Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1996-03-20
2001-02-20
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S324000, C530S399000, C536S023510
Reexamination Certificate
active
06191106
ABSTRACT:
DESCRIPTION
TECHNICAL FIELD
This invention relates to the molecular biology of cellular growth factors and recombinant DNA technology. More specifically, this invention relates to epidermal growth factor (EGF) modified to increase its binding activity at low pH, and the therapeutic uses of modified EGF of the invention.
BACKGROUND OF THE INVENTION
Epidermal growth factor (EGF) is a 53-amino acid protein synthesized in the duodenum and salivary glands of normal humans, and normally excreted in the urine. For its effect in reducing gastric acid secretion, and its first isolation source, it was formerly termed urogastrone. After it was sequenced, it was recognized that urogastrone was homologous to murine EGF, and that urogastrone additionally stimulated the proliferation of certain cell types, prompting a change in nomenclature to EGF. The biological and chemical properties of hEGF and mEGF are reviewed in G. Carpenter et al,
Ann Rev Biochem
(1979) 48:193-216.
The amino acid sequence of human EGF is:
Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg (SEQ ID NO:1)
This sequence was published by H. Gregory et al,
Int J Peptide Protein Res
(1977) 9:107-18, who isolated the protein as urogastrone from human urine (1 mg/ 1000 L), and disclosed its sequence homology with mEGF. Murine EGF has the sequence (differences from hEGF underlined):
Asn Ser Tyr Pro Gly Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Asn Gly Gly Val Cys Met His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn Cys Val Ile Gly Tyr Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu Arg Trp Trp Glu Leu Arg, (SEQ ID NO:2)
which conserves the disulfide bonds. Gregory also discloses that urogastrone may be maintained in aqueous solution at pHs of 1-11 for at least 20 hours without loss of activity, and that the six C-terminal amino acids may be removed without loss of biological activity.
Preparation of an hEGF gene is described in EPO 046,039, and cloning and expression of EGF is disclosed in commonly-owned copending U.S. patent application Ser. No. 004,212, filed Jan. 5, 1987, now U.S. Pat. No. 5,096,825, a continuation of Ser. No. 457,412, filed Jan. 12, 1983, now abandoned. U.S. Ser. No. 004,212 now U.S. Pat. No. 5,096,825, is incorporated herein by reference in full. Other disclosures of EGF preparation are M. S. Urdea et al,
Proc Nat Acad Sci USA
, (1983) 80:7461-65 (chemical synthesis of gene and expression in yeast), T. Oka et al,
Proc Nat Acad Sci USA
, (1985) 82:7212-16 (fusion protein in
E. coli
); Cohen et al, U.S. Pat. No. 4,743,679 (recombinant fusion protein); Sugimoto, U.S. Pat. No. 4,621,052 (human hybridoma cell culture); Nishimura et al, U.S. Pat. No. 4,528,186 (adsorption from urine); and Cohen et al, U.S. Pat. No. 3,948,875 (purification from murine submaxillary glands). Pharmaceutical compositions containing EGF are disclosed in Finkenaur, U.S. Pat. No. 4,717,717 (stabilized against degradation by moisture with water-soluble cellulose derivatives); U.S. Pat. No. 4,703,108 (cross-linked collagen sponge); and Camble et al, U.S. Pat. No. 3,917,824 (lyophilized solid or dextrose solution).
C. George-Nascimento et al,
Biochem
(1988) 27:797-802 reported the isolation from recombinant yeast culture of four distinct forms of EGF, termed A, B, C, and D, each of which exhibit full EGF activity. EGF-D represents the 52-amino acid sequence obtained by removing the C-terminal arg, while EGF-B corresponds to EGF-D wherein the C-terminal arg-leu has been removed. EGF-C appears to be EGF-D in which Met
21
has been oxidized, while EGF-A appears to be EGF-B with Met
21
oxidized. EGF-D is reported to be stable when stored as a lyophilized powder.
George-Nascimento et al, U.S. Ser. No. 07/351,773, now U.S. Pat. No. 5,158,935, disclosed EGF muteins in which the Asp
11
residue is replaced, preferably with Glu, in order to prevent degradation by isomerization of the Asp residue. Asp
11
may isomerize to iso-Asp, which disturbs the peptide backbone and impairs the peptide's activity.
The physical structure of a recombinantly-produced hEGF has been partially elucidated using COSY and NOESY by K. Makino et al,
Proc Nat Acad Sci USA
(1987) 84:7841-45. Makino disclosed that amino acids 19-32 form an antiparallel beta-pleated sheet, placing His
10
in close proximity to Tyr
22
and Tyr
29
. Makino also suggested that amino acids 45-53 may form an alpha helix which crosses the surface of the beta-pleated sheet, creating a hydrophobic pocket comprising His
10
, Tyr
22
, Tyr
29
, Trp
49
, and Trp
50
. Removal of amino acids 49-53 altered the NMR chemical shift and pKa of the ring protons on His
10
, His
19
, Tyr
22
, and Tyr
29
, and drastically reduced the activity, suggesting that these amino acids may participate in the EGF binding site.
Nestor et al, U.S. Pat. No. 4,686,283 disclosed the preparation of polypeptides and polypeptide analogs homologous to amino acids 34-43 of EGF and TGF-&agr;, which are useful as EGF antagonists and for preparing anti-EGF antibodies.
DISCLOSURE OF THE INVENTION
Contrary to the teachings in the art, we have found that His
16
is not a residue essential for receptor binding activity. Surprisingly, we discovered that EGF muteins in which His
16
is replaced with a neutral or acidic amino acid exhibit activity equal or superior to wild-type EGF at neutral and basic pH, and which retain their activity at pHs as low as 4.0, whereas wtEGF exhibits substantially reduced binding activity below pH 6.5.
We have now invented EGF muteins having neutral or acidic residues at position 16, which exhibit activity at low pH which is enhanced with respect to wild-type EGF. We have also invented therapeutic methods of treatment comprising administering the EGF muteins of the invention to a subject in need thereof.
REFERENCES:
patent: 3917824 (1975-11-01), Camble et al.
patent: 3948875 (1976-04-01), Cohen et al.
patent: 4528186 (1985-07-01), Nishimura et al.
patent: 4621052 (1986-11-01), Sugimoto
patent: 4686283 (1987-08-01), Nestor, Jr. et al.
patent: 4703108 (1987-10-01), Silver et al.
patent: 4717717 (1988-01-01), Finkenaur
patent: 4743679 (1988-05-01), Cohen et al.
patent: 5096825 (1992-03-01), Barr et al.
patent: 5130982 (1992-07-01), Cini et al
patent: 5158935 (1992-10-01), Nascimento et al.
patent: 46039 (1982-02-01), None
Carpenter and Cohen,Ann. Rev. Biochem. 48:193-216 (1979).
George-Nascimento et al.,Biochem. 27:797-802 (1988).
Greenfield et al.,Biophysical Journal59:293a (1991).
Gregory and Preston,Int. J. Peptide Protein Res. 9:107-118 (1977).
Hommel et al.,Biochemistry30:8891-8898 (1991).
Makino et al.,Proc. Natl. Acad. Sci. USA 84:7841-7845 (1987).
Moy et al.,PNAS86:9836-9840 (1989).
Oka et al.,Proc. Natl. Acad. Sci. USA 82:7212-7216 (1985).
Urdea et al.,Proc. Natl. Acad. Sci. USA 80:7461-7465 (1983).
Blaney Jeffrey M.
Mullenbach Guy T.
Rosenberg Steven
Blackburn Robert P.
Chiron Corporation
Hayes Robert C
Kunz Gary L.
Morley Kimberlin L.
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