Human Chondroitinase Glycoprotein (CHASEGP), Process for Preparing the Same, and Pharmaceutical Compositions Comprising Thereof

• US 20100015698A1
• Filed: 05/29/2009
• Published: 01/21/2010
• Est. Priority Date: 12/16/2002
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Frost, Gregory I., Kundu, Anirban, Bookbinder, Louis H.
• Granted Patent: US 8,815,558 B2
• Time in Patent Office: Days
• US Class Current: 435/320.100
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12Y 402/02004 Chondroitin ABC lyase (4.2.2.4), i.e. chondroitinase C12Y402/0202, C12Y402/02021 take precedence

Abstract

The invention relates to the discovery of novel Chondroitinase Glycoproteins (CHASEGPs), methods of manufacture, and potential uses in conditions where removal of chondroitin sulfates may be of therapeutic benefit. Chondroitinase Glycoproteins require both a substantial portion of the catalytic domain of the CHASEGP polypeptide and asparagine-linked glycosylation for optimal chondroitinase activity. The invention also includes carboxy-terminal deletion variants of CHASEGP that result in secreted variants of the protein to facilitate manufacture of a recombinant CHASEGP. Further described are suitable formulations of a substantially purified recombinant CHASEGP glycoprotein derived from a eukaryotic cell that generate the proper glycosylation required for its optimal activity. CHASEGP is useful for the degradation of glycosaminoglycans and chondroitin sulfate proteoglycans under clinical conditions where their removal is of therapeutic value.

Claims

1. A substantially purified chondroitinase glycoprotein comprising, a CHASEGP polypeptide and at least 1 N-linked sugar moiety, wherein said N-linked sugar moiety is covalently attached to an asparagine residue of said polypeptide.

16. A glycoprotein of claim 1 that is a mutein, wherein:

up to about 50% of the amino acids are replaced with another amino acid; and

the resulting polypeptide is a single chain or two chain polypeptide that has catalytic activity of at least 10% of the unmutated polypeptide.

22. A nucleic acid molecule, comprising a sequence of nucleotides that encodes the polypeptide of claim 1.

23. The nucleic acid molecule of claim 22 that comprises a sequence of nucleotides selected from the group consisting of:

(a) a sequence of nucleotides set forth as nucleotides 726-1995 in SEQ ID No. 3;

(b) a sequence of nucleotides that hybridizes under high stringency along its length or along at least about 70% of the full-length to the sequence of nucleotides set forth as nucleotides 726-1995 in SEQ ID No. 3 or as SEQ ID No. 5;

(c) a sequence of nucleotides that is a splice variant of a or b;

(d) a sequence of nucleotides that encodes the chondroitinase domain or a catalytically active portion thereof that includes a sequence of nucleotides having at least about 60%, 70%, 80%, 90% or 95% sequence identity the sequence set forth in SEQ ID Nos. 3,4 or 5; and

(e) a sequence of nucleotides comprising degenerate codons of (a), (b),(c) or (d).

24. An isolated nucleic molecule that encodes a mutein of claim 16.

25. A vector comprising the nucleic acid molecule of claim 22.

26. The vector of claim 25 that is an expression vector.

27. The vector of claim 25 that is a eukaryotic vector.

28. The vector of claim 25 that includes a sequence of nucleotides that directs secretion of any polypeptide encoded by a sequence of nucleotides operatively linked thereto.

2-15. -15. (canceled)

17-21. -21. (canceled)

29-55. -55. (canceled)