Deamidated interferon-beta

This application claims priority to U.S. Provisional Patent Application No. 60/626,837, filed Nov. 10, 2004, titled DEAMIDATED INTERFERON-BETA, the disclosure of which is incorporated herein by reference in its entirety and for all purposes.

1. Technical Field

This invention is in the general area of biologically active protein chemistry. More specifically it relates to mutationally and chemically altered Interferon-β analogs that differ from the native protein by substitutions, deletions or modifications of cysteine, asparagine and other residues.

2. Background Art

Interferon-β has been found to be useful in the treatment of human disease, in particular multiple sclerosis. Multiple sclerosis (MS) is a chronic, often disabling disease of the central nervous system that occurs when a protective sheath surrounding nerve fibers breaks down. About thirty percent of MS patients suffer from a relapsing-remitting form of the disease in which symptoms disappear totally or partially after a flare-up and are followed by a period of stability that can last for months or years. Administration of beta interferon (Interferon-β or IFN-β) has been demonstrated to reduce the frequency of MS flare-ups. As a result, Interferon-β based pharmaceuticals have become a valuable tool in management and treatment of MS.

Recombinant DNA (rDNA) techniques have been developed to facilitate the large scale manufacturing of Interferon-β based pharmaceuticals. One problem in particular that needed to be addressed by these techniques was that human beta interferon, the amino acid sequence of which is provided in FIG. 1 (SEQ ID NO: 1), contains cysteine residues at positions 17, 31, and 141, Gene (1980) 10:11-15 and Nature (1980) 285:542-547), at least some of which are nonessential to their activity but are free to form undesirable intermolecular or intramolecular links. In the course of the microbial preparation of IFN-β by rDNA techniques, it has been observed that dimers and oligomers of IFN-β are formed in extracts containing high concentrations of IFN-β due to this intermolecular linking. This multimer formation renders purification and isolation of IFN-β very laborious and time-consuming and necessitates several additional steps in purification and isolation procedures such as reducing the protein during purification and reoxidizing it to restore it to its original conformation, thereby increasing the possibility of incorrect disulfide bond formation. In addition, this multimer formation has been associated with low specific biological activity.

In order to address these issues, refined rDNA techniques have been developed to alter microbially produced biologically active IFN-β protein analogs in a manner that does not affect their activity adversely, but reduces or eliminates their ability to form intermolecular crosslinks or intramolecular bonds that cause the protein to adopt an undesirable tertiary structure (e.g., a conformation that reduces the activity of the protein). Directed mutagenesis techniques have been successfully used to form mutationally altered biologically active protein analogs (a “protein analog” refers herein to a synthetic protein in which one or more amino acids has been genetically and/or chemically modified and that retains a biological activity of the parent protein) that retain a desired activity of their parent proteins but lack the ability to form intermolecular links or undesirable intramolecular disulfide bonds. Synthetic protein analogs of IFN-β biologically active protein which have the cysteine residue at position 17 deleted or replaced by another amino acid have been found to have the desired activity and characteristics.

In particular, Interferon-β 1b (IFN-β 1b), a synthetic, recombinant protein analog of IFN-β, is a biologically active protein which has the cysteine residue at position 17 replaced by a serine residue has been made. As a microbially produced protein, IFN-β 1b is unglycosylated. It also has an N-terminal methionine deletion. IFN-β 1b has been formulated into a successful pharmaceutical marketed as Betaseron® that has been shown to be effective for treatment and management of MS. This protein analog, materials and techniques for its manufacture, its formulation as a therapeutic and its use to treat MS are described and claimed in a number of U.S. patents and applications including application Ser. No. 435,154, filed Oct. 19, 1982; U.S. Pat. No. 4,588,585, issued May 13, 1986; U.S. Pat. No. 4,737,462, issued Apr. 12, 1988; and U.S. Pat. No. 4,959,314, issued Sep. 25, 1990; each of which is incorporated by reference herein for their disclosure of these features.

Large scale manufacturing of IFN-β for pharmaceuticals is also conducted from mammalian sources, in particular Chinese hamster ovary (CHO) cells. This IFN-β analog, referred to as IFN-β 1a, lacks the Ser17 mutation of IFN-β 1b and is glycolsylated. IFN-β 1a is formulated into therapeutic products marketed as Avonex® and Rebith®.

As with most therapeutics, there is a continual desire to identify and manufacture more potent biologically active agents. It the case of IFN-β based pharmaceuticals, a IFN-β analog with increased biological activity would be desirable.

In addition, some IFN-β pharmaceutical formulations, including Betaseron®, contain human albumin (HA or HSA), a common protein stabilizer. HA is a human blood product and is in increasingly low supply. Accordingly, more recently there has been a desire for HA-free drug formulations, and a stable and effective HA-free IFN-β formulation would be desirable.

The present invention addresses these needs by providing deamidated Interferon-β. The deamidated IFN-β is a human interferon-β protein analog in which the asparagine at position 25, numbered in accordance with native interferon-β, is deamidated. The deamidated product exhibits a biological activity of native human interferon-β at an increased level and does not require HA for protein stabilization.

In a specific embodiment, the deamidated IFN-β is a synthetic human interferon-β 1b protein analog in which cysteine at position 17, numbered in accordance with native interferon-β, is deleted or replaced by a neutral amino acid, in particular serine, and the asparagine at position 25, is deamidated such that it becomes a cyclic imide, aspartate or iso-aspartate residue. The deamidated product exhibits the desired biological activity of native human interferon-β (e.g., cellular cytopathic effects or antiproliferative activity, such as have been shown to be correlated with reducing the frequency of multiple sclerosis flare-ups) at an increased level relative to its IFN-β parent protein. In addition, the enhanced biological activity is observed in an HA-free formulation of the protein analog.

Formulations of the active protein analog compounds in therapeutic compositions and methods of making and use are also provided.

In addition, an endoproteinase-C peptide map technique that produces a fingerprint profile for proteins using an enzymatic digest of a reduced protein sample at relatively low pH, followed by chromatographic resolution of peptide fragments, useful in quality control as an ID test for the deamidated products is provided.

These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.