a cysteine residue and a histidine residue at a spacing typically found in cysteine proteases. For example, in SEQ ID NO: 4, these residues are found at a spacing of about 130 amino acids, which is typically found in cysteine proteases.
[00101] The polypeptide Id fragment used in the invention is typically at least 10, for example, at least 15, 20, 25, 30, 40, 50 or more amino acids in length, up to 100, 150, 200, 250 or 300 acidic amino acids in length, as long as it retains the IgE cysteine protease activity of Ides. Preferably, the fragment of the polypeptide Ides used in the present invention comprises residues of Lys-55 and / or Cys-65 and / or His-233 and / or Asp-255 and / or Asp-257 of SEQ ID NO :
[00102] [00102] IDEs polypeptides for use, in accordance with the invention display the immunoglobulin cysteine protease activity, and in particular the IgG cysteine protease activity. Preferably, the IgG cleaves polypeptide hinge region and, more particularly in the region of the heavy chain hinge. Cleavage results in the production of IgG Fab and Fc fragments. Preferably, the activity is specific for IgG. The cysteine protease activity can be determined using an appropriate assay. For example, a test polypeptide can be incubated with IgG at an appropriate temperature, such as 37 ° C. The starting materials and reaction products can then be analyzed by SDS-PAGE to determine whether the desired IgG cleavage product is present. Typically, this cleavage product is the 3 lkDa fragment. There is usually no further degradation of IgG after this first cleavage. The cleavage product can be subjected to N-terminal sequencing to verify that the cleavage has occurred in the IgG hinge region. Preferably, the N-terminal sequence comprises the GPSVFLFP sequence.
[00103] [00103] The activity of polypeptide cysteine protease can be further characterized by inhibition studies. Preferably, the activity is inhibited by the peptide derived Z-LVG-CFIN 2 and / or by iodoacetic acid both of which are protease inhibitors. However, activity in general is not inhibited by E64
[00104] [00104] The polypeptide cysteine protease activity is generally specific IgG in which the polypeptides cannot degrade the other Ig classes, that is, IgM, IgA, IgD and IgE, when incubated with these immunoglobulins under conditions that allow cleavage IgG. The Ides polypeptide is capable of cleaving human IgG. In preferred embodiments, the polypeptide has the ability to cleave human, rabbit, rat or goat IgG.
[00105] [00105] Polypeptide ides for use in the present invention can be isolated from any appropriate organism that expresses an Ides polypeptide. Normally, the IDEs polypeptide is isolated from the appropriate IDES expressing strains of S. pyogenes. Suitable organisms and stresses can be identified by a number of techniques. For example, strains of S. pyogenes can initially be tested for the presence of a gene for ides. The presence of the ides gene can then be verified by PCR using the primers or by hybridizing the probes to the genomic DNA of the S. pyogenes strain.
[00106] [00106] S. pyogenes strains expressing active Ides can be identified by assay for IgG cysteine protease activity in the culture supernatant. Preferably E64 inhibitor is added to the supernatant to inhibit any SpeB cysteine protease activity. At least five strains express active IDEs: API, AP12, AP55, KTL3 and SF370 strains. Preferably, the strain it expresses is selected from API, API 2 and AP55.
[00107] [00107] The isolation and purification of Ides from an expression of S. pyogenes culture, or from cell cultures expressing other Ides is typically based on IgG cysteine protease activity. Preferably, the purification method involves an ammonium sulfate precipitation step and an ion exchange chromatography step. According to a method, the culture medium is fractionated by adding increasing amounts of ammonium sulfate. The amounts of ammonium sulfate can be from 10 to 80%.
[00108] [00108] Preferably, the culture medium is fractionated with 50% ammonium sulphate, and the resulting supernatant is further precipitated with 70% ammonium sulphate. Granulated polypeptides can then be subjected to ion exchange chromatography, for example, on a FPLC Mono Q column. Eluted fractions can be analyzed for IgG cysteine protease activity and peak activity fractions can be pooled. Fractions can be analyzed by SDS-PAGE. For example, an N-terminal sequence can be obtained from the SDS-PAGE protein band. Fractions can be stored at -20 ° C. Methods using endoglycosidase activity EndoS49
[00109] [00109] As described herein, EndoS49 has endoglycosidase activity and is capable of hydro Ylse the glycan of glycoproteins including IgG and alpha-1-microglobulin. The present invention thus provides methods for the deglycosylation of glycoproteins, and in particular, the hydrolysis of glycan of glycoproteins, and in particular, from IgG and alpha-1-microglobulin. Typically, such a method comprises incubating a sample containing the glycoprotein with EndoS49 with a glycoprotein, under conditions that allow for endoglycosidase activity. Suitable conditions include the use of EndoS49 at a concentration of at least 1 ug / ml, 2 ng / ml, 4 ng / ml, 6 mg / ml, 8 ug / ml, 10 ug / ml, 12 ug / ml, 15 µg / ml or 20 µg / ml, preferably at least 10 µg / ml. Suitable conditions include incubating the sample with EndoS49 for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, preferably at least 60 minutes. The incubation takes place preferably at room temperature, more preferably at about 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C or 45 ° C, and more preferably at about 37 ° C.
[00110] [00110] These methods can be used to provide deglycosylated glycoproteins, which can themselves be useful in research or therapy. These methods can also be used to characterize glycans in glycoproteins, for example, in or glycomapping. Such glycomapping is glyco-profile and is particularly useful for antibody molecules, such as IgG molecules, for example, in the analysis of monoclonal IgG molecules. Typically, the methods involved in incubating the protein with EndoS49 hydrolase the glycans of the protein. Subsequently, the glycans and the protein or polypeptide are separated, for example, using any suitable technique, such as HPLC or gel chromatography. The separate portions can then be analyzed by any suitable method, such as mass spectrometry, HPLC, gel chromatography, gel electrophoresis, spectrometry, capillary electrophoresis and other standard laboratory techniques for the analysis of glycans and / or proteins.
[00111] [00111] According to other methods of the present invention, the methods may also comprise the use of additional enzymes, such as Ides so that the glycans on the Fc portion of the antibody can be analyzed in more detail, using the methods and techniques here described.
[00112] [00112] An example is to analyze the fucosylation of an immunoglobulin. The degree of fucosylation on the Fc glycans in an IgG molecule is important for the therapeutic potential of an IgG drug candidate. Afucosylated IgG molecules enhance the ADCC (nn) effect of the therapeutic IgG molecule. Thus, according to the present invention, a method is provided for analyzing the amount of fucose in the Fc glycans of an IgG, using EndoS49.
[00113] [00113] Typically, such a method comprises incubating a glycoprotein, in this case an immunoglobulin, with EndoS49 under conditions that allow EndoS49 endoglycosidase activity. Suitable conditions include the use of EndoS49 at a concentration of at least 1 ug / ml, 2 ng / ml, 4 ng / ml, 6 mg / ml, 8 ug / ml, ug / ml, 12 ug / ml, 15 ug / ml or 20 µg / ml, preferably at least 10 µg / ml. Suitable conditions include incubating the sample with EndoS49 for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, preferably at least 60 minutes. Incubation takes place preferably at room temperature, more preferably at about 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C or 45 ° C, and more preferably at about 37 ° C. IDEs can be added after reaction with EndoS49, or in the same reaction mixture, to induce proteolysis, divide the immunoglobulin molecule into F (ab ') 2 and Fe. The two fragments are separated using a separation method before injection on a mass spectrometer. After glycan cleavage, a GlcNAc residue and Fuc nucleus remain bound to Asn in the Fc / 2 local glycosylation consensus. Since an immunoglobulin is not a fucosylated nucleus, some Fc / 2 will contain only one GlcNAc after digestion. The characteristic mass difference (-146 Da) resulting from the absence of fucose is easily noticeable in the mass spectrum. Use of EndoS49, therefore, facilitates direct estimation of the degree of IgG afucosylation nucleus. Method for determining the presence or absence of IgG in a sample, or for isolating IgG from a sample containing IgG.
[00114] [00114] Isolation and / or detection of IgG antibodies is typically carried out in the art, using agents such as Protein G or Protein A. Bacterial proteins interact well with IgG. However, protein A does not bind to all four IgG subclasses (IgG 1-4), and both protein A and protein G are not able to discriminate between non-glycosylated and / or denatured, inactive IgG and glycosylated and / or native, functionally active IgG. In contrast, the present inventors have identified that EndoS49s polypeptide that lack IgG endoglycosidase activity typically bind all four IgG subclasses with high affinity, and are selective for normally glycosylated IgG, i.e., IgG, in its native, functionally active form.
[00115] [00115] Therefore, the present invention provides an improved method for determining the presence or absence of IgG in a sample, which method comprises contacting said sample with a polypeptide that lacks IgG EndoS49 endoglycosidase activity, separating said EndoS49 to from the sample in contact, and thus determining the presence or absence of IgG and, optionally, in which IgG is present, obtaining isolated IgG. Therefore, the invention also provides a method for isolating IgG from a sample containing IgG, the method of which comprises contacting said sample with a polypeptide that lacks EndoS49 IgE endoglycosidase activity, separating said EndoS49 from the contact sample, and, thus, obtaining isolated IgG.
[00116] [00116] The above samples are contacted with EndoS49 polypeptide under conditions suitable for the interaction between the polypeptide and the sample to take place and the IgG binding activity to occur, that is, to allow the formation of an IgG-EndoS49 polypeptide complex. Suitable conditions include the use of EndoS49 at a concentration of at least 1 ug / ml, 2 ng / ml, 4 ng / ml, 6 mg / ml, 8 ug / ml, 10 ug / ml, 12 ug / ml, 15 µg / ml or 20 µg / ml, preferably at least 10 µg / ml. Suitable conditions include incubating the sample with EndoS49 for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, preferably at least 60 minutes. The incubation takes place preferably at room temperature, more preferably at about 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C or 45 ° C, and more preferably at about 37 ° C.
[00117] [00117] A particular advantage of EndoS49 in these methods is that it specifically binds to normally glycosylated IgG EndoS49. IgG-binding activities of other IgG-binding agents usually require or are enhanced by denaturation of the IgG glycoprotein. This is typically accomplished by treating a sample containing IgG with acid. This treatment can damage or denature some antibodies (antibodies sensitive to acids). Since the method of the invention does not require such treatment, the method is particularly suitable for isolating sensitive to IgG acid in its native form from a sample.
[00118] [00118] The EndoS49 can be separated from the contacted sample by any suitable method. A preferred method for removing EndoS49 from a sample comprises using an EndoS49 that is derivatized or modified, as described above.
[00119] [00119] A preferred modification comprises the addition of a histidine marker. The presence of a histidine marker means that the polypeptide binds with a high affinity to a reagent or separation medium containing chelating groups on its surface, which carry a nickel, copper or zinc ions. The histidine marker binds strongly to these metal ions. Such a reagent can therefore be used to separate a sample of EndoS49.
[00120] [00120] Another preferred modification comprises the addition of a biotin tag. The presence of a biotin tag means that the polypeptide binds with a high affinity for a reagent or separation comprising streptavidin media. The biotin tag binds strongly to streptavidin. Such a reagent can therefore be used to separate a sample of EndoS49.
[00121] [00121] Preferred reagents or separation media are populations of magnetic particles capable of binding to the EndoS49 polypeptide. For example, where the EndoS49 polypeptide is derivatized with a histidine marker, the magnetic particles contain in their groups chelating surfaces that carry a nickel, copper or zinc ions. Alternatively, when the EndoS49 polypeptide is derivatized with a biotin tag, the magnetic particles contain streptavidin on its surface.
[00122] [00122] Thus, a preferred method of removing EndoS49 from a sample comprises using a population of magnetic particles as described above and carrying out magnetic field separation over the sample. The magnetic particles are preferably magnetic nanoparticles, and the separation of the magnetic field is preferably the largest magnetic field gradient separation.
[00123] [00123] It should be understood that any suitable means of separation can be used. For example, the alternative means described in the previous section.
[00124] [00124] The EndoS49 of the contacted sample can be evaluated for the presence or absence of IgG bound by any appropriate means. For example, the molecular weight of EndoS49 can be analyzed.
[00125] [00125] IgG-bound EndoS49 have a higher molecular weight than IgG-unbound EndoS49. Consequently suitable methods include any method capable of discriminating protein species by weight, for example, SDS-PAGE and Western Blot, mass spectrometry, etc. Alternatively, the Western Blot above can be directly analyzed for the presence of IgG using antibodies or IgG antibodies specific to a particular IgG subclass. Detecting proteins from a stain in this manner is a technique widely used in the art.
[00126] [00126] Other suitable means for detecting the presence or absence of IgG linked to EndoS49 include incubating EndoS49 with antibodies to IgG or IgG-binding proteins with coupled enzymes (eg, horseradish peroxidase, alkaline phosphatase), followed by the addition of fluorogenic / chromogenic substrates. In this example, the development of a color signal indicates the presence of IgG antibodies, with the amount of IgG being proportional to the signal strength. Detecting proteins in this manner is a technique widely used in the art.
[00127] [00127] In addition, suitable means for detecting the presence or absence of EndoS49-bound IgG comprise first separation of EndoS49-bound IgG so that it can be analyzed / detected independently of EndoS49 by any of the above methods or any other suitable method . IgG can be separated from EndoS49 by any suitable means.
[00128] [00128] Suitable means include eluting IgG from EndoS49 by contacting EndoS49 from the sample in contact with a suitable elution buffer. The choice of the elution buffer will normally depend on whether or not the IgG bound to EndoS49 is known or suspected of being inactivated by contact with acid-sensitive acids, or is denatured.
[00129] [00129] When the antibody is not acid sensitive, an elution protocol using a low pH elution buffer can typically be employed. Elution protocols of this type are well known in the art. Such elution buffers typically have a pH below about pH 3, more preferably below about pH 2. Preferred examples include 0.1 M glycine at pH 2. In addition, or, optionally, such elution buffers can typically comprise at least one of the following:
[00130] [00130] - Sodium or potassium salts, preferably in a concentration of about 0.5 M and about 1 M;
[00131] [00131] - Mono-, di- or polysaccharides with structures similar to the glycan associated with Asn-297 in native IgG;
[00132] [00132] or any combination thereof. However, as described above, the methods of the invention are particularly suitable for the detection / isolation of acid sensitive antibodies. Where EndoS49-bound IgG is known or suspected to be sensitive to acids, therefore, it is preferable to use buffers and protocols that do not require a low elution pH. Such protocols are also known in the art and are based on the principle of providing a buffer that comprises a molecule that competes with IgG bound by binding to EndoS49, thereby leading to the release of bound IgG. Suitable competition elution buffers, therefore, typically comprise one or more mono-, polysaccharides with glycan-like structures associated with Asn-297 in native di-, or IgG. Particularly preferred elution buffers comprise about 0.25 M to about 0.5 M sucrose, preferably with a pH of about 5.3 to about 8.3. Examples of preferred specific elution buffers include, for example:
[00133] [00133] 0.25 M sucrose, in PBS pH 7.4; 0.5 M sucrose, in PBS pH 7.4; 0.25 M sucrose, in PBS pH5.3; 0.25 M sucrose, in PBS pH 8.5 and 0.25 M sucrose, 0.25 M maltose, in PBS pH 7.4.
[00134] [00134] In addition, or, optionally, such competitive elution buffers may typically comprise the sodium or potassium salts, preferably in a concentration of about 0.5 M and about 1 M.
[00135] [00135] Also can be used the means of separating IgG bound from EndoS49 as described above, to obtain IgG isolate.
[00136] [00136] Method for assessing the state of glycosylation and / or functional quality of a sample containing IgG
[00137] [00137] The EndoS49 polypeptides of the invention in unmodified form have the ability to hydrolyze the IgG glycan.In addition, as described above, the EndoS49 polypeptides devoid of IgG endoglycosidase activity and having IgG binding activity are specific for glycosylated and / or native, functionally active IgG.
[00138] [00138] Thus, the EndoS49 polypeptides can be used to analyze the glycosylation status of a glycoprotein, and, in particular, an IgG antibody.
[00139] [00139] According to one aspect of the invention, an IgG antibody is incubated with an EndoS49 polypeptide of the invention that has endoglycosidase activity.
[00140] [00140] The products obtained can be analyzed using any suitable techniques, including HPLC, mass spectrometry, gel chromatography, gel electrophoresis, spectrophotometry, capillary electrophoresis. Such analysis methods can be carried out at any stage of the preparation of a protein, for example, during the screening of candidate drugs, during the development of biological drug production processes, as well as quality control in release assays and, during the production.
[00141] [00141] EndoS49 polypeptides that have been modified to remove or reduce endoglycosidase activity, or that retain the ability to bind IgG in its native form can also be useful in glycomapping, and in particular glycoprotein analysis, and in particular the IgG structure.
[00142] [00142] For example, using said EndoS49 polypeptides, optionally in combination with an alternative Igg binding reagent, the present invention provides a method for assessing the state of glycosylation or the functional quality of an IgG containing the sample, which comprises, having a first and a second sub-sample of the sample containing IgG, contacting the first sub-sample with an EndoS49 polypeptide as described in the previous section, and the second sub-sample with an alternative Igg binding reagent that is capable of non-binding glycosylated and / or denatured, inactivates IgG, and then quantifies the amount of IgG bound to the EndoS49 polypeptide in the first sub-sample, and the amount of IgG bound to the alternative Igg binding reagent in the second sub-sample. Finally, by comparing both amounts of bound IgG determined in the first and second sub-samples, the state of glycosylation or the functional quality of an IgG containing the sample can be assessed.
[00143] [00143] The alternative Igg binding reagent is typically Protein A or Protein G, which bind to all forms (native or denatured) of IgG. In this example, the amount of IgG bound to said reagent therefore represents the total IgG present in the second sub-sample. The EndoS49 polypeptide binds only to the glycosylated and / or native, functionally active IgG, and therefore the amount of IgG bound to EndoS49 represents only the glycosylated and / or native, present functionally active IgG in the first sub-sample. By comparing the concentration of total IgG antibodies from the second sub-sample to the concentration of native IgG in the first example, the person skilled in the art will recognize that a proportion is obtained that reflects the proportion of IgG in the original sample that is present in its glycosylated and / or native form, functionally active.
[00144] [00144] In another embodiment, the alternative Igg binding reagent could be specific for the non-glycosylated and / or denatured IgG. Such a reagent can be, for example, an antibody. Therefore, in this embodiment, the proportion of IgG in the original sample that is present in its glycosylated and / or native, functionally active form can be assessed by the formula: Amount of IgG in the first sub-sample / (Amount of IgG in the first sub-sample + IgG quantity in the second sub-sample)
[00145] [00145] The examples of the above methods are contacted with EndoS49 polypeptide or alternative Igg binding reagent under conditions suitable for the interaction between the polypeptide or the reagent and the sample to occur and IgG binding activity to occur. Suitable conditions are, for example, equivalent to those established in the previous section. Method for the isolation of IgG Fab or Fe fragments from a sample containing IgG
[00146] [00146] The methods of the present invention can be used to isolate Fab fragments from samples containing IgG. In one embodiment, the present invention provides a method for isolating Fab IgG fragments from a sample containing IgG, the method of which: (a) contacting said sample containing IgG with Ides, and an EndoS49 polypeptide; (b) separating said IDEs and said EndoS49 polypeptide from the contacted sample, and isolating Fab fragments.
[00147] [00147] Preferred methods for separating the Ides and EndoS49 polypeptide from a sample using an Ides comprise and / or EndoS49 polypeptide which is derivatized or modified as described above. Modification of the same or a different one can be used in each of the Ides and the EndoS49 polypeptide.
[00148] [00148] A preferred modification comprises the addition of a histidine marker. The presence of a histidine marker means that the polypeptide binds with a high affinity to a reagent or separation medium containing chelating groups on its surface, which carry a nickel, copper or zinc ions. The histidine marker binds strongly to these metal ions. Such a reagent can therefore be used for separate Ides and / or EndoS49 polypeptide from a sample.
[00149] [00149] Another preferred modification comprises the addition of a biotin tag. The presence of a biotin tag means that the polypeptide binds with a high affinity for a reagent or separation comprising streptavidin media. The biotin tag binds strongly to streptavidin. Such a reagent can therefore be used for separate Ides and / or EndoS49 polypeptide from a sample. Preferred reagents or separation media are populations of magnetic particles capable of binding the EndoS49 polypeptide. For example, where the Ides and / or EndoS49 polypeptide polypeptide is derivatized with a histidine marker, the magnetic particles contain in their chelating surface groups that carry a nickel, copper or zinc ions. Alternatively, where the Ides and / or EndoS49 polypeptide polypeptide is derivatized with a biotin tag, the magnetic particles contain streptavidin on its surface.
[00150] [00150] Thus, a preferred method of removing EndoS49 from a sample comprises using a population of magnetic particles as described above and carrying out magnetic field separation over the sample. The magnetic particles are preferably magnetic nanoparticles, and the separation of the magnetic field is preferably the largest magnetic field gradient separation.
[00151] [00151] Thus, step (a) of the method above preferably comprises additionally the contact of the sample with a population of magnetic nanoparticles capable of binding Ides and the EndoS49 polypeptide, and in which step (b) comprises conducting magnetic field separation over the sample.
[00152] [00152] The EndoS49 polypeptide is preferably a modified EndoS49 polypeptide deficient in endoglycosidase activity.
[00153] [00153] In the above embodiment of the present invention, the sample containing IgG typically comprises purified or isolated IgG. By "purified or isolated IgG" is meant a fraction of normal commercial grade IgG purity. IgG is typically isolated from a sample such as serum or, in the case of recombinant IgG, from cell lysates. Isolation can be carried out according to any suitable method, preferably according to the method described above for the isolation of IgG using a modified EndoS49 polypeptide deficient in endoglycosidase activity. Thus, an embodiment of the invention includes the method described above, comprising, prior to step (a): (i) contacting said IgG-containing sample with a polypeptide that lacks EndoS49 IgE endoglycosidase activity, thus allowing the formation of an IgG-EndoS49 polypeptide complex; (ii) separating said IgG-EndoS49 polypeptide complex from the contacted sample; (iii) IgG eluting from the IgG-EndoS49 polypeptide complex thus obtaining a sample containing IgG, and in which steps (a) and (b) the sample containing IgG obtained in step (iii) is carried out. The separation of the IgG-EndoS49 polypeptide complex from a sample is preferably carried out according to the methods described in the previous section related to the methods for determining the presence or absence of IgG in a sample, or to isolate IgG from a sample. containing IgG.
[00154] [00154] In an alternative embodiment of the invention, the methods are adapted to isolate Fab fragments from IgG-contained samples, without the need to purify the IgG before performing the method. These methods can be performed on a sample containing unpurified IgG, for example, total serum, cell lysate or cell culture medium. In this embodiment of the invention, the method comprises: (a) contacting said IgG-containing sample with an EndoS49 polypeptide to thereby allow the formation of an IgG-EndoS49 polypeptide complex; (b) separating said IgG-EndoS49 polypeptide complex from the contacted sample; (c) adding to the polypeptide IgG-EndoS49 complexes obtained in step (b) Ides, and (d) separating said Ides and said EndoS49 polypeptide from the mixture obtained in (c); and isolating Fab fragments.
[00155] [00155] The methods for separating the Ides and / or EndoS49 polypeptide from the above samples / mixtures comprise, preferably using an Ides and / or EndoS49 polypeptide which is derivatized or modified as described above. Modification of the same or a different one can be used in each of the Ides and the EndoS49 polypeptide. Preferred reagents or separation media are populations of magnetic particles capable of binding to IDEs and / or EndoS49 polypeptide. For example, where the Ides and / or EndoS49 polypeptide polypeptide is derivatized with a histidine marker, the magnetic particles contain in their groups chelating surfaces that carry a nickel, copper or zinc ions. Alternatively, where the Ides and / or EndoS49 polypeptide polypeptide is derivatized with a biotin tag, the magnetic particles contain streptavidin on its surface.
[00156] [00156] Thus, step (a) of the method above preferably comprises additionally the contact of the sample with a population of magnetic nanoparticles capable of binding to the EndoS49 polypeptide, step (c) additionally comprises the contact of the complexes of IgG-EndoS49 polypeptide obtained in step (b) with a population of magnetic nanoparticles capable of binding Ides and the polypeptide
[00157] [00157] It should be understood that any suitable means of separation can be used. For example, the alternative means described in the section related to methods for the isolation of a population of cells, which are substantially free of IgG molecules bound to FcyRs could be adapted for the separation of Ides and / or EndoS49 polypeptide.
[00158] [00158] The EndoS49 polypeptide in the above embodiment is preferably a modified EndoS49 polypeptide deficient in endoglycosidase activity.
[00159] [00159] The previous methods of the invention can also be used to isolate the Fc fragments from samples containing IgG. In such an embodiment of the invention, the method comprises: (a) contacting said sample containing IgG with Ides; (b) separation of Ides from the mixture obtained in step (a), thus isolating the Fab and Fc fragments; (c) contacting said Fab and Fc fragments with an EndoS49 polypeptide to thereby allow the formation of an Fc-EndoS49 fragment polypeptide complex; (d) separating the complex polypeptide Fc-EndoS49 fragments from the mixture obtained in step (c); and (e) isolating Fc fragments from the complex EndoS49 fragment of Fc polypeptide obtained in step (d).
[00160] [00160] It should be understood that any appropriate separation medium can be used, as described above, however, the methods for separating the Ides and / or EndoS49 polypeptide from the above samples / mixtures comprise, preferably using a
[00161] [00161] Preferably, step (a) of the above method additionally comprises the contact of the sample with a population of magnetic nanoparticles capable of binding to Ides, step (c) additionally comprises the contact of the Fab and Fc fragments , obtained in step (b) with a population of magnetic nanoparticles capable of binding to the EndoS49 polypeptide, and in which steps (b) and (d) comprise the accomplishment of magnetic field separation on the sample of (a) and the mixture obtained in (c), respectively. The EndoS49 polypeptide is preferably a modified EndoS49 polypeptide deficient in endoglycosidase activity.
[00162] [00162] In an alternative embodiment, the Fc fragments can be isolated from a sample containing IgG by a method comprising: (a) contacting said sample containing IgG with Ides and an EndoS49 polypeptide (b) separating the EndoS49 polypeptide from the mixture obtained in (a); isolating Fc fragments.
[00163] [00163] It should be understood that any appropriate means of separation can be used, as described above. However, preferably, step (a) of the above method additionally comprises contacting the sample with a population of magnetic nanoparticles capable of binding to the EndoS49 polypeptide but not Ides, and in which step (b) comprises carrying out magnetic field separation over the mixture obtained in (a). Preferably, the Ides and / or the polypeptide are derivatized or modified EndoS49, as described above, with the proviso that a different modification is applied to each. For example, where Ides is modified by the addition of a histidine marker, such that it binds to a population of magnetic particles, containing chelating groups on its surface that carry a nickel, copper or zinc ions, the EndoS49 polypeptide can be modified by the addition of a tin bio tag in such a way that it binds to a population of magnetic particles, containing streptavidin on its surface. The EndoS49 polypeptide is preferably a modified EndoS49 polypeptide deficient in endoglycosidase activity.
[00164] Similar to the methods of isolating Fab fragments, it will be appreciated that in methods for separating Fc fragments from the sample containing IgG it typically comprises purified or isolated IgG. A preferred method of isolating IgG is described in steps (i) to (iii) as set out above. Kits
[00165] [00165] The present invention provides - A kit to isolate IgG from a sample containing IgG, comprising: (a) an EndoS49 polypeptide according to the invention, which has no endoglycosidase activity, and optionally (b) means to separate the said EndoS49 polypeptide from a sample. a kit for determining the presence or absence of IgG in a sample, comprising: (a) an EndoS49 polypeptide according to the invention, which has no endoglycosidase activity, and optionally (b) means for separating said EndoS49 polypeptide from a sample . - A kit to assess the glycosylation status and / or the functional quality of an IgG containing the sample, comprising: (a) an EndoS49 polypeptide according to the invention, which has no endoglycosidase activity, and optionally; (b) an alternative Igg binding reagent that is capable of binding denatured and / or deglycosylated IgG;
[00166] [00166] The alternative Igg binding reagent comprises protein G and / or protein A and / or protein A / G.
[00167] [00167] The present invention also provides: - A kit for isolating IgG Fab or Fc fragments comprising: (a) IDEs; (b) an EndoS49 polypeptide; and (c) The means for separating said IDEs and said EndoS49 polypeptide from a sample.
[00168] [00168] In a preferred embodiment, the kit additionally comprises an EndoS49 polypeptide according to the invention, which has no endoglycosidase activity and a means for separating said EndoS49 polypeptide from a sample. The polypeptide is preferably an EndoS49 polypeptide EndoS49 according to the invention, which has no endoglycosidase activity.
[00169] [00169] Preferred embodiments of the above kits further comprise instructions for using the kit of a method of the invention. Other preferred embodiments include those in which the means for separating an EndoS49 polypeptide, an alternative Igg binding reagent, an Ides polypeptide, or a polypeptide from an EndoS49 sample are populations of magnetic nanoparticles, where each population is capable of bind to at least one of the indicated polypeptides / reagents / proteins. In this embodiment, the kit typically additionally comprises instructions for carrying out the magnetic field separation on the sample.
[00170] [00170] In preferred embodiments of the above methods and kits, the
[00171] [00171] polypeptides / proteins / reagents used are derivatized with an affinity tag, preferably a histidine marker, to aid the separation of said polypeptides. The following examples illustrate the invention: Example 1 MATERIALS AND METHODS Bacterial strains and growth
[00172] [00172] The genome of the NZ131 GAS strain, serotype M49, was sequenced and this strain was therefore selected as the reference strain in the present work (McShan et al. 2008) (Chaussee et al., 1999). NZ131 GAS strain was propagated on blood agar andEscherichia coli Top 10 (Invitrogen) and BL21 pLysS (Invitrogen) were propagated on lysogen agar broth (LB). For selection in E. coli Top 10 cells, carbenicillin was used at 100 ug / mL and for E. coli BL21 pLysS, 100 µg / ml carbenicillin and 34 µg / ml chloramphenicol were used. E. coli overnight cultures were performed in LB medium at 37 ° C with aeration. NZ131 genomic DNA preparation of GAS strain was performed using DNA Puregene Purification Kit (Qiagen).
[00173] [00173] The transformation was carried out using heat shock at 42 ° C for 30 s. Plasmid preparations from E. coli were performed using the miniprep plasmid Kit I (EZNA). All primers used in this work are listed in Table 2. The recombinant expression of EndoS49.
[00174] [00174] EndoS49 recombinant expression in E. coli was created by PCR amplification of the strep ndoS49 gene from group A strain NZ131, serotype M49 with the primers ndoS49-F-Bamffl, CTGTAAGGATCCAGGAGAAGACTG, and ndoS49-R-XTTTAGATGATGATGATGACT . The ndoS49 gene fragment was digested with the restriction enzymes BamHI and XhoI and ligated to the expression vector pGEX-5X-3 (Amersham Biosciences) using T4 DNA ligase (Fermentas) creating the plasmid pGEX-ndoS49. The expression vector was used to transform E. coli Top 10 chemically competent cells and recombinant cells were cultured on carbenicillin plates at 100 µg / ml and screened by PCR using the ndoS49-F-BamHI and ndoS49-R-XhoI primers. Positive clones were isolated and plasmid pGEX-ndoS49 was purified and transformed into E. coli strain BL21 pLysS expression as described above.
[00175] [00175] A recombinant clone was grown overnight at 37 ° C with antibiotics and diluted 1:20 in LB medium with antibiotics and cultured for 3 h. EndoS49 protein expression was induced with 0.1 mM IPTG for 3 h. The cells were harvested and lysed with BugBuster Protein Extraction Reagent (Novagen). Recombinant GST-EndoS49 was purified in column with glutathione-Sepharose 4B (GE Healthcare) and eluted with reduced glutathione. The mutagenesis of EndoS49.
[00176] [00176] Site-directed mutagenesis from glutamic acid 186 (Glu-186) to leucine (E186L) was performed using II QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. The mutagenesis primer used was CTAGATATTGATATTCTTCACGAATTTACGAAC in combination with the antisense from the above sequence and plasmid pGEX-ndoS49 (underlined mutation). This generated plasmids pGEX-ndoS49 (El% 6V) and, after sequencing, recombinant EndoS49 (E186L) was expressed and purified as described by EndoS49.The truncated versions of EndoS49 were constructed by amplifying parts of the NZ131 GAS ndoS49 gene with primersndoS49 (truncl-5) which contains local BamHI and XhoI restrictions (Table 2). The fragments were digested and ligated into the pGEX vector as described above, and transformed into E. coli Top 10 and, subsequently, BL21 pLysS and cultured with antibiotics. The proteins were produced as above and the 80 kDa EndoS49 (truncl), 70 kDa EndoS49 (trunc2) proteins, EndoS49
[00177] [00177] 1 µg recombinant EndoS49and its mutants were incubated with 3 µg of each glycoprotein in 20 PBS overnight at 37 ° C. Glycol hydrolysis was analyzed on a 10% SDS-PAGE gel and then analyzed with LCAlectin blot as previously described (Collin and Olsen, 2001). Slot-blot analysis
[00178] [00178] EndoS49 and its mutants were immobilized on a PVDF membrane activated methanol at 4, 2, 1 mg in PBS per slot using Millipore blot slot equipment. The membrane was blocked with 5% skim milk (Disco) for 1 h at room temperature.
[00179] [00179] Washing has always been done for 3x10 minutes in PBST. The membrane was incubated with 10 µg of human IgG (Sigma) in 0.5% skimmed milk for 1 hour at 37 ° C and then washed. 5 µg of horseradish peroxidase conjugated to protein G (Invitrogen) was added to the membrane and incubated for 1 h at 37 ° C. After washing the membrane, it was developed with Supersignal West Pico substrate chemiluminescence (Thermo Scientific). Bioinformatics analysis
[00180] [00180] The ndoS49 and NDOS genes were translated into EndoS49 and EndoS and compared by the ClustalW algorithm within the MacVector software (MacVector Inc.). The phylogenetic tree was constructed with MacVector using NCBI PubMed protein sequences with the following access numbers: EndoS (AF296340), EndoE (AAR20477), EndoH (NP 631673), Endoc (ADC53484), EndoF2 (P36912), EndoF3 (P36913 ) (Collin and Olsen, 200 lb) (Collin and Fischetti, 2004) (Tarentino and Plummer, 1974) (Tarentino et al., 1993). Screening PCR for ndoS49
[00181] [00181] Primers amplifying the GAS ndoS49 gene were designed and denoted ndoS49-F (AAAACGCGGACCACTATATGC) and ndoS49-R (AAACGTTGTCCGAGGATTTG). 42 gas samples were propagated on blood agar and grown overnight at 37 ° C with 5% CO 2. Single colonies were harvested and lysed in sterile 20 H 20 at 99 ° C for 10 minutes. These lysates were used as a template for a PCR reaction to accurately detect ndoS49 in GAS 42 strains. As a positive control, primers for amplification of the recA gene were designed, recA-F (AGCCCTTGATGATGCTTTG) and recA-R (AACAATTCTGGGTGATCGG). As positive controls, both PCR reactions used the NZ131 genomic DNA strain GAS (M49) and API (Ml) as a model.
[00182] [00182] The ndoS49 and NDOS genes were in silica translated into proteins and compared by ClustalWalgorithm. At the gene level the identity is 50% and 37% in relation to the protein level. The ClustalW analysis revealed a (nearly) identical signal peptide sequence and a conserved family of 18 lasecatalytic hydro domain glycoside (DGLDIDIE) (Figure 1). Experimental analysis of EndoS has shown that tryptophanes are essential for hydrolysis-glycan activity (Allhorn et al., 2008). These tryptophans are also conserved in EndoS49.EndoS49. Recombinants show glycan hydrolyzing activity in human glycoproteins
[00183] [00183] The 90 kDa EndoS49 was successfully recombinantly expressed in E. coli BL21 and purified from the soluble fraction using the GST marker. EndoS49 (E186L), a catalytic mutant with the GH18 motif glutamic acid (El 86) replaced by a leucine (L), was constructed and purified in the same way. To map protein activity, 5 carboxy-terminal truncated versions of the enzymes were constructed and denoted EndoS49 (truncl) 80 kDa, EndoS49 (trunc2) 70 kDa, EndoS49 (trunc3) 60 kDa, EndoS49 (trunc4) 50 kDa and 42 kDa EndoS49 (trunc5). This collection of enzymes was used to analyze the hydrolysis activity of EndoS49 glycan in human glycoproteins. First, the enzymes were incubated with human IgG overnight and analyzed on an SDS-PAGE gel and with ACV lectin blot, the detection of the mannose structures in the IgG glycans. The gel revealed a 4 kD deviation from the heavy chain of IgG treated with EndoS49 and the LCA lectin blot confirmed this shift as a lack of N-linked glycans (Figure 2A). EndoS49 (E186L) showed no change and no change in glycans composition suggesting that E186 has a crucial role in the catalytic activity of EndoS49. Regarding the truncated enzymes, EndoS49 (truncl-4) showed an activity on the IgG glycans but EndoS49 (trunc5), the smallest of the enzymes (42 kDa), did not reveal any hydrolysis glycan activity (Figure 2A).
[00184] [00184] Additional analysis of IgG deglycosylation by EndoS49 was performed by incubating IgGi_ 4 with EndoS49 and EndoS49 (E 186L), overnight. The IgG subclasses were analyzed as described above and showed that EndoS49 has activity in all four IgG subclasses, and in line with the previous results, the catalytic mutant did not reveal any activity (Figure 2B). The enzyme collection incubation with alpha-1-microglobulin, the heavy glycosylated human whey protein, and the SDS-PAGE analysis showed the EndoS49on glycan hydrolysis activity of this glycoprotein (Figure 2C). To further elucidate the specificity of EndoS49, a model substrate consisting of an N-acetyl-beta-D-glucosaminide coupled to the 4-methylumbelliferylwas fluorescents incubated with EndoS49 for 1, 2, 3, 4 and 16 hours and the measured fluorescence. Fluorescence will increase if sugar is cleaved, but no increase in intensity has been observed (data not shown), suggesting that EndoS49 has activity only on glycoprotein substrates. EndoS49 connection to IgG
[00185] [00185] The finding that EndoS49 has glycans hydrolysis activity in IgG led us to believe that the enzyme binds IgG. This was evaluated by splotch analysis where EndoS49 and its catalytic mutant and truncated versions were immobilized on a PVDF membrane and incubated with IgG and the binding detected with protein G coupled to horseradish peroxidase (HRP). The groove stain exhibits increased IgG binding by the catalytic mutant EndoS49 (E186L) (Figure 3). The ndoS49 gene is present in the GAS M49 serotype
[00186] [00186] To elucidate whether ndoS49 is present in all serotypes other than M49, a rigorous PCR was implanted to analyze the presence of the ndoS49 gene in a selection of GAS strains. The primers ndoS49-F and ndoS49-R were used in a PCR on the lyses of the GAS colonies together with the positive control of amplifying the recA gene, present in all GAS strains. The ndoS49 gene was amplified in all selected M49 gas serotypes and also of M60 serotype, while no other serotype gave a PCR product (Table 1).
[00187] [00187] In the sequenced genomes of GAS strains NZ131 (M49) and MGAS5005 (Ml) from the genes surrounding ndoS49 and NDOS were compared revealing that the genes are located in the same genomic context and that the surrounding genes are highly conserved (Figure 4). The total length EndoS49 was compared to a selection of endoglycosidases previously described, EndoS, Endoc, EndoH, EndoE, EndoF2, EndoF3) and a phylogenetic tree was reconstructed (Figure 5). This revealed that EndoS and Endoc are more closely related than EndoS and EndoS49 and that Streptococcus endoglycosidases are closely related compared to enzymes from other bacteria.
[00188] [00188] Monoclonal Ig molecules may show small variations in the Fc glycans and as a consequence the Fc glycans may appear as a non-homogeneous pool of Fc glycans. The vast majority of glycans are identical, but a minority may show variable carbohydrate structure or composition. The variety arises both from the origin of the Fe part, which can be human, humanized or from another species, and from the choice of a cell culture cell line and the production conditions. In this example two well-known IgG-based drugs, Avastin and Erbitux, have been deglycosylated with both EndoS and EndoS49. The samples were incubated with EndoS or EndoS49 as set out in the Table below.
[00189] [00189] The results are shown in Figure
[00190] [00190] However EndoS49 shows a complete deglycosylation profile and is, therefore, the most favorable enzyme when it comes to glycan immunoglobulin profiles.

权利要求:
Claims (9)
[1]
1. Isolated polypeptide characterized by comprising: (a) the amino acid sequence SEQ ID NO: 1; (b) a variant thereof having at least 95% identity with the amino acid sequence SEQ ID NO: 1 more than at least 810 contiguous amino acids of SEQ ID NO: 1 and having the endoglycosidase activity of a polypeptide consisting of an amino acid sequence of SEQ ID NO: 1.
[2]
2. Polypeptide according to claim 1, characterized by the fact that it consists of the amino acid sequence of SEQ ID NO: 1.
[3]
3. Polynucleotide characterized by comprising a sequence that encodes a polypeptide as defined in claim 1 or claim 2.
[4]
Polynucleotide according to claim 3, characterized in that it comprises: (i) SEQ ID NO: 3 or a sequence complementary thereto; (ii) a sequence that hybridizes under stringent conditions to the sequence defined in (i); (iii) a sequence that is degenerated as a result of the genetic code for a sequence, as defined in (i) or (ii); (iv) a sequence having at least 60% identity with a sequence as defined in (i), (ii) or (iii); or (v) a fragment of any of the sequences (i), (ii), (iii) or (iv), and which encodes a polypeptide containing endoglycosidase activity, or the polypeptide of SEQ ID NO: 1.
[5]
Expression vector characterized by comprising a polynucleotide as defined in claim 3 or 4.
[6]
6. Method for the hydrolysis of glycan of a glycoprotein characterized in that it comprises incubating the glycoprotein with a polypeptide as defined in claim 1 or 2.
[7]
7. Method for assessing the glycosylation status of a glycoprotein, comprising incubating a glycoprotein with a polypeptide according to claim 1 or 2, and analyzing the products produced.
[8]
Method according to claim 7, characterized in that it comprises: (a) contacting the glycoprotein with said polypeptide for the glycan hydrolysis of the glycoprotein; (b) separating the glycan from the deglycosylated protein; (c) analyzing the glycan and / or deglycosylated protein produced.
[9]
Method according to any one of claims 6 to 8, characterized in that the glycoprotein includes an IgG antibody or an IgG monoclonal antibody.
FIGURE 1. IW Clustal Alignment of Endo S49 (NZ131) and EndoS (5005)
1/4
FIGURE 2.
Endo activity S49 IgG Blue stain LCA stain Endo activity S49 on IgG subclasses Stain blue LCA stain Endo activity S49 Alpha-1-microglobulin (A1m) Blue stain
FIGURE 3.
Connection of Endo S49 to IgG FIGURE 4.
Similarity: Similarity: Similarity: 50% Similarity: Similarity: 99% 98% 94% 99%
FIGURE 5. Phytogenetic tree of endoglycosidases
Method: Neighbor Joining; Bootstrap (10,000 reps); tie break = systematic Distance: Not correct (“p”) Intervals distributed proportionally
FIGURE 6.

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WO2017124084A1|2016-01-15|2017-07-20|University Of Maryland, College Park|Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins|

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US20210228738A1|2017-07-17|2021-07-29|INSERM |Compositions and methods for increasing or enhancing transduction of gene therapy vectors and for removing or reducing immunoglobulins|

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JP2021528406A|2018-06-19|2021-10-21|グリコス バイオメディカル オーワイ|Conjugate|

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EP3813883A1|2018-06-29|2021-05-05|Glykos Biomedical Oy|Conjugates|

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WO2021123506A1|2019-12-18|2021-06-24|Glykos Biomedical Oy|Stabile conjugate|

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CN111044731B|2019-12-19|2021-05-07|北京科技大学|Method for separating and enriching peptide impurities in polypeptide medicament by pulse incubation immunoreaction|

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WO2021142199A1|2020-01-09|2021-07-15|Mersana Therapeutics, Inc.|Site specific antibody-drug conjugates with peptide-containing linkers|

法律状态:
2021-01-19| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-08-10| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE A 9A ANUIDADE. |

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2021-09-08| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO EM FUNCAO DA REVOGACAO DO ART. 229-C DA LEI NO 9.279, DE 1996, POR FORCA DA LEI NO 14.195, DE 2021 |

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2021-11-30| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2640 DE 10-08-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

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2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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GB1115841.7|2011-09-13|

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GB201115841A|GB201115841D0|2011-09-13|2011-09-13|Protein and method|

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PCT/EP2012/067841|WO2013037824A1|2011-09-13|2012-09-12|Endoglycosidase from streptococcus pyogenes and methods using it|

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