Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-09-22
2001-05-22
Allen, Marianne P. (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S388100, C530S389100, C530S389200
Reexamination Certificate
active
06235884
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to growth factors.
Growth factors play a central role in mediating cell proliferation and differentiation, for example, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), transforming growth factor-alpha (TGF-&agr;), and transforming growth factor-beta (TGF-&bgr;) have been implicated in the proliferation of connective tissue cells and the induction of angiogenesis characteristic of wound repair (Van Brunt and Klausner, 6
Biotechnology
25, 1988). In addition, Agel et al. (146
J. Pathol.
197, 1985) suggest that growth factors are involved in the etiology of atherosclerosis; for example, the smooth muscle cell (SMC) hyperplasia that accompanies atherosclerosis has been attributed to PDGF, a potent SMC mitogen.
Heparin affinity chromatography has been used extensively for purifying and characterizing a variety of these growth factors. Acidic FGF (aFGF) and basic FGF (bFGF) bind to immobilized heparin columns and are eluted with 1.0M to 1.2M NaCl and 1.5M to 1.8M NaCl, respectively (Folkman and Klagsbrun, 235
Science
442, 1987; Lobb et al., 261
J. Biol. Chem.
1924, 1986). Several growth factors which are structurally homologous to aFGF and bFGF also have an affinity for heparin (see, for example, Rubin et al., 86
Proc. Natl. Acad. Sci. USA
802, 1989). PDGF binds to immobilized heparin, but with relatively low affinity, being eluted with only 0.5M NaCl. Epidermal growth factor (EGF) does not bind heparin to any substantial extent under the conditions described in the cited references on heparin binding growth factors. Lobb et al. (261
J. Biol. Chem.
1924, 1986) report the partial purification by heparin affinity of two classes of growth factors mitogenic for endothelial cells. Gospodarowicz et al. (81
Proc. Natl. Acad. Sci. USA
6963, 1984) report the use of heparin affinity in the purification of bovine brain and pituitary fibroblast growth factors. Shing et al. (29
J. Cell Biochem.
275, 1985) report a chondrosarcoma-derived growth factor purified by heparin-Sepharose affinity chromatography and Bio Rex 70 cation exchange chromatography. Bohlen et al. (185
FEBS Lett.
177, 1985) report a fibroblast growth factor, derived from human brain, which is purified by cation-exchange chromatography, heparin-Sepharose affinity, and reverse-phase HPLC. Shing et al. (223
Science
1296, 1984) report a heparin-binding tumor cell-derived capillary endothelial cell factor. Besner et al. (107
J. Cell Biol.
481a, 1988) report the detection of a heparin-binding, mononuclear cell-derived growth factor(s) which is cationic, is of 6000-14,000 MW, is inactivated by heat (100° C., 10 min), is inactivated by dithiothreitol (5 mM), and is resistant to incubation with 4M guanidine or 0.1M HCl.
SUMMARY OF THE INVENTION
In one aspect, the invention generally features a novel growth factor which we have termed heparin binding EGF-homologous mitogen (HB-EHM). By “EGF homologous” is meant having a segment structurally related to epidermal growth factor in that it contains six cysteine residues spaced in a manner characteristic of any of the EGF family of proteins (e.g., human amphiregulin (AR), Shoyab et al., 243
Science
1074, 1989; human TGF-&agr;, Derynck et al., 38
Cell
287, 1984; and human EGF, Gregory, 257
Nature
325, 1975), which participates in binding as described below to one or more of the EGF family of receptors (e.g., on A-431 cells or smooth muscle cells). In general, an EGF-homologous segment renders the protein including such a domain at least partially resistant to heat (e.g., following exposure to a temperature of 90° for 5 minutes) and sensitive to dithiothreitol (DTT) (e.g., following exposure to 5mM DTT for 2 hours). “Heparin binding” means having a specific affinity for heparin (i.e., an affinity beyond that predicted only by ionic interactions) as evidenced by binding to heparin at NaCl concentrations above those which elute similarly charged proteins having no specific affinity. In general, heparin binding factors remain bound to heparin up to NaCl concentrations of at least 0.6M (most preferably, at least 0.9M).
In a second aspect, the invention features polypeptides which bind heparin, which include an EGF-homologous segment, and which stimulate growth of fibroblast cells, epithelial cells, and smooth muscle cells, but not endothelial cells.
In preferred embodiments, these polypeptides are human HB-EHM, and, more preferably, they include a characteristic EGF-homologous segment sequence (substantially, amino acids 108 to 143 of SEQ ID NO:2: C L R K Y K D F C I H G E C K Y V K E L R A P S C I C H P G Y H G E R C). One particular such peptide includes the above described EGF-homologous segment and all or part of amino acids 1 to 208 of SEQ ID NO:2: M K L L P S V V L K L F L A A V L S A L V T G E S L E R L R R G L A A G T S N P D P P T V S T D Q L L P L G G G R D R K V R D L Q E A D L D L L R V T L S S K P Q A L A T P N K E E H G K R K K K G K G L G K K R D P C L R K Y K D F C I H G E C K Y V K E L R A P S C I C H P G Y H G E R C H G L S L P V E N R L Y T Y D H T T I L A V V A V V L S S V C L L V I V G L L M F R Y H R R G G Y D V E N E E K V K L G M T N S H. In a mature form, HB-EHMs may have an amino-terminus between aspartic acid residue 63 and alanine residue 82 (as shown in
FIG. 3
, SEQ ID NO:2) and a carboxy-terminus between serine residue 147 and proline residue 149 (also as shown in
FIG. 3
, SEQ ID NO:2). The invention also encompasses smaller polypeptides, for example, those having an amino-terminus between arginine residue 73 and alanine residue 82 (as shown in
FIG. 3
, SEQ ID NO: 2) and a carboxy-terminus at serine residue 147 (also as shown in
FIG. 3
, SEQ ID NO: 2). These polypeptides are preferably acid stable. The isolated polypeptide may contain a sequence substantially identical to the amino acid sequence shown in
FIG. 1
(amino acids 82 to 147 in SEQ ID NO:2) or may contain a sequence substantially identical to the amino acid sequence shown in
FIG. 4
(amino acids 63 to 148 in SEQ ID NO: 2). The polypeptides according to the invention are preferably cationic, may have a pI of between 7.2 and 7.8 (e.g., when produced by a eukaryotic cell), and may be, but need not be, glycosylated. Preferred polypeptides according to the invention have an apparent molecular weight of approximately 22,000 on a non-reducing polyacrylamide gel and include at least 66 amino acid residues. In addition, these polypeptides, preferably, are sufficiently isolated from other co-purifying substances to be suitable for therapeutic use.
In other aspects, the invention features: purified nucleic acid which encodes the polypeptides described above; vectors (preferably, pMTN-HBEGF, pAX-HBEGF, pNA28, and pNA51) which direct expression of this nucleic acid in eukaryotic (preferably, mammalian) or prokaryotic (preferably,
Escherichia coli,
most preferably,
E. coli B
or
E. coli
W3110) cells. The invention also features cells containing such vectors; such cells may be eukaryotic cells (for example, mammalian cells capable of secreting a mature form of the protein into the growth medium) or prokaryotic cells which are capable of producing a polypeptide which includes the primary sequence of a mature form of the protein and which may also include additional amino acids at the amino or carboxy terminus which facilitate improved expression, stability, or ease of isolation of the HB-EHM. The expression vectors or vector-containing cells of the invention can be used in a method of the invention to produce HB-EHM and equivalent polypeptides. These polypeptides can be used in a method of the invention for healing a wound in a patient, involving applying to the wound a wound healing amount of the polypeptides described above. The polypeptides can also be used in a method of the invention for the in vitro culturing of a cell whose proliferation is stimulated by HB-EHM, preferably, fibroblasts, epithelial cells, or smooth muscle cells, involving contacting the cells with a growth-stimulatory amount of the poly
Abraham Judith A.
Besner Gail E.
Higashiyama Shigeki
Klagsbrun Michael
Allen Marianne P.
Fish & Richardson P.C.
Scios Nova Inc.
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