WO2012136768A1 - Use of mutants of human hyaluronidase ph-20 with increased chondroitinase activity for axonal regrowth - Google Patents

Use of mutants of human hyaluronidase ph-20 with increased chondroitinase activity for axonal regrowth

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WO2012136768A1
WO2012136768A1 PCT/EP2012/056287 EP2012056287W WO2012136768A1 WO 2012136768 A1 WO2012136768 A1 WO 2012136768A1 EP 2012056287 W EP2012056287 W EP 2012056287W WO 2012136768 A1 WO2012136768 A1 WO 2012136768A1
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ph
human
hyaluronidase
chondroitinase
sulfate
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PCT/EP2012/056287
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French (fr)
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Hans-Dieter Haubeck
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Hans-Dieter Haubeck
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2474Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01035Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

Chondroitin sulfate proteoglycans are axon growth inhibitory molecules present in the glial scar that are responsible (at least in part) for regeneration failure after CNS or spinal cord injury. Removal of chondroitin sulfate glycosaminoglycan chains using the bacterial enzymes chondroitinase-ABC or AC in models of CNS injury promotes both axon regeneration and plasticity. The present invention relates to the use of mutants of human hyaluronidases and especially human hyaluronidase PH-20 (members of the human hyaluronidase family, endo-beta-acetyl-hexosaminidase enzymes, E.C. 3.2.1.35) with increased chondroitinase activity for the degradation of chondroitin sulfate (proteoglycans) in the glial scar to promote axonal regrowth in human CNS or spinal cord injury.

Description

Use of mutants of human

hyaluronidase PH-20 with increased chondroitinase activity for axonal

regrowth

Provisional

The present invention refer to mutants of human hyaluronidase PH-20 with an increased chondroitinase activity compared to the wild-type. These mutants can be used in medicine, particularly in the treatment of injuries and diseases of the nervous system. Further, the present invention discloses recombinant nucleic acids and vectors encoding a mutant hyaluronidase PH- 20 and recombinant cells being transformed or transfected with the nucleic acid or the vector.

FIELD AND STATE OF THE ART

[0001] Injury to the central nervous system (CNS) or to the spinal cord (SC) induces tissue damage and profoundly alters nerve function.

Spinal cord injury (SCI) often induces loss of motor and/or sensory function below the level of injury. Depending on the severity of the injury these deficits will persist, which causes enormous strain and distress to patients. Furthermore, treatment of these patients is extremely expensive for public health care systems.

Therefore CNS and SC injury represent a clinical problem of major importance and novel therapeutic approaches are urgently needed.

[0002] However, the mechanisms underlying the lack of recovery in the adult CNS (and SC) have been clarified only partially at the molecular level. Knowledge of these mechanisms should therefore provide the basis for the development of new therapeutic strategies for CNSI and SCI. [0003] In the adult CNS, a standard response to injury is the formation of a glial scar, which quickly seals off the injured site from healthy tissue - preventing uncontrolled tissue damage, for example by bacterial invasion. However, glial scarring also blocks the long-term regeneration of damaged axons. Scar tissue is made up of reactive astrocytes, oligodendrocyte precursors, microglia/macrophages, meningeal cells and vascular endothelial cells and contains axon growth inhibitory molecules.

[0004] Following injury to the mammalian CNS, those axons that attempt to regenerate face several inhibitory challenges. The result is failure of regeneration or limited local sprouting (Fawcett J W. Adv. Exp. Med. Biol. 557, 11-24, 2006; Rhodes KE, Fawcett JW. J. Anat. 204, 33-48, 2004; Silver J., Miller JH., Nat. Rev. Neurosci. 5: 146-156, 2004).

[0005] Increased expression of certain extracellular matrix (ECM) molecules after CNS injury is believed to inhibit/restrict axonal regeneration.

[0006] Chondroitin sulfate proteoglycans (CS-PG) are a class of ECM molecules that inhibit neurite outgrowth and are rapidly upregulated after CNS injury (Silver J., Miller JH. Nat. Rev. Neurosci. 5: 146-156, 2004; Busch SA, Silver J. Curr. Opin. Neurobiol. 17, 120-127, 2007; Jones LL, et al. J. Neurosci. 23, 9276-9288, 2003; Lu P, et al. Exp. Neurol. 203, 8-21 , 2007). The role of CS-PG in reducing axon growth after injury has been studied extensively (Levine JM, J Neurosci. 14: 4716- 4730,1994; Jones LL, et al. J. Neurosci. 23, 9276-9288, 2003, Ughrin YM, et al., J. Neurosci. 23: 175-186; Tan AM et al., J. Anat. 207: 717-725, 2005) and it has been shown in vitro and in-vivo (in several animal models), that CS-PG, produced mainly by reactive astrocytes and oligodendrocyte precursor cells (OPC) in glial scars, restrict neurite outgrowth.

[0007] CS-PG are complex macromolecules of the ECM that are expressed in a wide variety of tissues and cells of human and nonhuman origin. The principal structure of CS-PG consists of a core protein, to which one or several chondroitin (or dermatan) sulfate chains are covalently attached. The linear polysaccharide chains generally consist of repeating disaccharide subunits composed of a glucuronic acid (or in dermatan sulfate alternatively iduronic acid) and galactosamine. These disaccharides are substituted to a varying degree with sulfate groups and N-linked acetyl residues. In the past, chondroitin sulfates have been subdivided into different forms, e.g. chondroitin sulfate A (chondroitin-4-sulfate), chondroitin sulfate C (chondroitin-6-sulfate) and chondroitin sulfate B (dermatan sulfate, that contains the C-5 epimeric iduronic acid instead of glucuronic acid residues) (Hoffman et al. Fed. Proc. 17, 1078-1082, 1958).

However, in the following years a more detailed analysis has shown that chondroitin sulfates may have more heterogenous structures (Suzuki S, et al. J. Biol. Chem. 243: 1543-1550, 1968).

[0008] After CNS injury, the expression and synthesis of several CS-PG is increased and the developing glial scar contains large amounts of CS-PG. The relative increase during post-injury periods is different for these CS-PG, e.g. Neurocan, Brevican, Versican, Phosphacan and NG2. Various lines of evidence demonstrate that much of the axon growth inhibitory activity of CS- PG comes from their glycosaminoglycan (GAG) chains (Levine JM, J Neurosci. 14: 4716-4730,1994; Jones LL, et al. J. Neurosci. 23, 9276-9288, 2003, Ughrin YM, et al., J. Neurosci. 23: 175-186; Tan AM et al., J. Anat. 207: 717-725, 2005).

[0009] Direct evidence for the important role of CS-PG in the impaired axonal regeneration after CNS or spinal cord injury (but also peripheral nerve injury) comes from several in-vitro and in-vivo experiments.

In these experiments degradation of chondroitin sulfate glycosaminoglycan chains by the bacterial enzymes chondroitinase ABC or chondroitinase AC can overcome the inhibition of axonal growth by CS-PG and promote nerve regeneration and functional recovery (Groves ML et al., Exp. Neurol. 195: 278-292, 2005; Lu P et al., Exp. Neurol. 203, 8-21 , 2007; Bradbury EJ et al., Nature 416, 636-640, 2002; Snow DM et al., Exp. Neurol. 109, 1 10-130, 1990).

[0010] Endoglycosidases are enzymes capable of cleaving polymers comprising glycosaminoglycan chains, for example, chondroitin sulfate glycosaminoglycans.

[0011] The catabolism of glycosaminoglycans (GAG) and especially of chondroitin sulfate has been studied in detail in the past (Glaser JH, Conradt HE, J Biol. Chem. 254, 2316-2325, 1979; Ingmar B, Wasteson A., Biochem. J. 179, 7-13, 1979) and the sequential degradation of a chondroitin sulfate trisaccharide by different lysosomal exoglycosidases was shown (Glaser JH, Conradt HE, J Biol. Chem. 254, 2316-2325, 1979). However, a human homologue to the bacterial enzymes chon-droitinase ABC or chondroitinase AC (see below [0012] - [0014]) has not been described.

[0012] Bacterial enzymes have been isolated and characterized, that degrade chondroitin sulfates (Yamagata T et al., J. Biol. Chem. 243, 1523- 1535, 1968; Suzuki S, et al. J. Biol. Chem. 243 : 1543-1550, 1968; Sanderson PN et al., Biochem. J. 257, 347-354, 1989).

[0013] According to its specificity for the different chondroitin sulfate types (see above [0007]) the enzyme purified from extracts of Proteus vulgaris was given the name chondroitinase ABC. A second chondroitinase, chondroitinase AC, has been purified from Flavobacter heparinum (Yamagata T et al., J. Biol. Chem. 243, 1523-1535, 1968; Suzuki S, et al. J. Biol. Chem. 243 : 1543-1550, 1968). In contrast to chondroitinase AC, chondroitinase ABC will cleave also dermatan sulfate (chondroitin sulfate B; Sanderson PN et al., Biochem. J. 257, 347-354, 1989).

Both enzymes are endoglycosidases and carry out an elimination reaction, 243, 1523-1535, 1968).

[0014] The strong upregulation of chondroitin sulfate synthesis after CNS or spinal cord injury and the high content of chondroitin sulfate (proteoglycans) within glial scars have suggested that CS-PG are axon growth inhibitory molecules that play a major role in regeneration failure after damage to the CNS and which restrict CNS plasticity.

[0015] Enzymatic removal of the glycosaminoglycan (GAG) side chains by chondroitinase ABC (or chondroitinase AC), has been shown to render CS- PG less inhibitory to axon growth, and to render glial cells more permissive. Chondroitinase ABC therefore can contribute to local degradation of chondroitin sulfate and thus facilitate cell migration and passage of growth cones across the glial scar (Zuo J et a!., Exp. Neurol. 154, 654-662, 1998; Smith-Thomas L, et al. J. Cell Sci. 107, 1687 - 1695, 1994, Smith-Thomas L, et al. J. Cell Sci. 108, 1307 -1315, 1995, Curinga GM et al. J. Neurochem. 102, 275-288, 2007). These data show that much of the axon growth inhibitory activity of CS-PG comes from their glycosaminoglycan (GAG) chains.

[0016] Application of chondroitinase ABC to mammalian brain and spinal cord injury in vivo (in different animal models), has shown increased regeneration of injured nerve fibres, increased plasticity and recovery of function (Moon et al. Nat. Neurosci. 4, 465 -466, 2001 ; Bradbury EJ et al., Nature 416, 636-640, 2002; Houle JD et al., J. Neurosci. 26, 7405-7415, 2006).

[0017] In most of these studies chondroitinase ABC was infused several times as a bolus (for example on alternate days). The rationale for this regimen was that extracellular matrix turnover in the CNS has been shown to be relative slow. Therefore it may be (probably) sufficient to achieve an enzyme concentration that degrades CS GAG chains. [0018] In a recent study the immediate and long-term effects of a single injection of chondroitinase ABC on CS-PG, GAG and axon regeneration were analyzed (Lin R et al. J. Neurochem. 104, 400-408, 2008). In this study a single dose of chondroitinase ABC injected adjacent to the damaged adult nigrostriatal tract of the rat showed that this promoted axon regeneration to a similar extent as in the aforementioned previous studies. In this study it was also shown that the active enzyme persisted in the injured CNS for at least 10 days and prevented the rise in GAG concentration that occurs normally following CNS damage.

[0019] Taken together, there is strong evidence for the important role of CS (-PG) for the impaired axonal regeneration after CNS injury (but also spinal cord and peripheral nerve injury). The data described so far, strongly suggest that enzymatic removal of the CS GAG side chains by chondroitinase ABC (or chondroitinase AC) will lead to improved regeneration of injured nerve fibres, increased plasticity and recovery of nerve function.

[0020] Therefore, the use of enzymes that degrade CS at the site of CNS and SC injury might represent an interesting novel therapeutic approach.

[0021] At present, several approaches have been described, that use enzymes that can degrade CS. These approaches have concentrated on the use of the bacterial enzyme(s) chondroitinase ABC (and / or AC), either as the native enzyme(s) or of deletion mutants (to reduce the size of chondroitinase ABC and to facilitate access to the site of injury). Further approaches involve the use of fusion proteins of chondroitinase ABC with other molecules that can overcome inhibition of axon regeneration, like soluble NOGO antagonists and neurotrophic factors, or facilitate diffusion or transduction, like the TAT-peptide (from the human immunodeficiency virus (HIV)).

[0022] Application EP-A-1 911 460 A1 describes the use of [0022] Application EP-A-1 91 1 460 A1 describes the use of chondroitinase ABC for promoting axonal regrowth and behaviour recovery in spinal cord injury.

[0023] Application WO 2004/103299 A2 particularly refers to the bacterial chondroitinase ABC (and chondroitinase AC). However, it also indicates other enzymes, that might have chondroitin sulfate degrading activity, e.g. hyaluronidase 1 , hyaluronidase 2, hyaluronidase 3 and hyaluronidase 4 and fragments thereof.

[0024] Application WO 2004/1 10360 A2 refers to mutants and mutants of chondroitinase ABC, but also to mutants and mutants of hyaluronidases 1 -4. It is suggested that deletion mutants of chondroitinase ABC reduce the size while retaining the activity. Furthermore, it is alleged that the. reduction in size might (eventually) reduce the immunogenicity of chondroitinase ABC.

[0025] Application US2007/0274979 A1 describes the use of chondroitinase AC and/or chondroitinase B for the degradation of CS to promote neuronal outgrowth.

[0026] Application US2009/0028829 A1 describes the use of fusion proteins of chondroitinase ABC and/or TAT-peptides with the Nogo-Receptor or functional domains of the Nogo-Receptor, the neural cell adhesion molecule L1 , or the neuregulin glial growth factor 2 (GGF2) to promote axon regeneration and neuronal outgrowth. However, they also claimed fusion proteins using other enzymes, that could have chondroitin sulfate degrading activity, e.g. Hyal-1 , Hyal-2, Hyal-3 and Hyal-4 and PH-20.

[0027] Application WO 03/074080 describes the use of chondroitinase ABC to reduce the inhibitory properties of CS for axonal regrowth, behaviour recovery in spinal cord injury and increased plasticity. It further describes other enzymes that might be able to degrade chondroitin sulfate, including the hyaluronidases, e.g. Hyal-1 , Hyal-2, Hyal-3 and Hyal-4. [0028] A major problem for the use of chondroitinase ABC (or chondroitinase AC) in the human will come from the fact that chondroitinase ABC is a bacterial enzyme (purified from extracts of Proteus vulgaris; Yamagata T et al., J. Biol. Chem. 243, 15231535, 1968).

[0029] After CNS or SC injury, the blood-brain barrier (e.g. the basement membrane) is frequently damaged. Therefore, injection of chondroitinase ABC to the site of injury will lead to an immune reaction against the bacterial enzyme, for example to the generation of anti-chondroitinase-ABC antibodies and as a consequence to a loss of enzyme activity. This will become even worse after repeated injections.

[0030] Furthermore, it cannot be excluded, that the immune reaction (and/or inflammation) leads to a severe damage of nerve tissue near the site of injection in the injured areas. Therefore, nerve tissue, that has not been affected by the primary injury, may be damaged, too.

[0031] Another important disadvantage of chondroitinase ABC, for the use in human brain and spinal cord injury (e.g. in vivo), is the (big) size of the enzyme (for example chondroitinase ABC I from Proteus vulgaris has an apparent molecular weight of about 110 - 120 kDa). To overcome the resulting problems with diffusion of the enzyme to the site of action/injury WO2004/1 10360 has suggested that deletion mutants of chondroitinase ABC reduce the size while retaining the activity. It is suggested that the reduction in size might (eventually) reduce the immunogenicity of chondroitinase ABC.

[0032] In summary, the use of the bacterial enzyme chondroitinase ABC (and/or chondroitinase AC) has major risks for human patients and therefore cannot be applied in the human safely (see above, [0029]-[0031]).

[0033] Therefore, the availability of a human enzyme, that acts like the bacterial enzyme chondroitinase ABC, e.g. can degrade chondroitin sulfate, would be extremely valuable.

[0034] Hyaluronidases are enzymes capable of cleaving hyaluronan glycosaminoglycans (HA). In addition to the degradation of hyaluronan, vertebrate hyaluronidases are capable to catabolize chondroitin, chondroitin- 4-sulfate, chondrotin-6-sulfate and dermatan sulfate as alternative substrate (Meyer K, Rapport MM, Adv. Enzymol. Relat. Areas Mol. Biol. 13, 199-236, 1952). Therefore, they are potential candidates for the therapy of CNS and SC injury.

[0035] WO 2010/017912 relates to the use of human hyaluronidases for the degradation of chondroitin sulfate to promote axonal regrowth in central nervous system or spinal cord injuries. Since the identification of human hyaluronidases having chondroitinase activity without or little hyaluronidase activity is of great interest, assays for the examination of the chondroitinase activity of recombinantly expressed human hyaluronidases in vitro are provided. It is suggested to use those members of the human hyaluronidase family that have chondroitinase activity.

[0036] According to their catalytic mechanism hyaluronidases were classified into three major families (Meyer, 1971 , Hyaluronidases. in: The Enzymes, Vol V. ed Boyer PD, New York). Human hyaluronidases belong to the first group (vertebrate hyaluronidases), whereas the other two groups comprise bacterial and parasitic hyaluronidases.

[0037] Human hyaluronidases (E.C. 3.2.1 .35) are a group of five endo-beta- acetyl-D-hexosaminidase enzymes (Hyal-1 , -2, -3, -4 and PH-20; and one pseudogene HYAL-P1 ).

[0038] Hyaluronidases degrade hyaluronan by a hydrolytic mechanism. They cleave the β-(1 ->4) glycosidic bond of hyaluronan by an endolytic process (see below [0055]). [0039] Hyaluronan (previously called also hyaluronic acid) is a linear, negatively charged, high molecular weight glycosaminoglycan (GAG), that is found predominantly in the extracellular matrix (ECM) (Meyer K, Palmer JW, J.Biol.Chem. 107, 629-634, 1934; Fraser JRE, Laurent TC, in Extracellular Matrix, ed Comper WD, Amsterdam 1996).

[0040] Hyaluronan is composed of repeating disaccharides of β-D-glucuronyl (1->3)-N-acetyl-D-glucosamine. These disaccharides are then linked by β- (1 ->4) glycosidic bonds. Hyaluronan chains in the ECM can reach sizes of 100 - 10.000 kDa, however, they are not attached to a core-protein. Furthermore, hyaluronan chains are, in contrast to other GAG chains, not sulfated.

[0041] The structure and function of hyaluronan (Fraser JRE, Laurent TC, in Extracellular Matrix ed Comper WD, Amsterdam 1996) but also the catabolism of hyaluronan have been extensively studied (Stern R, Glycobiol. 13, 105-1 15, 2003).

[0042] Hyaluronan plays an important role in many different biological functions, including cell migration, invasion, differentiation and proliferation, for example by the interaction with hyaluronan-binding proteins and receptors (Fraser JRE, Laurent TC, in Extracellular Matrix ed Comper WD, Amsterdam 1996; Knudson CB, Knudson W. FASEB J. 7, 1233-1244, 1993, Toole BP et al., J. Biol. Chem. 277, 4593-4596, 2002; Toole BP. 2001 ., Semin. Cell Dev. Biol. 12, 79-87, 2001 ).

[0043] In addition to the degradation of hyaluronan, vertebrate hyaluronidases are capable to catabolize chondroitin, chondroitin-4-sulfate, chondrotin-6-sulfate and dermatan sulfate as alternative substrate, although at a slower rate (see below, Example 3).

[0044] The cleavage of chondroitin sulfate by vertebrate hyaluronidases is not a non-specific reaction but can be explained by the close relationship of chondroitin sulfate and hyaluronan. Comparison of the basic disaccharide subunit of both glycosaminoglycans, e.g. chondroitin sulfate and hyaluronan, shows that both glycosaminoglycans contain glucuronic acid as uronic acid. Furthermore, the N-acetyl-glucosamine residue in hyaluronan is the C-4 epimer of the N-acetyl-galacatosamine residue in chondroitin sulfate (e.g. they differ only in the configuration at C-4 with respect to the orientation of the hydroxyl group). Even though in chondroitin sulfate these disaccharides can be substituted to a varying degree with sulfate groups, it is evident that hyaluronan is quite similar to chondroitin sulfate.

[0045] Previously, two forms of hyaluronidase with different pH optimums have been described, one with an acidic and one with a neutral pH optimum. PH-20 was identified to be a hyaluronidase with neutral pH optimum. Although this protein was first identified as a sperm protein (alternatively named SPAM-1 for sperm adhesion molecule-1 ), PH-20 is also expressed in female tissues, like breast and urogenital tract. Based on their homology to PH-20 further hyaluronidases, like Hyal-2, have been identified.

[0046] Human plasma hyaluronidase has been purified and its cDNA was cloned in 1997 (Frost et al.Biochem. Biophys. Res. Commun. 236, 10-15). The gene encoding this enzyme, now called HYAL1 , was mapped to Chromosome 3p21.3.

[0047] PH-20, Hyal-1 and Hyal-2 differ with respect to the degradation of hyaluronan. Whereas hyaluronan is cleaved by hyaluronidase-2 to 20 kDa intermediate-size fragments (of about 50 disaccharides), PH-20 and hyaluronidase-1 cleave hyaluronan down to disaccharides or tetrasaccharides (Stern R, Glycobiol. 13, 105-115, 2003, Hofinger et al. Glycobiology 17, 963-971 , 2007, Hofinger et al. Glycoconj. J. 25, 101-109, 2008).

[0048] In the human, three genes (HYAL1 , HYAL2, HYAL3) are found tightly clustered on chromosome 3p21.3 coding for hyaluronidase-1 (hyal-1 ), hyal-2 and hyal-3. Another three genes HYAL4, PHYAL1 (a pseudogene) and SPAM1 (Sperm Adhesion Molecule 1 ) are clustered in a similar fashion on chromosome 7q31.3. They code for Hyal-4 and PH-20 (Csoka AB, et al. Matrix Biol. 20, 499-508, 2001 ; Csoka AB et al., Genomics 60, 356-361 , 1999).

[0049] Cloning of the mammalian hyaluronidases has facilitated a detailed analysis of their structure and function and their unique mechanism of hydrolysis (Jedrzejas MJ, Stern R. Proteins 61 , 227-238, 2005; Stern R. Glycobiol. 13, 105-115, 2003; Chao KL, et ai. Biochem. 46, 691 1 -6920, 2007).

[0050] Sequence analysis of the cloned enzymes (Hyal-1-4 and hPH-20) has shown that theses hyaluronidases are relative uniform in their size (chain length ranging from 435 amino acids (aa) (hyal-1 ) to 510 aa (hPH-20)).

[0051] Pairwise and multiple sequence alignments of these hyaluronidases have shown sequence identities ranging from 33 % to 41 % and indicate common structural properties. Furthermore, there are a number of absolutely conserved regions (Jedrzejas MJ, Stern R. Proteins 61 , 227-238, 2005), that are necessary for the function of hyaluronidases (see below, [0055], [0057]).

[0052] Although X-ray crystal structures are available at present only for bee venom hyaluronidase (BvHyal) and bovine PH-20 (bPH-20), based on their homology to the human hyaluronidases, these enzymes are thought to be structurally representative for all vertebrate hyaluronidases (Jedrzejas MJ, Stern R. Proteins 61 , 227-238, 2005).

[0053] For all human hyaluronidases (Hyal-1-4 and hPH-20) a two domain structure was shown. The major domain, that has catalytic activity, starts from the N-terminus of the protein and is followed by a smaller C-terminal domain. [0054] The active site was identified based on the homology to BVHyal and mutational analysis (Jedrzejas MJ, Stem R., Proteins 61 , 227-238, 2005). The active site is located within the substrate-binding cleft of the catalytic domain. This cleft is surrounded by a number of highly conserved positively charged and hydrophobic amino acid residues that correspond to the negatively charged and hydrophobic substrate, e.g. hyaiuronan or chondroitin sulfate.

[0055] The catalytic mechanism of the hyaluronidases involves double displacement at C1 next to the β-(1->4) glycosidic bond to be cleaved. Concomitant with cleavage of the glycosidic bond, a glutamic acid (Glu131 in hHyal-1 ) transfers a proton to the C-4 oxygen of the released hyaiuronan fragment. A water molecule replaces the released hyaiuronan fragment and completes hydrolysis.

[0056] Whereas Hyal-1 has been described first as a plasma hyaluronidase, three members of the hyaluronidase family are GPI-anchored to the cell membrane, e.g. Hyal-2, Hyal-4 and PH-20.

[0057] In summary, vertebrate and especially human hyaluronidases use a highly conserved mechanism for the degradation of hyaiuronan and also of chondroitin sulfate and therefore play an important role also in the catabolism of chondroitin sulfate (proteoglycans).

DESCRIPTION OF THE INVENTION

[0058] According to the present invention it was found that a mutant of human hyaluronidase PH-20 with an amino acid substitution in its catalytic center has an increased chondroitinase activity compared to the wild-type enzyme.

[0059] Thus, the present invention refers to mutants of human hyaluronidases (E.C. 3.2.1.35) with an increased chondroitinase activity compared to the wild type. The human hyaluronidase may be selected from Hyal-1 , Hyal-2, Hyal-3, Hyal-4 and PH-20. Preferably, the. human hyaluronidase is PH-20 (SEQ ID NO:1).

[0060] Surprisingly it was found that mutants of the human enzymes, particularly Hyal-1 , Hyal-2 and PH-20, more particularly PH-20, can be used as a therapeutic agent for the treatment of an injury or disease of the nervous system, although the chondroitinase activity of the native human enzymes is rather low. Due to their increased chondroitinase activity, the mutants of the invention can be used for the degradation of chondroitin sulfate to promote axonal regrowth, particularly in human CNS or spinal cord injury.

[0061] In a preferred embodiment, the present invention refers to a mutant of human hyaluronidase PH-20 which has a C5o value against chondroitin-4- sulfate of 1 pg/ml or less, preferably of 0.8 pg/ml or less, and most preferably of 0.6 pg/ml or less at pH 7.0 (under assay conditions as described in the present Examples).

[0062] In a further preferred embodiment, the invention refers to a mutant human hyaluronidase PH-20, which has a C5o value against chondroitiri-4- sulfate of 0.5 pg/ml or less, particularly of 0.4 pg/ml or less, and more particularly of at least 0.3 pg/ml or less at pH 5.8 (under assay conditions as described in the Examples of the present application).

[0063] In a still further preferred embodiments, the mutant human hyaluronidase PH-20 has a chondroitinase activity against chondroitin-4- sulfate, which is at least a factor 1.5, particularly at least a factor 2 higher than the corresponding non-mutant enzyme at pH 7.0 or which is at least a factor 2, particularly a factor 4 higher than the corresponding non-mutant enzyme at a concentration of 2.5 pg/ml enzyme (under assay conditions as described in the Examples). [0064] The mutant hyaluronidase, particularly the mutant hyaluronidase PH- 20, comprises at least one amino acid substitution, deletion and/or insertion within its catalytic center (amino acids 255-275). More preferably, the mutant hyaluronidase PH-20 comprises at least one amino acid substitution at position 264 (Tyr). Amino acid 264 may be replaced by another amino acid, particularly a small and/or hydrophobic amino acid such as Gly, Ala, Leu, lie, Ser, Thr, Met or Cys. More preferably, Tyr264 is replaced by Cys.

[0065] Alternatively, the invention encompasses a mutant human Hyal-1 enzyme, wherein amino acid Tyr247 is replaced by another amino acid or a mutant human Hyal-2 enzyme, wherein Tyr253 is replaced by another amino acid as described above, e.g. Cys.

[0066] In a further embodiment, the mutant hyaluronidase, particularly the mutant hyaluronidase PH-20, is a C-terminally truncated molecule. In some embodiments at least 10, at least 20, at least 30, at least 40 and up to 50 or up to 60 of C-terminal amino acids are deleted. In an especially preferred embodiment, at least 21 C-terminal amino acids including the GPI-anchor have been deleted.

[0067] Alternatively, the respective C-terminally truncated mutants for Hyal-1 and Hyal-2 (that is GPI-anchored like PH-20) can be prepared.

[0068] The C-terminally truncated mutant PH-20Cys264 and PH-20 enzymes were tested against the substrate chondroitin-4-sulfate (see Examples 3 and 4). The results show that the activity of the mutant enzyme PH-20Cys264 (C50= approx. 0.5 pg/rnl), compared to PH-20 (c50= approx. 2 pg/ml), is increased at least by a factor of 4 (FIG. 2 and FIG. 3).

[0069] Furthermore the activity of the mutant enzyme is dependent on the pH, e.g. the activity of the C-terminally truncated mutant PH-20Cys264 enzyme is increased further by a factor of 3-4 at pH 5.8 (compared to pH 7.0) (FIG.4). [0070] The activity of the C-terminally truncated human mutant PH-20Cys264 enzyme is comparable to the activity of chondroitinase ABC (purified from of Proteus vulgaris) against the substrate chondroitin-4-sulfate (see Example 2, Fig. 1 ). Whereas, the in-vitro activity of chondroitinase ABC (pH optimum 8.1 ), is approx. 5-fold higher at pH 7.0 (on a weight base), this advantage, e.g. higher activity, will be lost at a more acidic pH (see Yamagata T et al., J. Biol. Chem. 243, 1523-1535, 1968), that will be observed most probably at the site of CNS or SC injury.

Furthermore, equi-active concentrations of the enzymes in vitro will be pharmacologically not equi-effective in vivo, for example due to the much larger size of chondroitinase ABC (see below).

[0071] However, the broad pH activity range of the enzyme PH-20 is an important advantage, not only when compared with chondroitinase ABC, but also when compared with other members of the hyaluronidase family. For example, Hyal-1 with an acidic pH optimum (lysosomal enzyme), has nearly no activity at pH values > 4.0.

[0072] Whereas hyaluronan is cleaved by PH-20 and Hyal-1 down to di- saccharides or tetra-saccharides, by Hyal-2 it is cleaved only to 20 kDa intermediate-size fragments (of about 50 disaccharides).

[0073] The mutant human hyaluronidase of the present invention, particularly the mutant human hyaluronidase PH-20, has an identity on the amino acid level (over its entire length) with the human wild-type enzyme of at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98% or at least 99%.

[0074] In a further embodiment, the mutant hyaluronidase, particularly the mutant hyaluronidase PH-20 may have an N-terminal truncation, e.g. of up to 35, up to 40 or up to 50 amino acids. In a still further preferred embodiment, the mutant hyaluronidase, particularly the human hyaluronidase may have an N- and a C-terminal truncation.

[0075] The mutant hyaluronidase of the present invention is preferably a recombinant polypeptide, which is produced in a recombinant host cell or host organism. Depending on the host cell/host organism, the recombinant enzyme may be a glycosylated or unglycosylated molecule.

[0076] Preferred host cell/host organisms for the recombinant production of the enzyme are prokaryotic cells such as E.coli or eukaryotic cells, e.g. yeast cells, insect cells or mammalian cells, including human cells. Preferably, a mutant molecule of the invention is a glycosylated molecule which is obtainable from recombinant eukaryotic host cells/organisms. A suitable host cell is e.g. a mammalian cell line such as HEK293.

[0077] A further aspect of the present invention is a recombinant nucleic acid encoding a mutant human hyaluronidase, particularly a human hyaluronidase PH-20 as described above. The recombinant nucleic acid molecule is preferably a recombinant DNA-molecule.

[0078] Still a further aspect of the present invention is a recombinant vector comprising the nucleic acid molecule as described above, operatively linked to an expression control sequence, preferably to a heterologous expression control sequence. The recombinant vector may be a prokaryotic or eukaryotic vector. The vector may be a plasmid or a viral vector. The vector may be extrachromosomal or a vector which integrates into the cellular chromosome. Examples of suitable vectors are e.g. described by Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbour, 1989. The recombinant vector comprises an expression control sequence, e.g. a promoter optionally together with other expression elements and/or termination sequences operatively linked to the sequence encoding the mutant hyaluronidase. Suitable expression control sequences are well known to the skilled person and e.g. described in Sambrook et al, supra. [0079] Still a further aspect of the invention is a method for preparing a mutant human hyaluronidase, particularly a mutant human hyaluronidase PH-20, as described above, comprising the steps:

(a) culturing a recombinant cell as described above, in a culture medium under conditions, under which the mutant human hyaluronidase PH-20 is expressed, and

(b) isolating the mutant human hyaluronidase PH-20 from the cell or the culture medium.

[0080] Conditions for cultivating recombinant host cells/organisms and isolating recombinant expression products therefrom or from the culture medium are well known to the skilled person and are e.g. described in Sambrook et al., supra.

[0081] Still a further aspect of the invention is an agent selected from a mutant hyaluronidase as described above, a nucleic acid molecule coding therefor as described above, a vector comprising the nucleic acid molecule as described above, or a cell transformed or transfected with the nucleic acid molecule, or the vector for use in medicine, particularly for use in human medicine.

[0082] The agent may be used in the treatment of an injury or disease of the nervous system, e.g. the central nervous system, particularly the brain, the spinal cord or the peripheral nervous system. The injury or disease of the nervous system may e.g. be selected from partial or complete severance of neuronal strands including partial and/or complete loss of motor and/or sensory function. For example, the injury may be a spinal cord injury, involving a partial and/or complete severance of the spinal cord.

[0083] Administration of the agent of the invention promotes axon growth and/or sprouting, which may lead to an at least partial regeneration of neuronal function. [0084] The agent is administered in a therapeutically effective dose to a subject in need thereof, particularly a human subject.

[0085] The administration is preferably a local administration at the site of injury, e.g. by injection or by infusion, for example via an epidural intrathecal catheter. The agent may be administered either continuously for a predetermined time, or by one or multiple doses. The agent is preferably administered in a formulation comprising suitable pharmaceutical excipients, e.g. an isotonic solution.

[0086] The agent may be administered either alone or in combination with further agents. For example, the mutant enzyme may be administered together with a further neuronal active agent.

[0087] In CNS and spinal cord the degradation of hyaluronan within the extracellular matrix might facilitate axon sprouting (Moon LD, et al., Neurosci. Res., 71 , 23-37, 2003).

[0088] Therefore, axon regeneration might be enhanced, when in addition to a mutant enzyme such as PH-20cys264, with preferential and/or exclusive chondroitlnase activity, a second enzyme like wild-type PH-20 or a C- and/or N-terminally truncated PH-20 wild-type enzyme, that has also hyaluronidase activity, is used.

[0089] Therefore the ratio of the chondroitlnase activity / hyaluronidase activity could be adjusted not only by modification of the enzyme but in a pharmaceutical composition by the use of a mixture of at least two enzymes with different levels of chondroitlnase and/or hyaluronidase activity, wherein a first enzyme comprises an increased chondroitlnase activity with or without hyaluronidase activity (for example mutant PH-20cys264) and a second enzyme does not have increased chondroitlnase activity (for example wild- type PH-20). [0090] In addition; the described mutants of the human hyaluronidase family could be used together with various substances, that could enhance axonal regrowth, behaviour recovery in spinal cord injury and increased plasticity, for example growth factors, like the neuregulin glial growth factor 2 (GGF2), and inhibitors of the NOGO / NOGO-receptor complex.

[0091] The mutant hyaluronidase, particularly the human hyaluronidase may be present as a fusion polypeptide linked to heterologous peptide or polypeptide sequences, e.g. poly-His or FLAG sequences to facilitate purification. Furthermore, the described mutant hyaluronidase family may be linked to biologically functional peptides or polypeptides like the Nogo- Receptor or functional domains of the Nogo-Receptor, the neural cell adhesion molecule L1 , or GGF2 to promote axon regeneration and neuronal outgrowth.

[0092] In an especially preferred embodiment, the present invention refers to a therapeutic concept that may rely on a concerted action on several targets within the injured nervous system. For example, an efficient therapeutic concept may consist of a combination of two or three or more agents, selected from:

(i) a mutant human hyaluronidase, particularly a mutant human hyaluronidase PH-20 as described above;

(ii) an inhibitor of the NOGO/NOGO-receptor complex such as an antibody directed against NOGO or the NOGO-receptor, or a soluble NOGO-receptor,

(iii) one or more nerve growth factors such as GGF2, NT3, NGF or BDNF,

(iv) a micro-tubule stabilizing agent, e.g. a taxane, such as paclitaxel or docetaxel.

Further, the present invention shall be explained in more detail by the following Figures and Examples. BRIEF DESCRIPTION OF THE FIGURES

[0093] Figure 1 : The bar graph shows the dose-dependent degradation of biotin-labelled chondroitin sulfate by chondroitinase ABC at pH 7.0.

[0094] Figure 2 : The bar graph shows the dose-dependent degradation of biotin-labelled chondroitin-4-sulfate by recombinant C-terminally truncated non-mutant human hyaluronidase PH-20 at pH 7.0

[0095] Figure 3 : The bar graph shows the dose-dependent degradation of biotin-labelled chondroitin-4-sulfate by recombinant C-terminally truncated mutant human hyaluronidase PH-20264cys at pH 7.0.

[0096] Figure 4 : The bar graph shows the dose-dependent degradation of biotin-labelled chondroitin-4-sulfate by recombinant C-terminally truncated mutant human hyaluronidase PH-20264cys at pH 5.8.

EXAMPLES

[0097] The following examples illustrate in a nonlimiting manner the chondroitinase activity of mutants and mutants of human recombinant hyaluronidase and some of the preferred embodiments of the methods according to the invention:

Experimental Section

[0098] The following abbreviations are used:

CS: chondroitin sulfate, CS-4-S: chondroitin-4-sulfate

PBS: phosphate buffered saline

POD: horseradish-peroxidase

OPD: orthophenylenediamine

EXAMPLE 1

Expression and purification of mutants of recombinant human hyaluronidase PH-20

[0099] Reagents used : parental mammalian cell line HEK293

plasmid construct containing the respective gene sequences, C- terminal tagged (6 x His)

stable transfected cell lines

[0100] Transfected cell lines were cultured for up to 7 days under serum-free conditions. Thereafter, supernatants were harvested.

[0101] Supernatants were centrifuged at high speed (10.000 rcf) in order to remove cell debris from supernatant. Subsequently cleared supernatants were submitted to Ni-chelate chromatography and ConA-lectin

chromatography according to standard protocols.

[0102] Expressed and purified proteins were detected by Coomassie blue staining and Western blot using an anti-His mAb.

EXAMPLE 2

Assaying Activity of the Chondroitinase Type (Chondroitinase ABC)

[0103] Reagents used:

- biotin-labelled CS-4-S

- microplate, coated with protamine sulfate (from herring, grade III, Sigma P4505)

- avidin-POD (Sigma A 3151 )

- PBS/0.01 % Tween-20

- OPD (Dako S 204530)

- chondroitinase ABC from Proteus vulgaris (Sigma, C2905), specific activity 0,83 units / mg (contains BSA; for Chondroitinase ABC without BSA (Seikagaku) 1 unit = 9,1 Mg).

[0104] The enzyme reaction was carried out by mixing 50 μΙ of biotin-labelled CS-4-S at 0.04 pg/ml with increasing amounts of chondroitinase ABC, e.g. 0,16 - 20 mUnits of chondroitinase ABC. Samples were brought to a volume of 100 μΙ by addition of PBS.

[0105] This mixture was incubated at 37°C for 1 h. Thereafter the enzyme was inactivated by heating at 100°C for 10 min.

[0106] Samples (85 μΙ of the samples) were transferred to the CS-4-S binding (protamine)-coated microplate and incubated for 1 hour at room temperature.

[0107] Plates were washed 3 times with buffer (PBS/0.01 % Tween 20) and Avidin-POD (1 pg/rril in PBS/5 % BSA) was added.

[0108] After 15 min plates were washed 5 times with PBS 0.01 % Tween 20 and 100 μΙ OPD-solution (0,667 mg/ml) was added.

[0109] After 15 min the reaction was stopped by addition of 100 μΐ of 0,5 Mol H2S04. OD was measured at 490 nm using a microplate reader.

[01 10] The results are given in FIG. 1 , which shows the change in the signal for an increase in concentration of the enzyme chondroitinase ABC.

[01 11] The decrease in the signal correlates perfectly with the increase in the enzyme activity, i.e. the cleavage of the biotin-labelled CS substrate.

EXAMPLE 3

Assaying chondroitinase activity of a C-terminally truncated version of recombinant human hyaluronidase PH-20

[0112] Reagents used: same as in Example 2

C-terminally truncated human recombinant hyaluronidase PH-20.

[0113] The enzyme reaction was carried out by mixing 50 μΙ of biotin-labelled CS-4-S at 0.04 μg ml with 50 μΙ PBS containing increasing amounts of human recombinant hyaluronidase PH-20, e.g. 0,04 - 5,0 g / ml (final concentration). In this enzyme, the 21 C-terminal amino acids of human wild- type PH-20 had been deleted.

The following steps were identical to those described for Example 2 ([0104]- [01 11]). [01 14] The results are given in FIG. 2, which show the change in the signal for an increase in concentration of the enzyme.

EXAMPLE 4

Assaying chondroitinase activity of a C-terminally truncated mutant of recombinant human hyaluronidase PH-20

[01 15] Reagents used:

same as in Example 2

mutant human recombinant hyaluronidase PH-20cys264.

[01 16] The enzyme reaction was carried out by mixing 50 μΙ of biotin-labelled CS-4-S at 0.04 pg/ml with 50 μΙ PBS containing increasing amounts of human recombinant hyaluronidase PH-20cys264, e.g. 0,04 - 5,0 pg / ml (final concentration). In this enzyme amino acid Tyr264 was substituted by Cys and the 21 C-terminal amino acid of human PH-20 had been deleted.

Alternatively, the reaction was carried out in 50 mM citrate-phosphate buffer (pH5.8).

The following steps were identical to those described for Example 2.

[01 17] The results are given in FIG. 3 and FIG 4, which show the change in the signal for an increase in concentration of the enzyme.

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Claims

1. A mutant of human hyaiuronidase PH-20 with an increased chondroit- inase activity compared to the wild-type.
2. The mutant hyaiuronidase PH-20 of claim 1 , which has a C50 value against chondroitin-4-sulfate of 1 pg/ml or less at pH 7.0.
3. The mutant hyaiuronidase PH-20 of claim 1 or 2, which has C5o value against chondroitin-4-sulfate of 0.5 pg/ml or less at pH 5.8.
4. The mutant hyaiuronidase PH-20 of any one of claims 1-3, comprising at least one amino acid substitution, deletion and/or insertion within its catalytic center (amino acids 255-275).
5. The mutant hyaiuronidase PH-20 of any one of claims 1-4, comprising at least one amino acid substitution at position 264 (Y).
6. The mutant hyaiuronidase PH-20 of any one of claims 1-5, wherein the amino acid Y264 is replaced by Cys.
7. The mutant hyaiuronidase PH-20 of any one of claims 1-6, which is C- terminally truncated.
8. The mutant hyaiuronidase PH-20 of claim 7, wherein up to 60 C-ter- minal amino acid residues are deleted.
9. A recombinant nucleic acid molecule encoding a mutant human hyaiuronidase PH-20 of any one of claims 1-8.
10. A recombinant vector comprising the nucleic acid molecule of claim 9 operatively linked to a heterologous expression control sequence.
11. A recombinant cell transformed or transfected with a recombinant nucleic acid molecule of claim 9 or a recombinant vector of claim 10.
12. A method for preparing a mutant human hyaluronidase PH-20 of any one of claims 1-8, comprising the steps:
(a) culturing a recombinant cell of claim 11 in a culture medium under conditions under which the mutant human hyaluronidase PH-20 is expressed, and
(b) isolating the mutant human hyaluronidase PH-20 from the cell or the culture medium.
13. An agent selected from a mutant of human hyaluronidase PH-20 of any one of claims 1-8, the nucleic acid molecule of claim 9, the vector of claim 10 or the cell of claim 11 , for use in medicine, particularly for use in human medicine.
14. The agent for use according to claim 13 in the treatment of an injury or disease of the nervous system, e.g. the central nervous system, the spinal cord or the peripheral nervous system.
15. The agent for use according to claim 13 or 14 for local administration by injection or infusion.
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