Mammalian milk growth factor

Details Mammalian milk growth factor Mammalian milk growth factor

2000-05-10

2003-03-11

Eyler, Yvonne (Department: 1646)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Hormone or other secreted growth regulatory factor,...

C514S002600, C530S350000, C530S399000, C435S069100

Reexamination Certificate

active

06531134

ABSTRACT:

The present invention relates to a novel growth factor from bovine milk, milk products or milk product extracts. The present invention also relates to the use of recombinant DNA technology to isolate, clone and sequence nucleic acids encoding the mature and precursor forms of the growth factor and, in addition, to the use of these nucleic acids in the recombinant production of the growth factor.
Growth factors are implicated in a wide range of physiological and pathological processes such as cell communication, growth and development, apoptosis, embryogenesis, initiation of the immune response, cell survival and differentiation, wound healing, and cancer. Justifiably, there is a great deal of interest in isolating, characterising and defining the functional mechanisms of growth factors, not only in understanding the basic mechanisms behind normal growth control, but also because of their potential therapeutic use.
Growth factors may also comprise an essential component of defined media used in the growth of cells in culture by the biotechnology industry. For many years animal sera have been used to supplement culture media to provide essential components for cell growth. Foetal bovine serum (FBS) is most commonly used, however there are market and regulatory concerns about the safety of animal and human sourced proteins in pharmaceutical manufacturing processes.
Despite the considerable progress made using milk based alternatives to serum, the safety concerns surrounding mammalian body fluids as supplements for cell culture media (as a consequence of the potential for infection with latent pathogens (eg. bovine spongiform encephalopathy (BSE)) means that these are considered unsafe. Therefore attention has focussed on the development of completely defined cell culture media containing growth factors of known origin and defined purity. This means that the growth factor component of cell culture media is provided by purified native or recombinant molecules.
Milk is an important nutrient required for the growth and development of an infant. It is a source of nutrients such as casein, lactoferrin, lactalbumin, lactose and various other compounds such as vitamins, ions, enzymes etc. A number of growth factors have been identified in human and bovine milk. These include Insulin-like growth factor I and II, fibroblast growth factors, transforming growth factor &bgr;, and platelet-derived growth factor. Although epidermal growth factor is present in human milk it has not been convincingly demonstrated in bovine milk (lacopetta et al, Acta Paediatr, 81, 287, (1992)). Several roles have been proposed for milk-derived growth factors including development and differentiation of the mammary gland (Collier et al, Livestock Production Science, 35, 21, (1993)), regulation of the developing neonatal immune system (Ishizaka et al. Cellular Immunol, 159, 77, (1994)), gastrointestinal growth and maturation (Read et al, Pediatric Research, 18, 133, (1984)), and possible actions in other organs. Milk-derived growth factors have also been shown to support the growth of a variety of cells in culture. In addition, bovine milk whey, the by-product of cheese manufacture, can also support the growth of mammalian cells in the short or long term (Derouiche et al, Lait, 70, 313, (1990); Damerdji et al, Biotechnology Tech, 2, 253, (1988)) and has been shown to possess antitumor activity (Bounous et al, Clin. Invest. Med. 11, 312, (1988)). The prior art also includes Australian patent 645589 to the present applicant which describes the use of a bovine milk whey extract, containing a plurality of cell growth stimulating factors, as a replacement for serum in mammalian cell culture.
It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art. Accordingly, in a first aspect of the present invention there is provided a novel growth factor, hereinafter referred to as Bovine Milk Growth Factor (BMGF) obtainable from bovine milk, milk products or milk product extracts. The term “milk” as used herein refers to lactational secretions of human or animal origin.
The BMGF may be in a substantially pure or partially pure form. The term “substantially pure” as used herein to describe the purity of BMGF means at least 70% pure.
The term “partially pure” as used herein to describe the purity of BMGF means a specific activity greater than 0.3 units per milligram protein.
The term “one unit” as used herein as a measure of BMGF activity is defined as the amount of BMGF required to compete for 50% of the binding of
125
I-labelled recombinant human epidermal growth factor to AG2804 cells under the assay conditions described in Example 1 of this specification.
The native BMGF in its glycosylated form may have a molecular weight of approximately 21 to 25 kDa as determined by SDS-PAGE. It has the ability to stimulate the proliferation of fibroblasts and/or epithelial cells.
In a preferred form of this aspect of the invention the BMGF has an amino acid sequence as follows:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCIKGRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:2)
The BMGF disclosed herein may be a member of the epidermal growth factor family of growth factors. It contains eighty amino acid residues and eight half cysteines, a pattern that has also been described for an epidermal growth factor-like molecule purified from the conditioned media of mouse pancreatic beta tumor cells (Shing et al, Science, 259, 1604, (1993)). The amino acid sequence of the human form of this factor, termed, betacellulin, has also been deduced from a nucleotide sequence obtained from a human adenocarcinoma cell line (Sasada et al, Biochem. Biophys. Res. Commun. 190, 1173, (1993)). It is likely, based on the sequence homologies between BMGF and mouse betacellulin (58 identical residues) and with human betacellulin (72 identical residues), that BMGF is the bovine form of the betacellulin. Clearly, it is not the same molecule. The BMGF isolated from bovine cheese whey extract has a molecular mass of 21-25 kDa, which is substantially smaller in size than the 32 kDa reported for the natural mouse betacellulin.
The present invention also includes within its scope precursor forms of BMGF and functionally active fragments (peptides) of BMGF. Such fragments may have enhanced or diminished growth stimulatory activity and/or may expand or limit the range of cells responsive to BMGF's growth stimulatory activity. They may find useful applications in areas such as, but not limited to, the repair or prevention of gut damage or in wound healing.
They may be produced by methods known to those skilled in the art. Procedures at the genetic level such as (but not limited to) site-directed mutagenesis or at the protein level such as (but not limited to) chemical modification are within the scope of the invention.
In a preferred form of this aspect of the invention the fragments of BMGF may include the following amino acid sequences:
DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHF (SEQ ID NO:3); or
GYAGARCERVDLFY (SEQ ID NO:4); or
DGNSTRSPEDDGLLCGDHAENCPATTTQPK (SEQ ID NO:5); or
RRGHFSRCPK (SEQ ID NO:6); or
QYK (SEQ ID NO:7); or
HYCIK (SEQ ID NO:8); or
GRCRFVVAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO:9)
The present invention also provides BMGF that is substantially non-glycosylated. This may be prepared, for example, by subjecting glycosylated BIZGF to an enzymatic deglycosylation step and recovering the de-glycosylated form. The BMGF in its non-glycosylated or partially non-glycosylated form may have a molecular weight of approximately 9-14 kDa as determined by SDS-PAGE.
In a further preferred form of this aspect of the invention, the BMGF is obtained from bovine milk, bovine milk products or bovine milk product extracts. Moire preferably it is obtained from cheese whey, most preferably from bovine cheese whey extract.
In a further aspect of the present invention, there is provided a process for the isolation, in a substantially pure or partially pure form, of BMGF from bovine milk or milk

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