WO2013010660A1 - Enzymes having alpha2,9 endosialidase activity - Google Patents

Abstract

The invention relates to polypeptides, polynucleotides encoding the polypeptides, the use of such polynucleotides and polypeptides, and more specifically to polypeptides having alpha2,9 endosialidase activity.

Description

Enzymes having alpha2,9 endosialidase activity

This invention relates to polypeptides, polynucleotides encoding the polypeptides, the use of such polynucleotides and polypeptides, and more specifically to polypeptides having alpha2,9 endosialidase activity.

Sialic acid is a monosaccharide with a nine-carbon backbone, commonly occuring in nature ranging from bacteria, plants, fungi, and yeasts to human. To date, more than 50 structural variants of sialic acid are known to occur in nature, which variants include various substitutions like acylation, sulfation, and methylation and combinations thereof (Varki and Varki, 2007). The main derivatives of sialic acid occurring in nature are N-acetylneuraminic acid (Neu5Ac; Fig. 1A, compound 1), N-gylcolylneuraminic acid, (Neu5Gc; Fig. 1A, compound 2), and 3-deoxy-D-glycero-D-galacto-nonulosonicacid (KDN; Fig. 1A, compound 3). Sialic acid forms glycoconjugates, e.g. alpha2,3 or alpha2,6 linkages to galactose or galactosamine, and short oligomers, e.g. sialic acids connected via alpha2,8 linkages. Also, the following kinds of homopolymers of sialic acid - also referred to as polysialic acid (polySia) - are known: alpha2,8 linked polySia, alpha2,9 linked polySia, and alternating alpha2,8/alpha2,9 linked polySia (see, e.g. Fig. 1 B, compounds 4-6). Several modifications are known to occur on the homopolymers like O-acetylation at position 07 and 08 of NmC- polySia (Bergfeld et al., 2009).

In vertebrates, alpha2,8 linked polySia (Fig. 1B, compound 4), for example, is mainly found as a posttranslational modification of the neural cell adhesion molecule (NCAM). Here, polySia is widely expressed during . ontogenetic development and remains an important modulator of neuronal plasticity in the adult brain (Roth ef al., 1988; Rutishauser and Landmesser, 1996; Kleene and Schachner, 2004; Weinhold ef a/., 2005; Conchonaud et al., 2007). Studies investigating the role of polySia in vertebrates critically rely on the use of alpha2,8-specific endosialidases to positively identify the nature of this posttranslational modification.

In the bacterial domain all three so far known forms of polySia are expressed as extracellular capsules (Table 1). In general, bacterial capsules provide a physical barrier conferring resistance against attack from components of the immune system and bacteriophages alike. Moreover, polySia capsules are one of the major virulence factors of pathogens causing meningitis, septicaemia and urinary tract infections. The role of the capsule during infection is primarily to mask underlying bacterial surface antigens thereby conferring resistance against host immune defenses such as complement-mediated lysis, phagocytosis and opsonization (Johnson, 1991 ). However, specialised bacteriophages have developed tailspike proteins, which bind with high affinity to and enzymatically degrade the host capsular polySia. Table 1. Bacterial strains encapsulated with polySia in different linkages

PolySia linkage Bacterial strain Reference

alpha2,8 Escherichia coli K1 (Barry and Goebel, 1957;

McGuire and Binkley, 1964)

Neisseria meningitidis serogroup B (Bhattacharjee et al., 1975)

Moraxella nonliquefaciens (Devi et al., 1991)

Mannheimia (Pasteurella) haemolytica A2 (Adlam et al., 1987) alpha2,9 Neisseria meningitidis serogroup C (Bhattacharjee et al., 1975) alternating Escherichia coli Bos12 (016:K92:H-) (Furowicz and 0rskov, 1972) alpha2,8/alpha2,9

Escherichia coli K92 (Egan, 1977)

For instance, about 30 lytic bacteriophages have been isolated from sewage samples in relation to E. coli K1 (Gross et al., 1977; Kwiatkowski et al., 1982; Smith and Huggins, 1982; Kwiatkowski et al., 1983; Vimr et al., 1984; Miyake et al., 1997; Scholl et al., 2001 ; Bull et al., 2010). Additionally, several prophages have been found to be integrated into the genome of E. coli K1 strains (Deszo et al., 2005; Stummeyer et al., 2006; Chen et al., 2006; Johnson et al., 2007; Touchon et al., 2009; Moriel et al., 2010; Krause et al., 2010). The factor common to all known anti-K1 -bacteriophages is their tailspike protein with polysialic acid depolymerase activity: the endosialidase (or endo-N-acylneuraminidase, endoN). Phage- borne endosialidases are currently the only known enzymes that specifically cleave alpha2,8 linkages of K1 -polySia as well as K92-polySia. They catalyse a highly specific degradation of polySia that does not interfere with mono- or short oligosialylated structures (Finne and Makela, 1985; Schwarzer et a/., 2009). Endosialidases cleaving the alpha2,9-linkages in K92-polySia or NmC-polySia have not been described as yet.

In a study by Mushtaq et al. it has been shown that unmasking pathogenic bacteria by endosialidase, treatment of neonatal rats infected with a virulent strain of E. coli K1 was an effective therapeutic strategy preventing death in over 80% of animals (Mushtaq et al., 2005). Unfortunately, due to the presence of alpha2,8 linked polySia in humans, and the unknown effects of its sudden removal, the use of current endosialidases is not a viable therapeutic option against bacteria, because all currently known endosialidases catalyse hydrolysis of polySia in alpha2,8 linkages.

In contrast to endosialidases that exclusively cleave within the polySia chain, exosialidases release only sialic acid monomers from the non-reducing end of glycoconjugates. An exosialidase from Arthrobacter ureafaciens that non-specifically has been reported to release alpha2,3, alpha2,6, alpha2,8, and alpha2,9 linked sialic acid monomers from sialo- glycoconjugates and is commercially available from Sigma (N 3786) and amsbio (# 120057- 1). The relative rates of cleavage have been reported to be in the order a(2→6) > a(2→3) > a(2-→8) and a(2→9) (Uchida, 1979). The sequence of the A. ureafaciens sialidase (here also referred to as SiaAU) is available online under the Genbank accession number BAD66680. The protein is composed of 990 amino acids. When compared to known endosialidases, SiaAU shows about 10% sequence identity, which is a typical result for a comparison of endosialidase and exosialidase sequences, emphasizing that, despite structural similarities, the catalytic mechanism differs in both enzyme classes. The bacterium Helicobacter canadensis is an emerging pathogen that has been isolated from four Canadian patients with diarrhea and an Australian patient with bacteremia (Fox et al., 2000; Tee et al., 2001 ; Loman et al., 2009). Potentially, the strain is encapsulated by alpha2,9 linked polySia like the so far only known strain Neisseria meningitidis serogroup C (NmC).

Since alpha2,9 linked polySia does not exist in humans, an alpha2,9-specific endosialidase could find wide application in polySia research and also provide a promising therapeutic strategy against microorganisms with alpha2,9 linked polySia, e.g. NmC or H. canadensis, or alternating alpha2,8/alpha2,9 linked polySia, eg. E. coli K92. Accordingly, the problem underlying the present invention is to provide a polypeptide having alpha2,9 endosialidase activity, preferably alpha2,9-specific endosialidase activity.

The present invention relates to:

(1) An isolated, synthetic or recombinant nucleic acid comprising:

(i) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the sequence has a sequence identity to the full-length sequence of SEQ ID No. 1 , 3, 5, 7, 9, 10, 13, 15, 16, or 19 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity;

(ii) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the sequence hybridizes under highly stringent conditions to the complement of the sequence of (i), wherein the highly stringent conditions comprise heating the double stranded nucleic acid encoding a polypeptide having alpha2,9 endosialidase activity to 90°C-100°C and cooling to 25°C with a cooling rate of 0.2-4°C/s;

(iii) a sequence completely complementary to any nucleic acid sequence of (i) or (ii);

(iv) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide has a sequence identity to the full-length amino acid sequence of SEQ ID No. 2, 4, 6, 8, 11 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity.

(2) A vector comprising:

(a) a nucleic acid of (1); or

(b) the vector of (a), wherein the vector is a cloning vector, an expression vector or an artificial chromosome.

(c) the vector of (a), wherein the vector is a bacteriophage, a eukaryotic virus, especially adenovirus, retrovirus or baculovirus, or an archaeal virus, especially SH1.

(3) A host cell comprising:

(a) a nucleic acid of item (1 ), or the vector of item (2); or

(b) the host cell of (a), wherein the host cell is a prokaryotic cell, especially an E. coli, a Bacillus, e.g. Bacillus subtilis or Bacillus megaterium, or a Corynebacterium cell; or a eukaryotic cell, especially a slime mold, e.g. Dictyostelium discoideum, a yeast, e.g. Saccharomyces cerevisiae, an insect cell line, e.g. Sf-9, a cell of a mammalian cell line, e.g. a CHO cell, an embryonic stem cell, or an induced pluripotent stem cell.

(4) An isolated, synthetic or recombinant polypeptide having alpha2,9 endosialidase activity comprising:

(a) an amino acid sequence having a sequence identity to the full-length amino acid sequence of SEQ ID No. 2, 4, 6, 8, 1 1 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity;

(b) an amino acid sequence encoded by a nucleic acid of item (1 );

(c) the amino acid sequence of (a) or (b), wherein the polypeptide comprises one or more modifications and has a sequence identity to the full-length amino acid sequence of SEQ ID No. 2, 4, 6, 8, 11 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, and wherein the modification comprises acetylation, acylation, arylation, amidation, azidation, methylation, glycosylation, phosphorylation, SU Oylation, PEGylation, covalent attachment of a lipid, a peptide, polypeptide, or a fluorescence dye.

(5) A composition comprising:

(a) a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide comprises (i) an amino acid sequence encoded by the nucleic acid sequence of item (1), or (ii) an amino acid sequence of item (4);

(b) the composition of (a) further comprising a second polypeptide having fluorescence or an antibiotic activity, or a combination thereof; or

(c) the composition of (a) or (b), wherein the composition is a desialylation composition, a composition for the production of a Neisseria meningitidis serogroup C vaccine, or a composition for the production of a Helicobacter canadensis vaccine.

(6) Pharmaceutical composition comprising a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of item (1) or an amino acid sequence of item (4), and, optionally, at least one carrier and/or at least one adjuvant.

(7) A polypeptide having alpha2,9 endosialidase activity, a bacteriophage comprising a nucleic acid sequence of item (1 ), or a pharmaceutical composition of item (6) for the treatment of an infection with Neisseria meningitidis serogroup C or Helicobacter canadensis, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of item (1) or an amino acid sequence of item (4). (8) A method of producing a recombinant polypeptide having alpha2,9 endosialidase activity, comprising:

(a) the steps of introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide, and recovering the polypeptide, wherein the polypeptide comprises a polypeptide encoded by a nucleic acid of item (1);

(b) the method of (a), wherein the step of recovering the polypeptide is carried out using affinity chromatography, anion exchange chromatography, reversed phase chromatography, or precipitation. Precipitation is preferably carried out using ammonium sulfate or trichloroacetic acid. (9) A method of hydrolyzing an alpha2,9 polysialic acid (polySia) linkage comprising:

(a) contacting a sample comprising matter having alpha2,9 linked polySia with a polypeptide capable of hydrolyzing an alpha2,9 polySia linkage, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of item (1) or an amino acid sequence of item (4);

(b) the method of (a), wherein the sample is derived from Neisseria meningitidis serogroup C, Helicobacter canadensis, or comprises the isolated capsular polySia thereof.

(10) An isolated, synthetic or recombinant nucleic acid comprising a sequence according to SEQ ID No. 1 , 5, 9, 10, 15, or 16 encoding a polypeptide having alpha2,9 endosialidase activity, or a polypeptide having alpha2,9 endosialidase activity and a sequence according to SEQ ID No. 2, 6, 11 , 12, 17, or 18 for use in the method of item (9).

(11) An isolated, synthetic or recombinant nucleic acid comprising a sequence according to SEQ ID No. 1 or 5 encoding a polypeptide having alpha2,9 endosialidase activity, or a polypeptide having alpha2,9 endosialidase activity and a sequence according to SEQ ID No. 2 or 6 for use in the method of item (9).

The phrases "nucleic acid" or "nucleic acid sequence" as used herein refer to an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. The phrases "nucleic acid" or "nucleic acid sequence" includes oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss- Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156. "Oligonucleotide" includes either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated. A "coding sequence" of or a "nucleotide sequence encoding" a particular polypeptide or protein, is a nucleic acid sequence which is transcribed and translated into a polypeptide or protein when placed under the control of appropriate regulatory sequences. The nucleic acids used to practice this invention may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/ generated recombinantly. Techniques for the manipulation of nucleic acids, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993). A nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof. The term "isolated" as used herein means that the material, e.g., a nucleic acid, a polypeptide, a vector, a cell, is removed from its original environment, e.g., the natural environment if it is naturally occurring. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition and still be isolated in that such vector or composition is not part of its natural environment. The term "purified" does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library have been conventionally purified to electrophoretic homogeneity. The term "purified" also includes nucleic acids that have been purified from the remainder of genomic DNA or from other sequences in a library or other environment, e.g. nucleic acids derived from an amplification method, e.g. polymerase chain reaction, by at least one order of magnitude, typically two or three orders and more typically four or five orders of magnitude.

The term "synthetic" as used herein means that the material, e.g. a nucleic acid, has been synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661 ; Belousov (1997) Nucleic Acids Res. 25:3440- 3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22: 1859. The term "recombinant" means that the nucleic acid is adjacent to a "backbone" nucleic acid to which it is not adjacent in its natural environment. Backbone molecules according to the invention include nucleic acids such as cloning and expression vectors, self-replicating nucleic acids, viruses, integrating nucleic acids and other vectors or nucleic acids used to maintain or manipulate a nucleic acid insert of interest. Recombinant polypeptides, i.e. endosialidases of the invention, generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

The terms "homology", "sequence identity" and the like are used interchangeably herein. For the purpose of this invention, it is defined herein that in order to determine the degree of sequence identity shared by two amino acid sequences or by two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). Such alignment is carried out over the full lengths of the sequences being compared. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The degree of identity shared between sequences is typically expressed in term of percentage identity between the two sequences and is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences being compared are of the same or substantially the same length. The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For example, protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art, e.g. TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444- 2448, 1988; Altschul et al, J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al, Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al, Nature Genetics 3:266-272, 1993). Also, homology or identity is often measured using sequence analysis software, e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705. Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. The terms "homology" and "identity" in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.

As used herein a wording defining the limits of a range, such as, e. g., sequence identity "from at least 85% to at most 99.9%" means any reasonable value within in this range, i. e. 85%, 85.1%, 85.2%, 87%, 88%, 89%, 90.5%, 91%, 92.5%, etc. , wherein the minimal distance of the values is defined as one nucleotide per full-length polynucleotide sequence, or one amino acid per whole polypeptide sequence, respectively, e.g. if the polypeptide sequence is 1064 amino acids in length, the minimal distance between two values is 0,094%. Moreover, any range defined by two values explicitly mentioned is meant to comprise and disclose any value defining said limits and any value comprised in said range. In other words, the range defined by the limits from at least 85% to at most 99.9% is meant to also comprise ranges such as from at least 85%, 85.1%, 85.2%, 87%, 88%, 89%, 90%, 90.5%, 91%, 92.5%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 99.7% to at most 99.9% or, alternatively from at least 85% to at most 85.1%, 85.2%, 87%, 88%, 89%, 90%, 90.5%, 91%, 92.5%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.7%, or 99.9% thereby also encompassing ranges, such as, e. g. from at least 87% to at most 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%; 99.7%, or 99.9%; from at least 88% to at most 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 99.7%; from at least 87.5% to at most 97%, etc.

Methods to generate polynucleotides/polypeptides having a sequence identity from at least 85% to at most 99.9% to a known sequence are well-known in the art. For example, mutant libraries can be generated using error-prone PCR approaches as described in Methods in Molecular Biology, 2003, Volume 231.

The present invention thus provides polynucleotides encoding polypeptides, e.g. enzymes having alpha2,9 endosialidase activity. Enzymes are herein a subclass of polypeptides. Polypeptides or enzymes having alpha2,9 endosialidase activity according to the invention are able to hydrolize the Neisseria meningitidis serogroup C capsular polysaccharide which consists of sialic acid residues linked by alpha2,9-bonds only.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors, including cloning and expression vectors, comprise a polynucleotide of the invention encoding a polypeptide having alpha2,9 endosialidase activity or a functional equivalent thereof. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention 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 which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below. The vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. A variety of cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et at, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N. Y., (1989).

A vector according to the invention may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e. g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.

Vectors according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

A vector of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression. The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operationally linked to the nucleic acid sequence to be expressed.

Within a vector, such as an expression vector, "operationally linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), i.e. the term "operationally linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other expression regulation signal "operationally linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide. The term "regulatory sequence" or "control sequence" is intended to include promoters, operators, enhancers, attenuators and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

The term regulatory or control sequences includes those sequences which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences).

A vector or expression construct for a given host cell may thus comprise the following elements operationally linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the invention: (i) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the polypeptide in the given host cell; (ii) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture medium; (iii) optionally, a sequence encoding for a C-terminal, N-terminal or internal epitope tag sequence or a combination of the aforementioned allowing purification, detection or labeling of the polypeptide; (iv) a DNA sequence of the invention encoding a mature and preferably active form of a polypeptide having alpha2,9 endosialidase activity; and preferably also (v) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the polypeptide. Particular named bacterial promoters include lad, lacZ, T3, T7, SP6, K1 F, tac, tet, gpt, lambda P_R, P_L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Downstream of the nucleotide sequence according to the invention there may be a 3' untranslated region containing one or more transcription termination sites (e. g. a terminator). The origin of the terminator is less critical. The terminator can, for example, be native to the DNA sequence encoding the polypeptide. Preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG (or TUG or GUG in prokaryotes) at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and/or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression host and/or to provide for the inducible control of the expression of a polypeptide of the invention. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The vectors, such as expression vectors, of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. oxidoreductase proteins, mutant forms of oxidoreductase proteins, fragments, variants or functional equivalents thereof, fusion proteins, etc.).

The vectors, such as recombinant expression vectors, of the invention can be designed for expression of endosialidase proteins in prokaryotic or eukaryotic cells. For example, endosialidase proteins can be expressed in bacterial cells such as E. coli, Bacillus strains, insect cells (using baculovirus expression vectors), filamentous fungi, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Representative examples of appropriate hosts are described hereafter. Appropriate culture media and conditions for the above-described host cells are known in the art.

As set out above, the term "control sequences" or "regulatory sequences" is defined herein to include at least any component which may be necessary and/or advantageous for the expression of a polypeptide. Any control sequence may be native or foreign to the nucleic acid sequence of the invention encoding a polypeptide. Such control sequences may include, but are not limited to, a promoter, a leader, optimal translation initiation sequences (as described in Kozak, 1991 , J. Biol. Chem. 266:19867- 19870), a secretion signal sequence, a pro-peptide sequence, a polyadenylation sequence, a transcription terminator. At a minimum, the control sequences typically include a promoter, and transcriptional and translational stop signals. A stably transformed microorganism is one that has had one or more DNA fragments introduced such that the introduced molecules are maintained, replicated and segregated in a growing culture. Stable transformation may be due to multiple or single chromosomal integration(s) or by (an) extrachromosomal element(s) such as (a) plasmid vector(s). A plasmid vector is capable of directing the expression of polypeptides encoded by particular DNA fragments. Expression may be constitutive or regulated by inducible (or repressible) promoters that enable high levels of transcription of functionally associated DNA fragments encoding specific polypeptides. Expression vectors of the invention may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed, e.g., genes which render the bacteria resistant to drugs such as chloramphenicol, erythromycin, kanamycin, neomycin, tetracycline, as well as ampicillin and other penicillin derivatives like carbenicillin. Selectable markers can also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.

The appropriate polynucleotide sequence may be inserted into the vector by a variety of procedures. In general, the polynucleotide sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are disclosed in Ausubel et al. Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989). The polynucleotide sequence may also be cloned using homologous recombination techniques including in vitro as well as in vivo recombination. Such procedures and others are deemed to be within the scope of those skilled in the art. The vector may be, for example, in the form of a plasmid, a viral particle, or a phage. Other vectors include chromosomal, nonchromosomal and synthetic polynucleotide sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and bacteriophage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and pseudorabies.

The invention also provides host cells, i.e. transformed cells comprising a nucleic acid sequence of the invention, e.g. a sequence encoding a polypetide having alpha2,9 endosialidase activity, or a vector of the invention. The host cell may be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.

Preferred mammalian cells include e.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6 cells, hybridomas, Bowes melanoma or any mouse or any human cell line. Exemplary insect cells include any species of Spodoptera or Drosophila, including Drosophila S2 and Spodoptera Sf-9. Exemplary fungal cells include any species of Aspergillus. Preferred yeast cell include, e. g. a cell from a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain. More preferably from Kluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowia lipolytica, or Pichia pastoris. According to the invention, the host cell may be a prokaryotic cell. Preferably, the prokaryotic host cell is a bacterial cell. The term "bacterial cell" includes both Gram-negative and Gram-positive as well as archaeal microorganisms. Suitable bacteria may be selected from e.g. Escherichia, Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacte um, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus or Streptomyces. Preferably, the bacterial cell is selected from the group consisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans, Caulobacter crescentus CB 15, Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonas putida, Paracoccus zeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium melioti and Rhizobium radiobacter. The selection of an appropriate host is within the abilities of those skilled in the art.

The vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation (Davis, L, Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)). The nucleic acids or vectors of the invention may be introduced into the cells for screening, thus, the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid. The method of introduction is largely dictated by the targeted cell type. Exemplary methods include CaP0₄ precipitation, liposome fusion, lipofection (e.g., LIPOFECTIN™), electroporation, viral infection, etc. The candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction) or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets can be used.

Where appropriate, the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the nucleic acids of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells may be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof. Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification. Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art. The expressed polypeptide or fragment thereof can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps. The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Depending upon the host employed in a recombinant production procedure, the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated. Polypeptides of the invention may or may not also include an initial methionine amino acid residue. Cell-free translation systems can also be employed to produce a polypeptide of the invention. Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operationally linked to a nucleic acid encoding the polypeptide or fragment thereof. In some aspects, the DNA construct may be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.

Host cells containing the polynucleotides of interest, e.g., nucleic acids of the invention, can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes. The culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan. The clones which are identified as having the specified enzyme activity may then be sequenced to identify the polynucleotide sequence encoding an enzyme having the desired alpha2,9 endosialidase activity.

Recombinant DNA can be introduced into the host cell by any means, including, but not limited to, plasmids, cosmids, phages, yeast artificial chromosomes or other vectors that mediate transfer of genetic elements into a host cell. These vectors can include an origin of replication, along with c s-acting control elements that control replication of the vector and the genetic elements carried by the vector. Selectable markers can be present on the vector to aid in the identification of host cells into which genetic elements have been introduced. Means for introducing genetic elements into a host cell (e.g. cloning) are well known to the skilled artisan. Other cloning methods include, but are not limited to, direct integration of the genetic material into the chromosome. This can occur by a variety of means, including cloning the genetic elements described herein on non-replicating plasmids flanked by homologous DNA sequences of the host chromosome; upon transforming said recombinant plasmid into a host the genetic elements can be introduced into the chromosome by DNA recombination. Such recombinant strains can be recovered if the integrating DNA fragments contain a selectable marker, such as antibiotic resistance. Alternatively, the genetic elements can be directly introduced into the chromosome of a host cell without use of a non-replicating plasmid. This can be done by synthetically producing DNA fragments of the genetic elements in accordance to the present invention that also contain homologous DNA sequences of the host chromosome. Again if these synthetic DNA fragments also contain a selectable marker, the genetic elements can be inserted into the host chromosome.

The endosialidase of the invention may be favorably expressed in any of the above host cells.

"Amino acid" or "amino acid sequence" as used herein refer to an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these and to naturally occurring or synthetic molecules. "Amino acid" or "amino acid sequence" include an oligopeptide, peptide, polypeptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The term "polypeptide" as used herein, refers to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isoesteres and may contain modified amino acids other than the 22 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, SUMOylation, PEGylation, phosphorylation, prenylation, racemization, selenoylation, sulfation and transfer- RNA mediated addition of amino acids to protein such as arginylation. (See Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).

The peptides and polypeptides of the invention also include all "mimetic" and "peptidomimetic" forms, as described in further detail, below. "Recombinant" polypeptides or proteins refer to polypeptides or proteins produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or protein. "Synthetic" nucleic acids (including oligonucleotides), polypeptides or proteins of the invention include those prepared by any chemical synthesis, e.g., as described, below.

"Fragments" as used herein are a portion of a naturally occurring protein which can exist in at least two different conformations. Fragments may also include a "proteolytically matured endosialidase", i.e. the enzymatically active N-terminal portion of an endosialidase which is released from the precursor sequence SEQ ID (2) or (4) in an intramolecular proteolytic cleavage reaction. This intramolecular proteolytic cleavage reaction occurs after the folding of the precursor protein between the highly conserved residues, e.g. Thr-756 and Ser-757 in endoN92 or Thr-910 and Ser-9 1 in endoNF, respectively (Schulz et a/., 2010b). The C- terminal portion, the C-terminal intramolecular chaperone domain, is crucial for the folding process of endosialidases and cannot be truncated or mutated in conserved amino acid stretches (Schwarzer et a/., 2007; Muhlenhoff et a/., 2003; Schulz et a/., 2010a). The C- terminal chaperone domain is released to allow proper binding of endosialidase to polySia and processive degradation of polySia (Schwarzer et al., 2009). Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein. "Substantially the same" means that an amino acid sequence is largely, but not entirely the same, but retains at least the functional alpha2,9 endosialidase activity of the sequence to which it is related. In general two amino acid sequences are "substantially the same" or "substantially homologous" if they are at least about 85% identical. For instance, SEQ ID (11) and SEQ ID (12) may be referred to as fragments of SEQ ID (2), where M303 has been replaced with R (SEQ ID (11)) and K (SEQ ID (12)), respectively. Similarly, SEQ ID (17) and SEQ ID (18) may be referred to as fragments of SEQ ID (5), where M228 has been replaced with R (SEQ ID (17)) and K (SEQ ID (18)), respectively. Fragments which have different three dimensional structures as the naturally occurring protein are also included. An example of this is a "pro-form" molecule, such as a pro-protein with lower activity that can be modified by cleavage to produce a mature enzyme with significantly higher activity. The peptides and polypeptides of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic's structure and/or activity. As with polypeptides of the invention, which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that it's structure and/or function is not substantially altered. Thus, in one aspect, a mimetic composition is within the scope of the invention if it has an alpha2,9 endosialidase activity.

Polypeptide mimetic compositions of the invention can contain any combination of non- natural structural components. In alternative aspect, mimetic compositions of the invention include one or all of the following three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide of the invention can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N- hydroxysuccinimide esters, bifunctional maleimides, Ν,Ν'-dicyclohexylcarbodiimide (DCC) or Ν,Ν'-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., -C(=0)- CH₂- for -C(=0)-NH-), aminomethylene (CH₂-NH), ethylene, olefin (CH=CH), ether (CH₂-0), thioether (CH₂-S), tetrazole (CN₄-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357 ', "Peptide Backbone Modifications," Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non- natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphthylalanine; D- or L- phenylglycine; D- or L- 2 thieneylalanine; D- or L-l, -2, 3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D- (trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p- biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D- or L-2- indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso- pentyl, or a non-acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings. Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'-N-C-N-R") such as, e.g., 1- cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia- 4,4- dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative (e.g., containing the CN-moiety in place of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1 ,2- cyclo- hexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2- chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4- hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,- dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups. A residue, e.g., an amino acid of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but also can be referred to as the S- or R- form. The invention also provides methods for modifying the polypeptides of the invention by either natural processes, such as post-translational processing (e.g., phosphorylation, acylation, etc), or by chemical modification techniques, and the resulting modified polypeptides. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also a given polypeptide may have many types of modifications. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphatidylinositol, covalent attachment of a fluorescent dye, cross-linking cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. See, e.g., Creighton, T.E., Proteins - Structure and Molecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983). Solid-phase chemical peptide synthesis methods can also be used to synthesize the polypeptide or fragments of the invention. Such method have been known in the art since the early 1960's (Merrifield, R. B., J. Am. Chem. Soc, 85:2149-2154, 1963) (See also Stewart, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, III, pp. 11-12)) and have recently been employed in commercially available laboratory peptide design and synthesis kits (Cambridge Research Biochemicals). Such commercially available laboratory kits have generally utilized the teachings of H. M. Geysen et al, Proc. Natl. Acad. Sci., USA, 81 :3998 (1984) and provide for synthesizing peptides upon the tips of a multitude of "rods" or "pins" all of which are connected to a single plate. When such a system is utilized, a plate of rods or pins is inverted and inserted into a second plate of corresponding wells or reservoirs, which contain solutions for attaching or anchoring an appropriate amino acid to the pin's or rod's tips. By repeating such a process step, i.e., inverting and inserting the rod's and pin's tips into appropriate solutions, amino acids are built into desired peptides. In addition, a number of available FMOC peptide synthesis systems are available. For example, assembly of a polypeptide or fragment can be carried out on a solid support using an Applied Biosystems, Inc. Model 431 A™ automated peptide synthesizer. Such equipment provides ready access to the peptides of the invention, either by direct synthesis or by synthesis of a series of fragments that can be coupled using other known techniques. As used herein, the terms "NmC-polySia" and "alpha2,9-polySia" refer to all possible polymers of sialic acid, where the sialic acid units are connected via alpha2,9-linkages. These terms also encompass modified polymers including modifications of "NmC- polySia'V'alpha2,9-polySia" such as, for example, deoxylation, oxylation, oxidation, hydroxylation, decarboxylation, carboxylation, deamination, amination, formylation, acetylation, glycolylation, acylation, arylation, deamidation, amidation, azidation, methylation, sulfation, phosphorylation, iodination, fluoridation, bromination, racemization, glycosylation, PEGylation, myristolyation, prenylation, GPI anchor formation, covalent attachment of a lipid, a peptide, polypeptide, or a fluorescence dye. Preferably, the polymers have no alpha2,8- linkages.

The therapeutic use of the nucleic acids, polypeptides, vectors, host cells and also formulations and pharmaceutical compositions containing the same as active ingredient are within the scope of the present invention. The present invention also relates to their use as active ingredients in the preparation or manufacture of a medicament, especially, their use for the treatment of bacterial infections with pathogens encapsulated with alpha2,9 linked polySia, especially Neisseria meningitidis serogroup C or Helicobacter canadensis as well as their use for the preparation of medicaments for the treatment of bacterial infections with pathogens encapsulated with alpha2,9 linked polySia, especially Neisseria meningitidis serogroup C or Helicobacter canadensis.

The pharmaceutical compositions according to the present invention comprise at least one polypeptide having alpha2,9 endosialidase activity and, optionally, one or more carrier substances, excipients and/or adjuvants. Pharmaceutical compositions may additionally comprise, for example, one or more of water, buffers such as, e.g., neutral buffered saline or phosphate buffered saline, ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrates such as e.g., glucose, mannose, sucrose or dextrans, mannitol, proteins, adjuvants, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione and/or preservatives. Furthermore, one or more other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein. For instance, the endosialidases of the invention may advantageously be employed in combination with an antibiotic, anti-fungal, or anti-viral agent, an-anti histamine, a nonsteroidal anti-inflammatory drug, a disease modifying anti-rheumatic drug, a cytostatic drug, a drug with smooth muscle activity modulatory activity or mixtures of the aforementioned. Pharmaceutical compositions may be formulated for any appropriate route of administration, including, for example, topical such as, e.g., transdermal or ocular, oral, buccal, nasal, vaginal, rectal or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular such as, e.g., intravenous, intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injection, as well as any similar injection or infusion technique. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, compositions provided herein may be formulated as a lyophilizate. Formulation for topical administration may be preferred for certain conditions such as, e.g., in the treatment of skin conditions such as burns or itch.

Compositions intended for oral use may further comprise one or more components such as sweetening agents, flavoring agents, coloring agents and/or preserving agents in order to provide appealing and palatable preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as, e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate, granulating and disintegrating agents such as, e.g., corn starch or alginic acid, binding agents such as, e.g., starch, gelatin or acacia, and lubricating agents such as, e.g., magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed. A composition may further include one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in Martindale-The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.

For the treatment of a bacterial infection, the dose of the biologically active component according to the invention may vary within wide limits and may be adjusted to individual requirements. The required dose may be administered as a single dose or in a plurality of doses. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain a sufficient amount of active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination, i.e. other drugs being used to treat the patient, and the severity of the particular disease undergoing therapy.

Preferred compounds of the invention will have certain pharmacological properties. Such properties include, but are not limited to oral bioavailability, such that the preferred oral dosage forms discussed above can provide therapeutically effective levels of the compound in vivo.

Endosialidases provided herein are preferably administered to a patient such as, e.g., a human, orally or topically, and are present within at least one body fluid or tissue of the patient. Accordingly, the present invention further provides methods for treating patients suffering from a bacterial infection, e.g. with Neisseria meningitidis serogroup C or Helicobacter canadensis. As used herein, the term "treatment" encompasses both disease- modifying treatment and symptomatic treatment, either of which may be prophylactic, i.e., before the onset of symptoms, in order to prevent, delay or reduce the severity of symptoms, or therapeutic, i.e., after the onset of symptoms, in order to reduce the severity and/or duration of symptoms. Patients may include but are not limited to primates, especially humans, domesticated companion animals such as dogs, cats, horses, and livestock such as cattle, pigs, sheep, with dosages as described herein.

It is also within the present invention that the components according to the invention are used as or for the manufacture of a vaccine. For various applications, the components of the invention can be labelled by isotopes, fluorescence or luminescence markers, antibodies or antibody fragments, any other affinity label like nanobodies, aptamers, peptides etc., enzymes or enzyme substrates. These labelled components of this invention are, for example, useful for mapping the location of capsular polySia in vivo, ex vivo, in vitro and in situ such as, e.g. in tissue sections via autoradiography and as radiotracers for positron emission tomography (PET) imaging, single photon emission computerized tomography (SPECT) and the like to characterize those cells in living subjects or other materials. The labelled components according to the present invention may be used in therapy, diagnosis and other applications such as research tools in vivo and/or in vitro, in particular the applications disclosed herein.

Brief Description of the Figures

The following figures are illustrative of embodiments of the invention. Figure 1. Examples of sialic acid derivatives and polySia in different linkages. A, the three most common sialic acid derivatives found in nature, N-acetyl-neuraminic acid (Neu5Ac; (1)), N-glycolyl-neuraminic acid (Neu5Gc; (2)), and 3-deoxy-D-glycero-D-galacto-nonulosonicacid (KDN; (3)). B, homopolymers of Neu5Ac with different linkages found as capsular polysaccharides of different bacterial strains: alpha2,8 K1-polySia (4) (Escherichia coli K1 ; Neisseria meningitidis serogroup B; Moraxella nonliquefaciens; Mannheimia (Pasteurella) haemolytica A2); ; alternating alpha2,8/alpha2,9 K92-polySia (5) (Escherichia coli K92); alpha2,9 NmC-polySia (6) (Neisseria meningitidis serogroup C).

Figure 2 shows cleavage characteristics of the hydrolysis of NmC-polySia with the endosialidases AN-EndoN92, the inactive form AN-EndoN92-His180Ala, ΔΝ-EndoNF, the exosialidase SiaAU from SIGMA as well as Buffer as control. Anion-exchange chromatography was used to directly monitor the cleavage products of NmC-polySia. To visualize the digest with SiaAU, the chromatogram of SiaAU was included in a second more scaled version (SiaAU (zoomed in)). Numbers above peaks indicate the respective degree of polymerisation of sialic acid oligomers. As can be taken from Figure 2, EndoN92 hydrolyses alpha2,9 NmC-polySia. In contrast, the inactive form AN-EndoN92-His180Ala and ΔΝ- EndoNF do not cleave alpha2,9-linked polySia. Similarly, the exosialidase SiaAU releases only sialic acid monomers with weak activity (SiaAU (zoom in)).

Figure 3 illustrates the sequences of SEQ ID Nos. 1 to 8. Figure 3A shows SEQ ID No. 1 , which is the DNA sequence of the endosialidase N92 (endoN92 or Phi92_gp143) from the enterobacteria phage phi92 (EBI database accession number: FR775895). Figure 3B shows SEQ ID No. 2, which is the protein sequence of the endosialidase N92 (endoN92 or Phi92_gp143) from the enterobacteria phage phi92 (EBI database accession number CBY99572). Figure 3C shows SEQ ID No. 3, which is the DNA sequence of the endosialidase F (endoNF) gene (gene 17) from the enterobacteria phage K1 F (EBI database accession number: AM084414 or NC_007456). Figure 3D shows SEQ ID No. 4, which is the protein sequence of the endosialidase F (endoNF) from the enterobacteria phage K1 F (EBI database accession number: CAJ29390 or YP_338127). Figure 3E shows SEQ ID No. 5, which is the DNA sequence of the N-terminally truncated version of endoN92 (SEQ ID No. 1); Figure 3F shows SEQ ID No. 6, which is the protein sequence of the N-terminally truncated version of endoN92 (SEQ ID No. 2); Figure 3G shows SEQ ID No. 7, which is the DNA sequence of the N-terminally truncated version of endoNF (SEQ ID No. 3); Figure 3H shows SEQ ID No. 8, which is the protein sequence of the N-terminally truncated version of endoNF (SEQ ID No. 4).

Figure 4 illustrates the sequences of SEQ ID Nos. 9 to 20. Figure 4A shows SEQ ID No. 9, which is the T908G point mutant of SEQ ID No. 1. Figure 4B shows SEQ ID No. 10, which is the T908A point mutant of SEQ ID No. 1. Figure 4C shows SEQ ID No. 1 1 ; the protein sequence encoded by SEQ ID No. 9. Figure 4D shows SEQ ID No. 12; the protein sequence encoded by SEQ ID No. 10. Figure 4E shows SEQ ID No. 13; the T624A, G1230T, T1412G, and G1801A mutant of SEQ ID No. 3. Figure 4F shows SEQ ID No. 14; the protein sequence encoded by SEQ ID No. 13. Figure 4G shows SEQ ID No. 15, which is the T683G point mutant of SEQ ID No. 5. Figure 4H shows SEQ ID No. 16, which is the T683A point mutant of SEQ ID No. 5. Figure 41 shows SEQ ID No. 17; the protein sequence encoded by SEQ ID No. 15. Figure 4K shows SEQ ID No. 18; the protein sequence encoded by SEQ ID No. 16. Figure 4L shows SEQ ID No. 19, which is the G495T, T677G, and G1066A mutant of SEQ ID No. 7. Figure 4M shows SEQ ID No. 20; the protein sequence encoded by SEQ ID No. 9.

EXAMPLES

NmC-polySia can be purified from Neisseria meningitidis serogroup C strains like FAM18 (available from the American Tissue Culture Collection, number ATCC-700532) according to the protocol described previously for the purification of the K5 polysaccharide (Volpi, 2004) with the following modification: to avoid osmotic shock and cell lysis 150 mM NaCI can be added to the extraction buffer 50 mM Tris-HCI, pH 7.5, 5 mM EDTA. NmC-polySia can also be purified from FAM18 (ATCC-700532) as described in Rode et al. (2008). The exosialidase SiaAU from Arthrobacter ureafaciens has been obtained from SIGMA (N 3786).

Bacteria and Bacteriophage - Escherichia coli K92 (strain Bos-12, serotype 016:K92:H-, ATCC-35860) and enterobacteria phage phi92 (ATCC-35860-B1) have been obtained from the American Tissue Culture Collection (ATCC). E. coli BL21-Gold(DE3) were purchased from Stratagene. Expression vectors: Endosialidases endoN92 and the N-terminally truncated endoN92 (ΔΝ- endoN92) lacking the 75 amino acids N-terminal capsid binding domain were cloned from enterobacteria phage phi92 (ATCC-35860-B1 ) into expression vector pET22b-Strep vector (Stummeyer et al., 2005), a modified pET22b vector (Novagen). The inactive endosialidase mutant AN-endoN92-His180Ala was produced by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's guidelines and employing the plasmid containing the sequence encoding AN-endoN92 as a template. For endosialidase endoNF, the expression vector described in Stummeyer et al. (2005) has been used.

Protein Expression and Purification - Endosialidases were expressed in E. coli BL21- Gold(DE3) in the presence of 100 pg/ml Carbenicillin. Bacteria were cultivated in PowerBroth (Athena Enzyme Systems) at 30°C. Expression of endoNF was induced by adding 0.1 mM IPTG at an optical density (A600) of 1.5-2.0 and bacteria were harvested 6-7 h after induction. For the analysis of soluble and insoluble proteins, bacteria were lysed by sonication. Soluble and insoluble fractions were obtained after centrifugation (22,000 x g, 20 min, 4°C). Protein purification was performed as described previously (Schwarzer et al., 2009; Stummeyer et al., 2005). Concentration of protein was performed in Amicon Centrifugal Filter Units (Millipore, MWCO: 50,000) in 10 mM Tris-HCI buffer, pH 7.4; 50 mM NaCI.

Identification of alpha2,9 endosialidase activity - A bacterial lysis assay can be used to select bacteriophages carrying a polynucleotide sequence encoding a polypeptide sequence with alpha2,9 endosialidase activity. Bacterial lysis assays are known to those skilled in the art and have been described previously, e.g. Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N. Y., (1989).

Analysis of NmC-polySia cleavage products by anion-exchange chromatography - For the analysis of NmC-polySia cleavage products, 800 μg of NmC-polySia were incubated with 25 pmol of endosialidase (endoNF or endoN92 or endoN92-His180Ala) or 4 mlU of SiaAU in 100 mM sodium phosphate buffer, pH 5.1 for 40 hours at 37°C. For SiaAU one standard International milliunit (mlU) is the amount of activity that will release 1 nmol of 4- methylumbelliferone from 2-(4-methylumbelliferyl) a-D-N-acetylneuraminic acid per minute at pH 5.5 at 37° C. For endoNF the activity has been determined with a similar standard (TFMU-Sia3, trimeric sialic acid a-linked to trifluoromethylumbelliferyl) to be kcat = 77 nmol/(nmol^*min) (Morley et al., 2009). This can be converted into the standard International milliunit (mlU): 13 pmol endoNF correspond to 1 mlU to release 1 nmol of trifluoromethylumbelliferone from TFMU-Sia3 per minute at pH 4.5 at 37°C. With sialic acid tetramer (Sia4), the minimal substrate of endoNF, both endoNF and endoN92 showed similar activities. The used amount of 25 pmol of endoN therefore corresponds to approx. 2 mlU. And 4 mlU of SiaAU is two times as active as 25 pmol of endoN according to the standard definition. Samples were diluted with one volume of 10 mM Tris-HCI buffer, pH 8.0. After filtering (0.22 pm) and centrifugation (3 min at 19.000 x g) one half volume of the samples was loaded onto an AKTA design system equipped with a MonoQ HR 5/50 column (GE healthcare) using 10 mM Tris-HCI, pH 8.0 as running buffer. Separation on MonoQ HR 5/50 column was performed at a flow rate of 1 mL min^"1 using a segmented linear gradient: 2 ml 0%, 2 ml 0-8%, 7 ml 8-20%, 26 ml 20-45%, 1 ml 45-100%, and 5 ml 100% of 10 mM Tris- HCI, pH 8.0, 1 M NaCI.

Enzymatic activity assay NmC-polySia was used for the determination of the activity/substrate specifity of the endosialidases of the invention towards strictly alpha2,9 linked polySia. The assay was conducted as described previously (thiobarbituric acid (TBA) assay; Skoza and Mohos, 1976; Miihlenhoff et al., 2003). Buffer and the endosialidase endoNF were used as control.

It was found that an endoN92 effectively cleaves NmC-PSA, while no such activity was observed for endoNF and the buffer, respectively (see Figure 2).

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Claims

(1 ) An isolated, synthetic or recombinant nucleic acid comprising:

(i) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the sequence has a sequence identity to the full-length sequence of SEQ ID No. 1 , 3, 5, 7, 9, 10, 13, 15, 16, or 19 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity;

(ii) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the sequence hybridizes under highly stringent conditions to the complement of the sequence of (i), wherein the highly stringent conditions comprise heating the double stranded nucleic acid encoding a polypeptide having alpha2,9 endosialidase activity to 90°C-100°C and cooling to 25°C with a cooling rate of 0.2-4°C/s;

(iii) a sequence completely complementary to any nucleic acid sequence of (i) or (ii);

(iv) a sequence encoding a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide has a sequence identity to the full-length amino acid sequence of SEQ ID No. 2, 4, 6, 8, 11 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity.

(2) A vector comprising:

(a) a nucleic acid of claim (1 ); or

(b) the vector of (a), wherein the vector is a cloning vector, an expression vector or an artificial chromosome.

(c) the vector of (a), wherein the vector is a bacteriophage, a eukaryotic virus, especially adenovirus, retrovirus or baculovirus, or an archaeal virus, especially SH1.

(3) A host cell comprising:

(a) a nucleic acid of claim (1 ), or the vector of claim (2); or

(b) the host cell of (a), wherein the host cell is a prokaryotic cell, especially an £. coli, a Bacillus, e.g. Bacillus subtilis or Bacillus megaterium, or a Corynebacterium cell; or a eukaryotic cell, especially a slime mold, e.g. Dictyostelium discoideum, a yeast, e.g. Saccharomyces cerevisiae, an insect cell line, e.g. Sf-9, a cell of a mammalian cell line, e.g. a CHO cell, an embryonic stem cell, or an induced pluripotent stem cell. (4) An isolated, synthetic or recombinant polypeptide having alpha2,9 endosialidase activity comprising:

(a) an amino acid sequence having a sequence identity to the full-length amino acid sequence of SEQ ID No. 2, 4, 6, 8, 11 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, or an enzymatically active fragment thereof, wherein the fragment has alpha2,9 endosialidase activity;

(b) an amino acid sequence encoded by a nucleic acid of claim (1);

(c) the amino acid sequence of (a) or (b), wherein the polypeptide comprises one or more modifications and has a sequence identity to the full-length amino acid sequence of SEQ ID No. 2,

4, 6, 8, 11 , 12, 14, 17, 18, or 20 from at least 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to at most 99.9%, and wherein the modification comprises acetylation, acylation, arylation, amidation, azidation, methylation, glycosylation, phosphorylation, SUMOylation, PEGylation, covalent attachment of a lipid, a peptide, polypeptide, or a fluorescence dye.

(5) A composition comprising:

(a) a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide comprises (i) an amino acid sequence encoded by the nucleic acid sequence of claim (1), or (ii) an amino acid sequence of claim (4);

(b) the composition of (a) further comprising a second polypeptide having fluorescence or an antibiotic activity, or a combination thereof; or

(c) the composition of (a) or (b), wherein the composition is a desialylation composition, a composition for the production of a Neisseria meningitidis serogroup C vaccine, or a composition for the production of a Helicobacter canadensis vaccine.

(6) Pharmaceutical composition comprising a polypeptide having alpha2,9 endosialidase activity, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of claim (1) or an amino acid sequence of claim (4), and, optionally, at least one carrier and/or at least one adjuvant.

(7) A polypeptide having alpha2,9 endosialidase activity, a bacteriophage comprising a nucleic acid sequence of claim (1), or a pharmaceutical composition of claim (6) for the treatment of an infection with Neisseria meningitidis serogroup C or Helicobacter canadensis, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of claim (1) or an amino acid sequence of claim (4).

(8) A method of producing a recombinant polypeptide having alpha2,9 endosiaiidase activity, comprising:

(a) the steps of introducing a nucleic acid encoding the polypeptide into a host cell under conditions that allow expression of the polypeptide, and recovering the polypeptide, wherein the polypeptide comprises a polypeptide encoded by a nucleic acid of claim (1);

(b) the method of (a), wherein the step of recovering the polypeptide is carried out using affinity chromatography, anion exchange chromatography, reversed phase chromatography, or precipitation. Precipitation is preferably carried out using ammonium sulfate or trichloroacetic acid.

(9) A method of hydrolyzing an alpha2,9 polysialic acid (polySia) linkage comprising:

(a) contacting a sample comprising matter having alpha2,9 linked polySia with a polypeptide capable of hydrolyzing an alpha2,9 polySia linkage, wherein the polypeptide comprises an amino acid sequence encoded by the nucleic acid sequence of claim (1 ) or an amino acid sequence of claim (4);

(b) the method of (a), wherein the sample is derived from Neisseria meningitidis serogroup C, Helicobacter canadensis, or comprises the isolated capsular polySia thereof.

(10) An isolated, synthetic or recombinant nucleic acid comprising a sequence according to SEQ ID No. 1 , 5, 9, 10, 15, or 16 encoding a polypeptide having alpha2,9 endosiaiidase activity, or a polypeptide having alpha2,9 endosiaiidase activity and a sequence according to SEQ ID No. 2, 6, 11 , 12, 17, or 18 for use in the method of claim (9).