U.S. patent number RE38,240 [Application Number 09/538,305] was granted by the patent office on 2003-08-26 for dna encoding human endothelial cell growth factors and plasmids comprising said dna.
This patent grant is currently assigned to Aventis Pharmaceuticals, Inc.. Invention is credited to Wilson Burgess, William N. Drohan, Michael C. Jaye, Thomas Maciag.
United States Patent |
RE38,240 |
Jaye , et al. |
August 26, 2003 |
DNA encoding human endothelial cell growth factors and plasmids
comprising said DNA
Abstract
The present invention is directed to DNA encoding human
endothelial cell growth factors, and to plasmids comprising said
DNA. In particular, the invention relates to DNA encoding a
cleavable signal peptide and an endothelial cell growth factor,
wherein removal of said signal peptide yields a mature form of the
growth factor.
Inventors: |
Jaye; Michael C. (Glenside,
PA), Burgess; Wilson (Clifton, VA), Maciag; Thomas
(Freeport, ME), Drohan; William N. (Springfield, VA) |
Assignee: |
Aventis Pharmaceuticals, Inc.
(Bridgewater, NJ)
|
Family
ID: |
46252314 |
Appl.
No.: |
09/538,305 |
Filed: |
March 28, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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743261 |
Nov 4, 1996 |
5827826 |
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472964 |
Jun 7, 1995 |
5571790 |
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334884 |
Nov 3, 1994 |
5552528 |
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799859 |
Nov 27, 1991 |
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693079 |
Apr 29, 1991 |
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134499 |
Dec 18, 1987 |
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835594 |
Mar 3, 1986 |
4868113 |
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Reissue of: |
840088 |
Apr 11, 1997 |
05849538 |
Dec 15, 1998 |
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Current U.S.
Class: |
435/69.7;
435/320.1; 435/69.4; 435/69.8; 530/399; 536/23.4; 536/23.51 |
Current CPC
Class: |
C07K
14/501 (20130101); A61K 38/00 (20130101); Y10S
930/12 (20130101); Y02A 50/30 (20180101); Y02A
50/473 (20180101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/50 (20060101); A61K
38/00 (20060101); C12P 021/04 (); C12N 015/05 ();
C12N 015/63 (); C07H 021/04 (); C07K 014/50 () |
Field of
Search: |
;435/69.4,69.7,69.8,320.1,325,243,252.33 ;536/23.1,23.4,23.5,23.51
;530/399 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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437 281 |
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Apr 1994 |
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EP |
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WO 87/01728 |
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Mar 1987 |
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WO |
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Other References
Alberts et al., "The extracellular mMatrix" Molecular Biology of
the Cell, Garland Pub., pp. 692-715 (1983). .
Bohlen et al., "Acidic fibroblast growth factor (FGF) from bovine
brain: amino-terminal sequence and comparison with basic FGF," EMBO
J., vol. 4, pp. 1951-1956 (1985). .
Brake et al., "Alpha-factor-directed synthesis and secretion of
mature foreign proteins in Saccharomyces cerevisiae," Proc. Natl,
Acad. Sci., USA, vol. 81, pp. 4642-4646 (1984). .
Burgess et al., "Multiple forms of endothelial cell growth
factor-rapid isolation and biological and chemical
characterization," J. Biol. Chem., vol. 260, pp. 11389-11392
(1985). .
Collaborative Research Inc., Product Catalog (1983). .
Collaborative Research Inc., Product Catalog (1986). .
Conn et al., "The isolation and purification of two anionic
endothelial cell growth factors from human brain," Biochem.
Biophys. Res. Comm., vol. 124, pp. 262-268 (1984). .
de Ferra et al., "Alternative splicing accounts for the four forms
of myelin basic protein," Cell, vol. 43, pp. 721-727 (1985). .
Esch, "Primary structure of bovine brain acidic fibroblast growth
factor ("FGF")," Biochem. Biophys. Res. Comm., vol. 133, pp.
554-562 (1985). .
Esch et al., "Primary structure of bovine pituitary basic
fibroblast growth factor (FGF) and comparison with the
amino-terminal sequence of bovine brain acidic FGF," Proc. Natl.
Acad. Sci., USA, vol. 82, pp. 6507-6511 (1985). .
Gimenez-Gallego et al., "Brain-derived acidic fibroblast factor:
complete amino acid sequence and homologies," Science, vol. 230,
pp. 1385-1388 (1985). .
Hunkapiller et al., "High-sensitivity sequencing with a gas-phase
sequenator," Meth. Enzymol., vol. 91, pp. 399-413 (1983). .
Jaye et al., "Isolation of a human-anti-haemophilic factor IX cDNA
clone using a unique 52-base synthetic oligonucleotide probe
deduced from the amino acid sequence of bovine factor IX," Nucl.
Acids Res., vol. 11, pp. 2325-2335 (1983). .
Jaye et al., "Modulation of the sis gene transcript during
endothelial cell differentiation in vitro," Science, vol. 228, pp.
882-885 (1985). .
Klagsbrun et al., "Heparin affinity of anionic and cationic
capillary endothelial cell growth factors: analysis of
hypothalamus-derived growth factors and fibroblast growth factors,"
Proc. Natl. Acad. Sci. USA, vol. 82, pp. 805-809 (1985). .
Lathe, "Synthetic oligonucleotide probes deduced from amino acid
sequence data: theoretical and practical implications," J. Mol.
Biol., vol. 183, pp. 1-12 (1985). .
Lobb et al., "Purification and characterization of heparin-binding
endothelial cell growth factors," J. Biol. Chem., vol. 264, No. 4,
pp. 1924-1928 (1985). .
Lobb et al., "Comparison of human and bovine brain derived
heparin-binding growth factors," Biochemical and Biophysical
Research Communications, vol. 131, No. 2, pp. 586-592 (1985). .
Lobb et al., "Purification of two distinct growth factors from
bovine neural tissue by heparin affinity chromatography,"
Biochemistry, vol. 23, pp. 6295-6299 (1984). .
Maciag, "Heparin binds endothelial cell growth factor, the
principal endothelial cell mitogen in bovine brain," Science, vol.
225, pp. 932-935 (1984). .
Maciag et al., "Preparation of endothelial cell factor," Cell Cult.
Methods, Mol. Cell, Biol., vol. 1, pp. 195-205 (1984). .
Maciag, "High and low molecular weight forms of endothelial cell
growth factor," J. Biol. Chem., vol. 257, pp. 5333-5336 (1982).
.
Maciag, "An endothelial cell growth factor from bovine
hypothalamus: identification and partial characterization," Proc.
Natl. Acad. Sci., USA, vol. 76, pp. 5674-5678 (1979). .
Maciag, "Angiogenesis" Prog. Hemo. Thromb., vol. 7, pp. 167-182
(1984). .
Marglin et al., "Chemical synthesis of peptides and proteins," Ann.
Rev. Biohem., vol. 39, pp. 841-866 (1970). .
Mestre et al., "Comparative effects of heparin and PK 10169, a low
molecular weight fraction, in a canine model of arterial
thrombosis," Thrombosis Research, vol. 38, pp. 389-399 (1985).
.
Metzler, "Biochemistry," Academic Press, Inc. London (1977). .
Schreiber et al., "A unique family of endothelial cell mitogens:
the antigenic receptor cross-reactivity of bovine endothelial cell
growth factor, brain-derived acidic fibroblast growth factor, and
eye-derived growth factor-II," J. Cell. Biol., vol. 101, pp.
1623-1626 (1985). .
Schreiber et al., "Interaction of endothelial cell growth factor
with heparin: characterization by receptor and antibody
recognition," Proc. Natl. Acad. Sci. USA, vol. 82, pp. 6138-6142
(1985). .
Thomas et al., "Pure brain-derived acidic fibroblast growth factor
is a potent angiogenic vascular endothelial cell mitogen with
sequence homology to interleukin 1," Proc. Natl. Acad. Sci. USA,
vol. 82, pp. 6409-6413 (1985). .
Thomas, "Purification and characterization of acidic fibroblast
growth factor from bovine brain," Proc. Natl. Acad. Sci. USA, vol.
81, pp. 357-361 (1984). .
Thomas et al., "Fibroblast growth factors: broad spectrum mitogens
with potent angiogenic activity," Trends in Biochem Sci., vol. 11,
pp. 81-84 (1986). .
Watson, Molecular Biology of the Gene, Benjamin/Cummings, Menlo
Park, CA (1987). .
Webster's II New Riverside University Dictionary, Riverside Pub.
Co., Boston, MA., pp. 444 (1984)..
|
Primary Examiner: Saoud; Christine J.
Assistant Examiner: Turner; Sharon
Attorney, Agent or Firm: Finnegan, Henderson, Farabow,
Garrett & Dunner, L.L.P.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation of application Ser. No. 08/743,261 filed on
Nov. 4, 1996, .Iadd.now U.S. Pat. No. 5,827,826 .Iaddend.which is a
continuation-in-part of application Ser. No. 08/472,964, filed Jun.
7, 1995, now U.S. Pat. No. 5,571,790, which is a continuation of
application Ser. No. 08/334,884, filed Nov. 3, 1994, now U.S. Pat.
No. .[.5,552,628.]. .Iadd.5,552,528.Iaddend., which is a
continuation of application Ser. No. 07/799,859, filed Nov. 27,
1991, now abandoned, which is a continuation of application Ser.
No. 07/693,079, filed Apr. 29, 1991, now abandoned, which is a
continuation of application Ser. No. 07/134,499, filed Dec. 18,
1987, now abandoned, which is a continuation-in-part of application
Ser. No. 06/835,594, filed Mar. 3, 1986, now U.S. Pat. No.
4,868,113.
Claims
What is claimed is:
1. An isolated DNA encoding a cleavable signal peptide and an
endothelial cell growth factor, wherein removal of said signal
peptide yields a mature form of said endothelial cell growth
factor, and said endothelial cell growth factor either has the
amino acid sequence of .alpha.-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI
QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTYI S K K H A
E K N W F V G L K KNGSCKRGPRTHYGQKAILFLPLPVSSD) or comprises the
amino acid sequence of .[..alpha.-endothelial.].
.Iadd..beta.-.beta.endothelial .Iaddend.cell growth factor
(AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG
QYLAMDTDGLYYGSQTPNEECLFLERLEENHYNTYIS
KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
2. An isolated DNA according to claim 1, wherein said endothelial
cell growth factor is human .alpha.-endothelial cell growth factor
having the amino acid sequence N Y K K P K L L Y C S N G G H F L R
I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMNTDGLLYGSQ
TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK
KNGSCKRGPRTHYGQKAILFLPLPVSSD.
3. An isolated DNA according to claim 1, wherein said endothelial
cell growth factor is human .beta.-endothelial cell growth factor
having the amino acid sequence A E G E I T T F T A L T E K F N L P
P G N Y K K P K L LYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQT P N E E C L
F L E R L E E N H Y N T Y I S KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL
PVSSD.
4. An isolated DNA according to claim 1, wherein said signal
peptide is a heterologous signal peptide.
5. A DNA encoding a cleavable heterologous signal peptide and an
endothelial cell growth factor, wherein removal of said signal
peptide yields a mature form of said endothelial cell growth
factor, and said endothelial cell growth factor either has the
amino acid sequence of .alpha.-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI
QLQLSAEVSVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTY I S K K H
A E K N W F V G L K KNGSCKRGPRTHYCQKAILFLPLPVSSD) or comprises the
amino acid sequence of .beta.-endothelial cell growth factor
(AEGEITTFTALTAKFNLPPGNYKKPKLLYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG
QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS
KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
6. A DNA according to claim 5, wherein said endothelial cell growth
factor is human .alpha.-endothelial cell growth factor having the
amino acid sequence N Y K K P K L L Y C S N G G H F L R I L P
DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTFDGLLYGSQ
TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK
KNGSCKRGPRTHYGQKAILFLPLPVSSD.
7. A DNA according to claim 5, wherein said endothelial cell growth
factor is human .beta.-endothelial cell growth factor having the
amino acid sequence AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG QYLAMDTDGLLYGSQT P N E E C L
F L E R L E E N H Y N T Y I S KKHAEKNWFVGLKKNGSCKRGPRRTHYGQKAILFLPL
PVSSD.
8. A plasmid comprising a DNA encoding a cleavable signal peptide
and an endothelial cell growth factor, wherein removal of said
signal peptide yields a mature form of said endothelial cell growth
factor, and said endothelial cell growth factor either has the
amino acid sequence of .alpha.-endothelial cell growth factor
(NYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHI
QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ TPNEECLFLERLEENHYNTY I S K K H
A E K N W F V G L K KNGSCKRGPRTHYGQKAILFLPLPVSSD)
or comprises the amino acid sequence of .beta.-endothelial cell
growth factor (AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG
QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS
KKHAQEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD).
9. A plasmid according to claim 8, wherein said endothelial cell
growth factor is human .alpha.-endothelial cell growth factor
having the amino acid sequence N Y K K P K L L Y C S N G G H F L R
I L P DGTVDGTRDRSDQHI QLQLSAESVGEVYIKSTETGQYLAMDTDGLYYGSQ
TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK
KNGSCKRGPRTHYGQKAILFLPLPVSSD.
10. A plasmid according to claim 8, wherein said endothelial cell
growth factor is human .beta.-endothelial cell growth factor having
the amino acid sequence A E G E I T T F T A L T E K F N L P P G N Y
K K P K LLLYCSNGGHFLR ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG
QYLAMDTDGLLYGSQT P N E E C L F L E R L E E N H Y N T Y I S
KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD.
11. A plasmid according to claim 8, wherein said signal peptide is
a heterologous signal peptide.
12. A plasmid comprising a DNA encoding a cleavable heterologous
signal peptide and human .alpha.-endothelial cell growth factor
.[.comprising.]. having the amino acid sequence N Y K K K L L Y C S
N G G H F L R I L P DGTVDGTRDRSDQHI
QLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQ
TPNEECLFLERLEENHYNTYISKKHAEKNWFVGLK
KNGSCKRGRRTHYGQKAILFLPLPVSSD,
wherein removal of said signal peptide yields a mature form of said
human .alpha.-endothelial cell growth factor.
13. A plasmid comprising a DNA encoding a cleavable heterologous
signal peptide and human .beta.-endothelial cell growth factor
comprising the amino acid sequence
AEGEITTFTALTEKFNLPPGNYKKPKLLYCSNGGHFLR
ILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETG
QYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYIS
KKHAEKNWFVGLKKNGSCKRGPRTHYGQKAILFLPL PVSSD,
wherein removal of said signal peptide yields a mature form of said
human .beta.-endothelial cell growth factor..Iadd.
14. A process for expressing an endothelial cell growth factor in a
host cell, comprising introducing the plasmid according to claim 8
into the host cell..Iaddend..Iadd.
15. A process for expressing a human .alpha.-endothelial cell
growth factor in a host cell, comprising introducing the plasmid
according to claim 9 into the host cell..Iaddend..Iadd.
16. A process for expressing a human .beta.-endothelial cell growth
factor in a host cell, comprising introducing the plasmid according
to claim 10 into the host cell..Iaddend..Iadd.
17. A process for expressing an endothelial cell growth factor in a
host cell, comprising introducing the plasmid according to claim 11
into the host cell..Iaddend..Iadd.
18. A process for expressing a human .alpha.-endothelial cell
growth factor in a host cell, comprising introducing the plasmid
according to claim 12 into the host cell..Iaddend..Iadd.
19. A process for expressing a human .beta.-endothelial cell growth
factor in a host cell, comprising introducing the plasmid according
to claim 13 into the host cell..Iaddend..Iadd.
20. The process according to claim 14, wherein the host cell is a
prokaryotic cell..Iaddend..Iadd.
21. The process according to claim 20, wherein the prokaryotic cell
is E. coli..Iaddend..Iadd.
22. A process for preparing an endothelial cell growth factor,
comprising transforming a host cell with a plasmid according to
claim 8, culturing the host cell under conditions permitting
expression of the endothelial cell growth factor, and recovering
the endothelial cell growth factor..Iaddend..Iadd.
23. A process for preparing a human .alpha.-endothelial cell growth
factor, comprising transforming a host cell with a plasmid
according to claim 9, culturing the host cell under conditions
permitting expression of the human .alpha.-endothelial cell growth
factor, and recovering the human .alpha.-endothelial cell growth
factor..Iaddend..Iadd.
24. A process for preparing a human .beta.-endothelial cell growth
factor, comprising transforming a host cell with a plasmid
according to claim 10, culturing the host cell under conditions
permitting expression of the human .beta.-endothelial cell growth
factor, and recovering the human .beta.-endothelial cell growth
factor..Iaddend..Iadd.
25. A process for preparing an endothelial cell growth factor,
comprising transforming a host cell with a plasmid according to
claim 11, culturing the host cell under conditions permitting
expression of the endothelial cell growth factor, and recovering
the endothelial cell growth factor..Iaddend..Iadd.
26. A process for preparing a human .alpha.-endothelial cell growth
factor, comprising transforming a host cell with a plasmid
according to claim 12, culturing the host cell under conditions
permitting expression of the human .alpha.-endothelial cell growth
factor, and recovering the human .alpha.-endothelial cell growth
factor..Iaddend..Iadd.
27. A process for preparing a human .beta.-endothelial cell growth
factor, comprising transforming a host cell with a plasmid
according to claim 13, culturing the host cell under conditions
permitting expression of the human .beta.-endothelial cell growth
factor, and recovering the human .beta.-endothelial cell growth
factor..Iaddend..Iadd.
28. The process according to claim 23, wherein the host cell is a
prokaryotic cell..Iaddend..Iadd.
29. The process according to claim 28, wherein the prokaryotic cell
is E. coli..Iaddend.
Description
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates to recombinant DNA-directed synthesis of
certain proteins. More particularly, this invention relates to
endothelial cell growth factor (ECGF), its recombinant DNA-directed
synthesis, and ECGF's use in the treatment of endothelial cell
damage and/or regeneration.
(2) The Prior Art
Endothelial cell growth factor, referred to herein as "ECGF", is a
mitogen for endothelial cells in vitro. Growth of endothelial cells
is a necessary step during the process of angiogenesis [Maciag,
Prog. Hemostasis and Thromb., 7:167-182 (1984); Maciag, T., Hoover,
G. A., and Weinstein, R., J. Biol. Chem., 257: 5333-5336 (1982)].
Bovine ECGF has been isolated by Maciag, et al., [Science
225:932-935 (1984)] using streptomycin sulfate precipitation, gel
exclusion chromatography, ammonium sulfate precipitation and
heparin-.[.Sepharose.]. .Iadd.SEPHAROSE .Iaddend.affinity
chromatography. Bovine ECGF purified in this manner yields a
single-chain polypeptide which possesses an anionic iso-electric
point and an apparent molecular weight of 20,000 [Maciag, supra;
Schreiber, et al., J. Cell Biol., 101:1623-1626 (1985); and
Schreiber, et al., Proc. Natl. Acad. Sci., 82:6138-6142 (1985)].
More recently, multiple forms of bovine ECGF have been isolated by
Burgess, et al., [J. Biol. Chem. 260:11389-11392 (1985)] by sodium
chloride gradient elution of bovine ECGF from the
heparin-.[.Sepharose.]. .Iadd.SEPHAROSE .Iaddend.column or by
reversed-phase high pressure liquid chromatography (HPLC). The two
isolated polypeptides, designated alpha-and beta-ECGF have apparent
molecular weights of 17,000 and 20,000, respectively. Using this
procedure, the bovine ECGF contained in 8,500 ml of bovine brain
extract .[.(6.25.times.107.]. .Iadd.(6.25.times.10.sup.7 .Iaddend.
total units) is concentrated into a total of 6 ml of alpha-ECGF
.[.(3.0.times.106.]. .Iadd.(3.0.times.10.sup.6 .Iaddend. units) and
3 ml of beta-ECGF .[.(5.2.times.105.]. .Iadd.(5.2.times.10.sup.5
.Iaddend. units). This is a 9,300-fold purification of alpha-ECGF
and 16,300-fold purification of beta-ECGF (Burgess, supra.).
Recently, murine monoclonal antibodies against bovine ECGF have
been produced (Maciag, et al., supra.) which may be useful in
purifying bovine ECGF in a manner similar to the monoclonal
antibody purification of Factor VIIIC described by Zimmerman and
Fulcher in U.S. Pat. No. 4,361,509.
In general, recombinant DNA techniques are known. See Methods In
Enzymology, (Academic Press, New York) volumes 65 and 68 (1979);
100 and 101 (1983) and the references cited therein, all of which
are incorporated herein by reference. An extensive technical
discussion embodying most commonly used recombinant DNA
methodologies can be found in Maniatis, et al., Molecular Cloning,
Cold Spring Harbor Laboratory (1982). Genes coding for various
polypeptides may be cloned by incorporating a DNA fragment coding
for the polypeptide in a recombinant DNA vehicle, e.g., bacterial
or viral vectors, and transforming a suitable host. This host is
typically an Escherichia coli (E. coli) strain, however, depending
upon the desired product, eukaryotic hosts may be utilized. Clones
incorporating the recombinant vectors are isolated and may be grown
and used to produce the desired polypeptide on a large scale.
Several groups of workers have isolated mixtures of messenger RNA
(mRNA) from eukaryotic cells and employed a series of enzymatic
reactions to synthesize .[.doublestranded.]. .Iadd.double-stranded
.Iaddend.DNA copies which are complementary to this mRNA mixture.
In the first reaction, mRNA is transcribed into a singlestranded
complementary DNA .[.(cDNA).]. .Iadd.(ss-cDNA) .Iaddend.by an
RNA-directed DNA polymerase, also called reverse transcriptase.
Reverse transcriptase synthesizes DNA in the 5'-3' direction,
utilizes deoxyribonucleoside 5'-triphosphates as precursors, and
requires both a template and a primer strand, the latter of which
must have a free 3'-hydroxyl terminus. Reverse transcriptase
products, whether partial or complete copies of the mRNA template,
often possess short, partially double-stranded hairpins ("loops")
at their 3' termini. In the second reaction, these "hairpin loops"
can be exploited as primers for DNA polymerases. Preformed DNA is
required both as a template and as a primer in the action of DNA
polymerase. The DNA polymerase requires the presence of a DNA
strand having a free 3'-hydroxyl group, to which new nucleotides
are added to extend the chain in the 5'-3' direction. The products
of such sequential reverse transcriptase and DNA polymerase
reactions still possess a loop at one end. The apex of the loop or
"fold-point" of the double-stranded DNA, which has thus been
created, is substantially a single-strand segment. In the third
reaction, this single-strand segment is cleaved with the
single-strand specific nuclease S1 to generate a "blunt-end" duplex
DNA segment. This general method is applicable to any mRNA mixture,
and is described by Buell, et al., J. Biol. Chem., 253:2483
(1978).
The resulting double-stranded cDNA mixture (ds-cDNA) is inserted
into cloning vehicles by any one of many known techniques,
depending at least in part on the particular vehicle used. Various
insertion methods are discussed in considerable detail; in Methods
In Enzymology, 68:16-18 (1980), and the references cited
therein.
Once the DNA segments are inserted, the cloning vehicle is used to
transform a suitable host. These cloning vehicles usually impart an
antibiotic resistance trait .[.on.]. .Iadd.to .Iaddend.the host.
Such hosts are generally prokaryotic cells. At this point, only a
few of the transformed or transfected hosts contain the desired
cDNA The sum of all transformed or transfected hosts constitutes a
gene "library". The overall ds-cDNA library created by this method
provides a representative sample of the coding information present
in the mRNA mixture used as the starting material.
If an appropriate oligonucleotide sequence is available, it can be
used to identify clones of interest in the following manner.
Individual transformed or transfected cells are grown as colonies
on a nitrocellulose filter paper. These colonies are lysed; the DNA
released is bound tightly to the filter paper by heating. The
filter paper is then incubated with a labeled oligonucleotide probe
which is complementary to the structural gene of interest. The
probe hybridizes with the cDNA for which it is complementary, and
is identified by autoradiography. The corresponding clones are
characterized in order to identify one or a combination of clones
which contain all of the structural information for the desired
protein. The nucleic acid sequence coding for the protein of
interest is isolated and reinserted into an expression vector. The
expression vector brings the cloned gene under the regulatory
control of specific prokaryotic of eukaryotic control elements
which allow the efficient expression (transcription and
translation) of the ds-cDNA. Thus, this general technique is only
applicable to those proteins for which at least a portion of their
amino acid or DNA sequence is known for which an oligonucleotide
probe is available. See, generally, Maniatis, et al., supra.
More recently, methods have been developed to identify specific
clones by probing bacterial colonies or phage plaques with
antibodies specific for the encoded protein of interest. This
method can only be used with "expression vector" cloning vehicles
since elaboration of the protein product is required. The
structural gene is inserted into the vector adjacent to regulatory
gene sequences that control expression of the protein. The cells
are lysed, either by chemical methods or by a function supplied by
the host cell or vector, and the protein is detected by a specific
antibody and a detection system such as enzyme immunoassay. An
example of this is the lambda .[.gt11.]. .Iadd.gt.sub.11 .Iaddend.
system described by Young and Davis, Proc. Nat'l. Acad. Sci. USA,
80:1194-1198 (1983) and Young and Davis, Science 222:778
(1983).
SUMMARY OF THE INVENTION
The present invention has made it possible to provide readily
available, large quantities of ECGF or ECGF fragments. This has
been achieved with oligonucleotides whose design was based upon
knowledge of the amino acid sequence of bovine ECGF and which react
specifically with the ECGF cDNA. Production of ECGF is achieved
through the application or recombinant DNA technology to prepare
cloning vehicles encoding the ECGF protein and procedures for
recovering ECGF protein essentially free of other proteins of human
origin.
Accordingly, the present invention provides ECGF or its fragments
essentially free of other proteins of human origin. ECGF is
produced by recombinant DNA techniques in host cells or other
self-replicating systems and is provided in essentially pure form.
The invention further provides replicable expression vectors
incorporating a DNA sequence encoding ECGF and a self-replicating
host cell system transformed or transfected thereby. The host
system is usually of prokaryotic, e.g., E. coli or B. subtilis, or
eukaryotic cells.
The ECGF is produced by a process which comprises (a) preparing a
replicable expression vector capable of expressing the DNA sequence
encoding ECGF in a suitable host cell system; (b) transforming said
host system to obtain a recombinant host system; (c) maintaining
said recombinant host system under conditions permitting expression
of said ECGF-encoding DNA sequence to produce ECGF protein; and (d)
recovering said ECGF protein. Preferably, the ECGF-encoding
replicable expression vector is made by preparing a ds-cDNA
preparation representative of ECGF mRNA and incorporating the
ds-cDNA into replicable expression vectors. The preferred mode of
recovering ECGF comprises reacting the proteins expressed by the
recombinant host system with a reagent composition comprising at
least one binding .[.step.]. .Iadd.site .Iaddend.specific for ECGF.
ECGF may be used as a therapeutic agent in the treatment of damaged
or in regenerating blood vessels and other endothelial cell-lined
structures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a general procedure for enzymatic reactions to
produce cDNA clones.
FIG. 2 illustrates the production of a library containing DNA
fragments inserted into lambda .[.gt11.].
.Iadd.gt.sub.11.Iaddend..
FIG. 3 illustrates a partial amino acid sequence of bovine alpha
and beta ECGF. Line a: Amino-terminal amino acid sequence of bovine
alpha ECGF. Line b: Amino-terminal amino acid sequence of bovine
beta ECGF. The portion in parenthesis corresponds to NH2-terminal
segment for which sequence could not be determined; amino acid
composition is shown instead. The sequence beginning with PheAsnLeu
. . . was determined from trypsin-cleaved bovine beta ECGF. Line c:
Amino acid sequence of cyanogen bromide-cleaved bovine alpha ECGF.
Line d: Amino acid sequence of cyanogen bromide-cleaved bovine beta
ECGF.
FIG. 4 illustrates hydrogen-bonded base pairs.
FIG. 5 illustrates the design of an oligonucleotide probe for human
Endothelial Cell Growth Factor.
FIG. 6 illustrates a schematic diagram of human ECGF cDNA clones 1
and 29. The open reading box represents the open reading frame
encoding human beta ECGF. The EcoRI sites correspond to synthetic
oligonucleotide linkers used in the construction of the cDNA
library. The poly (A) tail at the 3' end of clone 1 is shown by
A17.
FIG. 7 illustrates homology between human ECGF cDNA sequence and
oligonucleotide probes. Line a: Bovine trypsin- or cyanogen
bromide-cleaved beta
ECGF amino acid sequence. Line b: Unique oligonucleotide probe.
Line c: Human ECGF cDNA sequence (determined from lambda ECGF
clones 1 and 29). Line d: Human ECGF amino acid sequence, deduced
from cDNA sequence analysis.
FIG. 8 illustrates the complete cDNA sequence of human ECGF. The
cDNA inserts from ECGF clones 1 and 29 were subcloned into M13mp18
and the ECGF-encoding open reading frame and flanking regions
sequenced by the chain termination method. In frame stop codons at
the 5' and 3' ends of the ECGF-encoding open reading frame are
indicated by the underlined sequence and trm, respectively. The
single-letter notation for amino acids is used: A, Ala; C, Cys; D,
Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M,
Met; N, Asn; P, Pro; Q, Gln; R. Arg; S, Ser; T, Thr; V, Val; W,
Trp; Y, Tyr.
FIG. 9 illustrates Northern blot analysis of ECGF mRNA. RNA was
denatured in 2.2M formaldehyde and 50% formamide and fractionated
by electrophoresis in a 1.25% agarose gel containing 2.2M
formaldehyde. This was transferred to .[.GeneScreen Plus.].
.Iadd.GENESCREEN PLUS .Iaddend.(New England Nuclear) by blotting
with 10X SSPE. Blots were hybridized to .[..sup.32 p-labeled.].
.Iadd..sup.32 P-labeled .Iaddend.nick-translated probes of ECGF
clone 1 at 65.degree. C. for 16 hours in a mixture containing 2X
SSPE, 20X Denhardt's solution, yeast transfer RNA (200 .mu.g/ml),
and 0.2% SDS. The membrane was subsequently washed at 65.degree.
C., twice with 2X SSPE and 0.2% SDS, then twice with 0.2 X SSPE and
0.2% SDS, air-dried, and exposed overnight to Kodak XAR film with
an intensifying screen. The migration of 28S and 18S RNA is noted.
Lane 1:10 .mu.g human brain poly(A)-containing RNA. Lane 2:10 .mu.g
human adult liver poly(A)-containing RNA.
FIG. 10 illustrates expressional cloning of human recombinant
.alpha.-ECGF. The expression vector pMJ26 was constructed as
indicated. The translation initiation codon provided by the
synthetic oligonucleotide is indicated by "ATG". The hybrid tac
promoter and the Shine-Dalgarno sequence provided by the vector
pKK223-3, are indicated by "Ptac" and S.D.", respectively.
Transcription terminators are indicated by "rrnBT.sub.1 T.sub.2 "
and "5S". The open arrow shows the direction of transcription from
the tac promoter.
FIG. 11 illustrates SDS-PAGE analysis of recombinant human
.alpha.-ECGF expression and purification. Cultures of pMJ26 in E.
coli JM103 were grown and induced with I mM IPTG. Lanes a and b,
samples lysed in Laemmli sample buffer. Lane a, uninduced pMJ26.
Lane b, induced pMJ26. Lanes c-f, purification of ECGF from induced
pMJ26. Lane c, supernatant, after removal of cell debris; Lane d,
material unabsorbed to .[.heparin-Sepharose.].
.Iadd.heparin-SEPHAROSE .Iaddend.in 250 mM NaCl; lane e, entire
cell debris pellet of lane c; Lane f, molecular weight standards.
Samples in lanes a-d contained 100 .mu.g protein.
FIG. 12 illustrates a comparison of human recombinant and bovine
brain-derived .alpha.-ECGF. --.smallcircle.--.smallcircle.--
.smallcircle.-- bovine .alpha.-ECGF;
--.circle-solid.--.circle-solid.--.circle-solid.-- recombinant
human .alpha.-ECGF; --.quadrature.--.quadrature.--.quadrature.--
reduced and alkylated recombinant human .alpha.-ECGF; .box-solid.
recombinant human .alpha.-ECGF, no heparin; .quadrature. bovine
.alpha.-ECGF, no heparin.
FIG. 12A. LE-II receptor binding competition assay. Receptor
competition assays were performed. Confluent cultures of LE-II
cells were incubated for 1.5 h at 4.degree. C. in the .[.present.].
.Iadd.presence .Iaddend.of approximately 5 ng/ml of .sup.25
I-bovine .alpha.-ECGF and the indicated amounts of unlabelled
HPLC-purified .alpha.-ECGF. Protein concentrations were determined
by amino acid analysis. Monolayers were washed three times with
DMEM containing 1 mg/ml BSA, lysed with 0.1 N NaOH, and the
cell-associated radioactivity determined. Binding observed in the
absence of competitor is defined as 100% control. Reduced and
alkylated recombinant .alpha.-ECGF was prepared as follows:
HPLC-purified ECGF in Tris-HCl pH 8.3, 6 M guanidine hydrochloride,
100 mM DTT was incubated for 60 minutes at 37.degree. C. under
nitrogen. .[.lodacetic.]. .Iadd.Iodacetic .Iaddend.acid was added
to 22 mM, and incubation continued in the dark for 60 minutes at
37.degree. C. The protein was isolated by reversed-phase HPLC.
Amino acid composition analysis indicated the presence of 2.9 mol
.[.s-carboxymethyl cystein, mol .alpha.-ECGF.].
.Iadd.S-carboxymethyl cysteine/mol .alpha.ECGF..Iaddend.
FIG. 12B. Stimulation of [.sup.3 H]-thymidine incorporation in
LE-II cells. Confluent, murine LE-II cells in DMEM containing 0.1%
fetal bovine serum were incubated with the indicated quantities of
bovine or recombinant human .alpha.-ECGF for 18 hours. Cells were
.[.labelled.]. .Iadd.labeled .Iaddend.for 4 hours in the presence
of 2.4 .[.uCi.]. .Iadd..mu.Ci .Iaddend.[.sup.3 H]-thymidine. Wells
containing 20% fetal calf serum (X) and 1 mg/ml bovine serum
albumin (BSA) served as controls.
FIG. 12C. Human umbilical vein endothelial cell (HUVEC) growth
assay. Costar 24 well tissue culture dishes (2 cm.sup.2 /well) were
precoated with human fibronectin (10 .mu.g/cm.sup.2) in PBS for
0.5-2 hours prior to seeding with 2.times.10.sup.3 HUVEC in Medium
199 containing 10% fetal bovine serum Cells were allowed to attach
for 2-4 hours at 37.degree. C., at which time the media was
aspirated and replaced with 0.75 ml Medium 199 containing 10% fetal
bovine serum and, unless otherwise indicated, 5 U/ml heparin.
Dilutions of HPLC-purified recombinant human .alpha.-ECGF and
bovine brain-derived .alpha.-ECGF in 1-50 .mu.l were added to
duplicate wells as indicated. Media were changed on days 2 and 4,
and on day 7 cells were harvested by trypsinization and cell number
was determined with a Coulter counter. Wells containing 20% fetal
calf serum (X) and 1 ng/ml BSA served as controls.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Introduction
As used herein, "ECGF" denotes endothelial cell growth factor or
its fragments produced by cell or cell-free culture systems, in
bioactive forms having the capacity to influence cellular growth,
differentiation, and migration in vitro as does ECGF native to the
human angiogenic process.
Different alleles of ECGF may exist in nature. These variations may
be characterized by differences in the nucleotide sequence of the
structural gene coding for proteins of identical biological
function. It is possible to produce analogs having single or
multiple amino acid substitutions, deletions, additions, or
replacements. All such allelic variations, modifications, and
analogs resulting in derivatives of ECGF which retain the
biologically active properties of native ECGF are included within
the scope of this invention.
The glycosaminoglycan heparin potentiates the mitogenic effect of
both bovine and recombinant human ECGF. Heparin naturally exists as
a heterogeneous mixture of polysaccharide chains ranging from about
6,000 to about 25,000 Da (Alberts et al. in "Molecular Biology of
the Cell" Garland Publishing, Inc. (1983) pp. 692-715). Low
molecular weight heparins (LMWH) having a variety of advantages
over natural heparin have been prepared (see U.S. Pat. No.
4,401,662; 4,446,314; 4,826,827; 5,032,679 and Mestre et al.
Thrombosis Research 38, 389-399 (1985)) and are also useful in the
practice of the present invention.
"Expression vectors" refer to vectors which are capable of
transcribing and translating DNA sequences contained therein, where
such sequences are linked to other regulatory sequences capable of
affecting their expression. These expression vectors must be
replicable in the host organisms or systems either as episomes,
bacteriophage, or as an integral part of the chromosomal DNA. One
form of expression vector which is particularly suitable for use in
the invention is the bacteriophage, viruses which normally inhabit
and replicate in bacteria. Particularly desirable phage for this
purpose are the lambda gt.sub.10 and gt.sub.11 phage described by
Young and Davis, supra. Lambda .[.gt11.]. .Iadd.gt.sub.11 .Iaddend.
is a general recombinant DNA expression vector capable of producing
polypeptides specified by the inserted DNA.
To minimize degradation, upon induction with a synthetic analogue
of lactose (IPTG), foreign proteins or portions thereof are
synthesized fused to the prokaryotic protein .[.B-galactosidase.].
.Iadd..beta.-galactosidase.Iaddend.. The use of host cells
defective in protein degradation pathways may also increase the
lifetime of novel proteins produced from the induced lambda
gt.sub.11 clones. Proper expression of foreign DNA in lambda
gt.sub.11 clones will depend upon the proper orientation and
reading frame of the inserted DNA with respect to the
.[.B-galactosidase.]. .Iadd..beta.-galactosidase .Iaddend.promoter
and translation initiating codon.
Another form of expression vector useful in recombinant DNA
techniques is the plasmid--a circular unintegrated
(extra-chromosomal), double-stranded DNA.[.loop.]. . Any other form
of expression vector which serves an equivalent function is
suitable for use in the process of this invention.
Recombinant vectors and methodology disclosed herein are suitable
for use in host cells covering a wide range of prokaryotic and
eukaryotic organisms. Prokaryotic cells are preferred for the
cloning of DNA sequences and in the construction of vectors. For
example, E. coli K12 strain HB101 (ATCC No. 33694), is particularly
useful. Of course, other microbial strains may be used. Vectors
containing replication and control sequences which are derived from
species compatible with the host cell or system are used in
connection with these hosts. The vector ordinarily carries an
origin of replication, as well as characteristics capable of
providing phenotypic selection in transformed cells. For example,
E. coli can be transformed using the vector pBR322, which contains
genes for ampicillin and tetracycline resistance [Bolivar, et al.,
Gene, 2:95 (1977)].
These antibiotic resistance genes provide a means of identifying
transformed cells. The expression vector may also contain control
elements which can be used for the expression of the gene of
interest. Common prokaryotic control elements used for expression
of foreign DNA sequences in E. coli include the promoters and
regulatory sequences derived from the .beta.-galactosidase and
tryptophan (trp) operons of E. coli, as well as the pR and pL
promoters of bacteriophage lambda. Combinations of these elements
have also been used (e.g., TAC, which is a fusion of the trp
promoter with the lactose operator). Other promoters have also been
discovered and utilized, and details concerning their nucleotide
sequences have been published enabling a skilled worker to combine
and exploit them functionally.
In addition to prokaryotes, eukaryotic microbes, such as yeast
cultures, may also be used. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among eukaryotic
microorganisms, although a number of other strains are commonly
available. Yeast promoters suitable for the expression of foreign
DNA sequences in yeast include the promoters for 3-phosphoglycerate
kinase or other glycolytic enzymes. Suitable expression vectors may
contain termination signals which provide for the polyadenylation
and termination of the mRNA transcript of the cloned gene. Any
vector containing a yeast-compatible promoter, origin or
replication, and appropriate termination sequence is suitable for
expression of ECGF.
Cell lines derived from multicellular organisms may also be used as
hosts. In principle, any such cell culture is workable, whether
from a vertebrate or invertebrate source. However, interest has
been greatest in vertebrate cells, and propagation of vertebrate
cells in culture (tissue culture) has become a routine procedure in
recent years. Examples of such useful hosts are the VERO, HeLa,
mouse C127, Chinese hamster ovary (CHO), WI138, BHK, COS-7, and
MDCK cell lines. Expression vectors for such cells ordinarily
include an origin of replication, a promoter located in front of
the gene to be expressed, RNA splice sites (if necessary), and
transcriptional termination sequences.
For use in mammalian cells, the control functions (promoters and
enhancers) on the expression vectors are often provided by viral
material. For example, commonly used promoters are derived from
polyoma. Adenovirus 2, and most frequently, Simian Virus 40 (SV40).
Eukaryotic promoters, such as the promoter of the murine
metallothionein gene [Paulakis and Hamer, Proc. Natl. Acad. Sci.
80:397-401 (1983)], may also be used. Further, it is also possible,
and often desirable, to utilize the promoter or control sequences
which are naturally associated with the desired gene sequence,
provided such control sequences are compatible with the host
system. To increase the rate of transcription, eukaryotic enhancer
sequences can also be added to the construction. These sequences
can be obtained from a variety of animal cells or oncogenic
retroviruses such as the mouse sarcoma virus.
An origin of replication may be provided either by construction of
the vector to include an exogenous origin, such as that provided by
SV40 or other viral sources, or may be provided by the host cell
chromosomal replication mechanism. If the vector is integrated into
the host cell chromosome, the latter is often sufficient.
Host cells can prepare ECGF which can be of a variety of chemical
compositions. The protein is produced having methionine as its
first amino acid. This methionine is present by virtue of the ATG
start codon naturally existing at the origin of the structural gene
or by being engineered before a segment of the structural gene. The
protein may also be intracellularly or extracellularly cleaved,
giving rises to the amino acid which is found naturally at the
amino terminus of the protein. The protein may be produced together
with either its own or a heterologous signal peptide, the signal
polypeptide being specifically cleavable in an intra- or
extracellular environment. Finally, ECGF may be produced by direct
expression in mature form without the necessity of cleaving away
any extraneous polypeptide.
Recombinant host cells refer to cells which have been transformed
with vectors constructed using recombinant DNA techniques. As
defined herein, ECGF is produced as a consequence of this
transformation. ECGF or its fragments produced by such cells are
referred to as "recombinant ECGF".
B. Recombinant and Screening Methodology
The procedures below are but some of a wide variety of well
established procedures to produce specific reagents useful in the
process of this invention. The general procedure for obtaining an
mRNA mixture is to obtain a tissue sample or to culture cells
producing the desired protein, and to extract the RNA by a process
such as that disclosed by Chirgwin, et al., Biochemistry, 18:5294
(1979). The mRNA is enriched .[.by poly(A)mRNA containing.].
.Iadd.for poly(A) mRNA-containing .Iaddend.material by
chromatography on oligo (dT) cellulose or poly(U) .[.Sepharose.].
.Iadd.SEPHAROSE.Iaddend., followed by elution of the poly(A)
containing mRNA fraction.
The above .Iadd.fraction enriched for .Iaddend.poly(A) containing
.[.mRNA-enriched fraction.]. .Iadd.mRNA .Iaddend.is used to
synthesize a single-strand complementary cDNA (ss-cDNA) using
reverse transcriptase. As a consequence of DNA synthesis, a hairpin
loop is formed at the 3' end of the DNA which will initiate,
second-strand DNA synthesis. Under appropriate conditions, this
hairpin loop is used to effect synthesis of the ds-cDNA in the
presence of DNA polymerase and deoxyribonucleotide
triphosphates.
The resultant ds-cDNA is inserted into the expression vector by any
one of many known techniques. In general, methods can be found in
Maniatis, et al., supra, and Methods In Enzymology, Volumes 65 and
68 (1980); and 100 and 101 (1983). In general, the vector is
linearized by at least one restriction endonuclease, which will
produce at least two blunt or cohesive ends. The ds-cDNA is ligated
with or joined into the vector insertion site.
If prokaryotic cells or other cells which contain substantial cell
wall material are employed, the most common method of
transformation with the expression vector is calcium chloride
pretreatment as described by Cohen, R. N., et al., Proc. Nat'l.
Acad. Sci. USA, 69:2110 (1972). If Cells without cell wall barriers
are used as host cells, transfection is carried out by the calcium
phosphate precipitation method described by Graham and Van der Eb,
Virology, 52:456 (1973). Other methods for introducing DNA into
cells such as nuclear injection, viral infection or protoplast
fusion may be successfully used. The cells are then cultured on
selective media, and proteins for which the expression vector
encodes are produced.
Clones containing part or the entire cDNA for ECGF are identified
with specific oligonucleotide probes deduced from a partial amino
acid sequence determination of ECGF. This method of identification
requires that the non-degenerate oligonucleotide probe be designed
such that it specifically hybridizes to ECGF ds-cDNA. Clones
containing ECGF cDNA sequences are isolated by radioactively
labeling the oligonucleotide probe with .[.32P-ATP.]. .Iadd..sup.32
P-ATP.Iaddend., hybridizing the radioactive oligonucleotide probe
to the DNA of individual clones of a cDNA library containing
ECGF-cDNA, and detection and isolation of the clones which
hybridize by autoradiography. Such a cloning system is applicable
to the lambda .[.gt11.]. .Iadd.gt.sub.11 .Iaddend. system described
by Young and Davis, supra. Clones containing the entire sequence of
ECGF are identified using as probe the cDNA insert of the ECGF
recombinants isolated during the initial screening of the
recombinant lambda .[.gt11.]. .Iadd.gt.sub.11 .Iaddend. cDNA
library with ECGF-specific oligonucleotides. Nucleotide sequencing
techniques are used to determine the sequence of amino acids
encoded by the cDNA fragments. This information may be used to
determine the identity of the putative ECGF cDNA clones by
comparison to the known amino acid sequence of the amino-terminus
of bovine ECGF and of a peptide derived by cyanogen bromide
cleavage of ECGF.
EXAMPLE
A. Preparation of Total RNA
Total RNA (messenger, ribosomal and transfer) was extracted from
fresh two-day old human brain stem essentially as described by
Chirgwin, supra. (1979). Cell pellets were homogenized in 5 volumes
of a solution containing 4 M guanidine thiocyanate, and 25 mM
Antifoam A (Sigma Chemical Co., St. Louis, Mo.). The homogenate was
centrifuged at 6,000 rpm in a .[.Sorvall.]. .Iadd.SORVALL
.Iaddend.GSA rotor for 15 minutes at 10.degree. C. The supernatant
fluid was adjusted to pH 5.0 by addition of acetic acid and the RNA
precipitated by 0.75 volumes of ethanol at -20.degree. C. for two
hours. RNA was collected by centrifugation and dissolved in 7.5 M
guanidine hydrochloride containing 2 mM sodium citrate and 5 mM
dithiothreitol. Following two additional precipitations using 0.5
volumes of ethanol, the residual guanidine hydrochloride was
extracted from the precipitate with absolute ethanol. RNA was
dissolved in sterile water, insoluble material removed by
centrifugation, and the pellets were re-extracted with water. The
RNA was adjusted to 0.2M potassium acetate and precipitated by
addition of 2.5 volumes of ethanol at -20.degree. C. overnight.
B. Preparation of Poly(A)-containing RNA
The total RNA precipitate, prepared as described above, was
dissolved in 20 mM Hepes buffer (pH 7.2) containing 10 mM EDTA and
1% SDS, heated at 65.degree. C. for 10 minutes, then quickly cooled
to 25.degree. C. The RNA solution was then diluted with an equal
volume of water, and NaCl was added to bring the final
concentration to 300 mM NaCl. Samples containing up to 240
.[.A260.]. .Iadd.A.sub.260 .Iaddend. units of RNA were
chromotagraphed on .[.poly(U)-Sepharose.]. .Iadd.poly(U)-SEPHAROSE
.Iaddend.using standard procedures. Poly(A)-containing RNA was
eluted with 70% formamide containing 1 mM Hepes buffer (pH 7.2),
and 2 mM EDTA. The eluate was adjusted to 0.24M NaCl and the RNA
was precipitated by 2.5 volumes of ethanol at -20.degree. C.
C. Construction of cDNA Clones in Lambda .[.gt11.]. .Iadd.gt.sub.11
.Iaddend.
The procedure followed for the enzymatic reaction is shown in FIG.
1. The mRNA (20 .mu.g) was copied into ds-cDNA with reverse
transcriptase and DNA polymerase I exactly as described by Buell,
et al., supra. and Wilkensen, et al., J. Biol. Chem., 253:2483
(1978). The ds-cDNA was desalted on .[.Sephadex.]. .Iadd.SEPHADEX
.Iaddend.G-50 and the void-volume fractions further purified on an
.[.Elutip-D.]. .Iadd.ELUTIP-D .Iaddend.column'(Schleicher &
Schuell, Keene, NH) following the manufacturer's directions. The
ds-cDNA was made blunt-ended by incubation with S1 nuclease [Ricca,
et al., J. Biol. Chem., 256:10362 (1981)]. The reaction mixture
consisted of 0.2M sodium acetate (pH 4.5), 0.4M sodium chloride,
2.5 mM zinc acetate and 0.1 unit of S1 nuclease per mg of ds-cDNA,
made to a final reaction volume of 100 .mu.l. The ds-cDNA was
incubated .[.to.]. .Iadd.at .Iaddend.37.degree. C. for one hour,
extracted with phenol:chloroform, and then desalted on a
.[.Sephadex.]. .Iadd.SEPHADEX .Iaddend.G-50 column as described
above. The ds-cDNA was then treated with .[.EcoRI.]. .Iadd.EcoRI
.Iaddend.methylase and Klenow fragment of DNA polymerase I using
reaction conditions described in Maniatis, et al., Molecular
Cloning, supra. The cDNA was again desalted on .[.Sepnadex.].
.Iadd.SEPHADEX .Iaddend.G-50 as described above and then ligated to
0.5 .mu.g of phosphorylated .[.EcoRI.]. .Iadd.EcoRI
.Iaddend.linkers using T4 DNA ligase (Maniatis, et al., supra). The
mixture was cleaved with .[.EcoRI.]. .Iadd.EcoRI .Iaddend.and
fractionated on an 8% acrylamide gel in Tris-borate buffer
(Maniatis, et al., supra). DNA with a size greater than 1 kilobase
was eluted from the gel and recovered by binding to an
.[.Elutip-D.]. .Iadd.ELUTIP-D .Iaddend.column, eluted with 1M NaCl
and then collected by ethanol precipitation.
As shown in FIG. 2, the DNA fragments were then inserted into
.[.EcoRI.]. .Iadd.EcoRI .Iaddend.cleaved and phosphatase-treated
lambda .[.gt11.]. .Iadd.gt.sub.11.Iaddend., using T4 DNA ligase. A
library of .[.5.7.times.106.]. .Iadd.5.7.times.10.sup.6 .Iaddend.
phage was produced, of which approximately 65% were recombinant
phage. The library was amplified by producing plate stocks at
42.degree. C. on E. coli Y1088 .[.[supE supF met: B trpR
hsdR-hsdM+tonA21 scrA lacU169 (proC::Tn5) (pMC9)].]. .Iadd.(supE
supF metB trpR hsdR.sup.31 hsdM.sup.+ tonA21 strA lacU169
(proC::Tn5) (pMC9)).Iaddend.. Amplification procedures are
described in Maniatis, et al., supra. Important features of this
strain, described by Young and Davis, supra, include (1) .[.supF.].
.Iadd.supF .Iaddend.(required suppression of the phage amber
mutation in the S gene), (2) .[.hsdR-hsdM+.]. .Iadd.hsdR.sup.-
hsdM.sup.+ .Iaddend. (necessary to prevent restriction of foreign
DNA prior to host modification), and (3) .[.lacU169 (proC::Tn5).].
.Iadd.lacU169 (proC::Tn5).Iaddend., and (4) (pMC9) .[.(a lac
I-bearing.]. .Iadd.(a lacI-bearing .Iaddend.pBR322 derivative which
represses, in the absence of an inducer, the expression of foreign
genes that may be detrimental to phage and/or cell growth)
D. Identification of Clones Containing ECGF Sequence
To screen the library for recombinant phage containing ECGF cDNA,
.[.1.5.times.106.]. .Iadd.1.5.times.10.sup.6 .Iaddend. phage were
plated on a lawn of E. coli Y1090 .[.[delta lacU169 proA delta Ion
araD139 strA supF (trpC22::TnIO) (pMC9)].]. .Iadd.(.DELTA.lacU169
proA .DELTA.lon araD139 strA supF (trpC22::TnIO) (pMC9))
.Iaddend.and incubated at 42.degree. C. for 6 hours. After the
plates were refrigerated overnight, a nitrocellulose filter was
overlaid on the plates. The position of the filter was marked with
a needle. The filter .Iadd.was .Iaddend.removed after one minute
and left to dry at room temperature. From each plate, a duplicate
filter was prepared exactly as described, except that the filter
was left in contact with the plate for 5 minutes. All filters were
then prepared for hybridization, as described in Maniatis, et al.,
supra. This involved DNA denaturation in 0.5M NaOH, 1.5M NaCl,
neutralization in 1M Tris-HCl, pH 7.5, 1.5M NaCl, and heating of
the filters for 2 hours at 80.degree. C. in vacuo.
To screen the human brain stem cDNA library for clones containing
ECGF inserts, a specific oligonucleotide was designed. This
oligonucleotide was based upon a partial amino acid sequence
analysis of the amino terminus of ECGF. As shown in FIG. 3, lines a
& b, bovine ECGF is isolated as two species, designated alpha
and beta ECGF, which differ only in the amino acids found at the
respective amino termini. As shown in FIG. 3, lines a & b,
beta-ECGF is a slightly larger species than alpha-ECGF. The exact
amino acid sequence at the amino terminus of beta-ECGF is
undetermined, however, a sequence derived from fast atom
bombardment mass spectral analysis and the amino acid composition
of the amino terminal tryptic peptide of bovine beta-ECGF is shown.
The amino terminal blocking group appears to be acetyl. If intact
beta-ECGF is cleaved by trypsin, a second amino amino acid sequence
found in beta but not alpha ACGF starting with PheAsnLeu . . . is
determined. This sequence is also found at the amino terminus of
acidic fibroblast growth factor [Thomas, K. A. et al., Prac. Natl.
Acad. Sci., 82:6409-6413 (1985)]. The amino terminus of alpha-ECGF
is AsnTyrLys . . . (FIG. 3, line a) and is the equivalent of
beta-ECGF minus an amino terminal extension. In FIG. 3, lines c and
d set forth for comparison the amino acid sequence of cyanogen
bromide-cleaved bovine alpha and beta ECGF, respectively.
For oligonucleotide design, the amino acid sequence
IleLeuProAspGlyThrValAspGlyThrLys, corresponding to alpha-ECGF
amino acids 19-29 inclusive, was chosen. Rather .[.then.].
.Iadd.than .Iaddend.design a mixture of oligonucleotides covering
all of the possible coding sequences (owing to the degeneracy of
the genetic code), a long unique oligonucleotide was designed. Such
oligonucleotide probes have been previously shown to be successful
probes in screening complex cDNA [Jaye, et al., Nucleic Acids
Research 11:2325-2335, (1983) and genomic [Gitschier, et al.,
Nature, 312:326-330 (1984)libraries. Three criteria were used in
designing the ECGF probe: (1) The dinucleotide CG was avoided. This
strategy was based upon the observed .[.underrepresentation.].
.Iadd.under representation .Iaddend.of the CG dinucleotide in
eukaryotic DNA Josse, et al., J. Biol. Chem. 236:864-875, (1961);
(2) preferred codon utilization data was used wherever possible. A
recent and comprehensive analysis of human codon utilization was
found in Lathe, J. Biol. 183:1-12 (1985); and (3) wherever the
strategies of CG dinucleotide and preferred codon utilization were
uninformative, unusual base pairing was allowed. This strategy was
based upon the natural occurrence of G:T, I:T, I:A and I:C base
pairs which occur in the interaction between tRNA anticodons and
mRNA codons Crick, J. Mol. Biol. 19:548-555, (1966). A diagram of
usual and unusual base pairs is shown in FIG. 4. Use of I (Inosine)
in a hybridization probe was first demonstrated, in a model
experiment, by Ohtsuka, et al., J. Biol. Chem. 260:2605-2608 1985).
The overall strategy and choice made in the design of the
oligonucleotide used to screen the human brain stem cDNA library
for .[.ECGE.]. .Iadd.ECGF .Iaddend.is shown in FIG. 5. In addition,
two other oligonucleotides, designed with the same strategy, were
constructed.
Approximately 30 pmole of the oligonucleotide shown in FIG. 5 were
radioactively labeled by incubation with .[.32P-gamma-ATP.].
.Iadd..sup.32 P-.gamma.-ATP .Iaddend.and T4 polynucleotide kinase,
essentially as described by .[.Maniatis, et al..]. .Iadd.Maniatis,
et al..Iaddend., supra. Nitrocellulose filters, prepared as
described above, were prehybridized at 42.degree. C. in
6.times.SSPE (1.times.SSPE=0.18M NaCl, 0.01M .[.NaHP04.].
.Iadd.NaHP0.sub.4 .Iaddend. pH 7.2, 0.001M EDTA), 2.times.
Denhardt's (1.times. Denhardt's-0.02% each .[.Ficoll.].
.Iadd.FICCOLL.Iaddend., polyvinylpyrrolidone, bovine serum
albumin), 5% dextran sulfate, and 100 .[.mu g/ml.]. .Iadd. .mu.g/ml
.Iaddend.denatured salmon sperm DNA. The .[.32P-labeled.].
.Iadd..sup.32 P-labeled .Iaddend.oligonucleotide was added
following four hours of prehybridization, and dehybridization
continued overnight at 42.degree. C. Unhybridized probe was removed
by sequential washing at 37.degree. C. in 2.times. SSPE, 0.1%
SDS.
From .[.1.5.times.106.]. .Iadd.1.5.times.10.sup.6 .Iaddend. plaques
screened, 2 plaques gave positive autoradiographic signals after
overnight exposure. These clones were purified to homogeneity by
repeated cycles of purification using the above oligonucleotide as
hybridization probe.
The two clones that were isolated, ECGF clones 1 and 29, were
analyzed in further detail. Upon digestion with .[.EcoRI.].
.Iadd.EcoRI.Iaddend., Clone 1 and 29 revealed cDNA inserts of 2.2
and 0.3 Kb, respectively. Nick translation of cloned cDNA and its
subsequent use as a radiolabeled probe in Southern blot analysis
(Maniatis, et al., supra) revealed that clones 1 and 29 were
related and overlapping clones. The overlapping nature of these two
clones is shown in FIG. 6.
Clones 1 and 29 were analyzed in further detail as follows: An
additional two oligonucleotides were designed, based upon the amino
acid sequence of bovine ECGF. These oligonucleotides were designed
based upon the same consideration as those used in the design of
the oligonucleotide used to isolate clones 1 and 29. These
oligonucleotides (ECGF oligonucleotides II and III) are shown in
FIG. 7. These two oligonucleotides as well as oligo(dT)18 were
radioactively labeled in a kination reaction as described above and
used as hybridization probes in Southern blotting experiments. The
results of these experiments showed that the 0.3 Kb cDNA insert of
clone 29 hybridized to ECGF oligonucleotides I and II but not to
ECGF oligonucleotide III or oligo (dT)18; the 2.2 Kb cDNA insert of
clone 1 hybridized to oligonucleotide I, II, III as well as
oligo(dT) 18. These data and subsequent nucleotide sequence
determination of clones 1 and 29 showed that the 3' end of clone 1
ends with a poly(A) tail. Hybridization of clone 1 to ECGF
oligonucleotide III, which is based on a cyanogen bromide cleavage
product of bovine ECGF, as well as to oligo (dT)18, strongly
suggested that this clone contains the rest of the coding sequence
for both alpha and beta ECGFs as well as a large (greater than 1
Kb) 3' flanking sequence.
The cDNA inserts from clones 1 and 29 were isolated, subcloned into
M13mp18, and the ECGF-encoding open reading frame and flanking
regions sequenced by the chain termination method [Sanger et al.,
Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)]. The nucleotide
sequence of these clones and the amino acid sequence deduced from
the nucleic acid sequence is shown in FIG. 8. Examination of the
nucleotide sequence reveals an open reading frame of 465
nucleotides encoding human ECGF. The 155 amino acids of human ECGF
were found to be flanked by translation stop codons. The NH.sub.2
-terminal amino acid of human beta ECGF deduced from the cDNA
sequence is methionine, which most likely serves as the translation
initiation residue. These data, together with the relatively
non-hydrophobic nature of the first 15-20 amino terminal residues,
strongly suggest that human beta ECGF is synthesized without a
.[.NH2-terminal.]. .Iadd.NH.sub.2 -terminal .Iaddend.signal
peptide. A comparison of FIGS. 3 and 8 shows that the amino
terminal amino acid sequence of trypsin-cleaved bovine beta ECGF as
well as that of bovine alpha ECGF are nearly identical to the amino
acid sequence predicted from the nucleotide sequence of lambda ECGF
clones 1 and 29. An overall homology between the two species of
over 95% is observed.
Northern blot analysis (Maniatis, et al, supra) reveals that ECGF
mRNA is a single molecular species which comigrates with 28S rRNA
(FIG. 9). Considering the variation in the estimated size of 28 S
rRNA, the approximate size of ECGF mRNA is 4.8.+-.1.4 Kb. All of
the sequence encoding the mature forms of both alpha and beta ECGF
is encoded within ECGF Clones 1 and 29, which together encompasses
approximately 2.3 Kb. Thus, these data demonstrate that the region
5' and flanking the ECGF-encoding sequences, is very large
(approximately 2.5.+-.1.4 Kb).
cDNA inserts from Clone 1 and Clone 29 were excised by digestion
with .[.EcoRI.]. .Iadd.EcoRI .Iaddend.and subcloned in pUC8 at the
.[.EcoRI.]. .Iadd.EcoRI .Iaddend.site. The plasmid formed from
Clone 1 was designated pDH15 and the plasmid formed from Clone 29
was designated pDH14.
Clone I was improved by inserting it into a vector allowing more
efficient expression of .alpha.-ECGF. This vector is pMJ26 and
places this gene under a high-efficiency tac promoter as described
in FIG. 10 and as done as follows. A double-stranded .[.Bam HI.].
.Iadd.BamHI .Iaddend.cohesive 66-mer oligonucleotide encoding
residues I19 of .alpha.-ECGF, preceded by initiator methionine, was
synthesized by the phosphoramoridite method and purified. The
oligonucleotide was ligated between the .[.Bam HI.]. .Iadd.BamHI
.Iaddend.sites of pDH15 creating pMJ25. In order to introduce
appropriate regulatory sequences, the .alpha.-ECGF-encoding open
reading frame was excised from pMJ25 by digestion with .[.EcoRI.].
.Iadd.EcoRI .Iaddend.and .[.Hinc II.]. .Iadd.HincII .Iaddend.and
cloned between the .[.Eco RI.]. .Iadd.EcoRI .Iaddend.and .[.Sma
I.]. .Iadd.SmaI .Iaddend.sites of pKK223-3 (PL Biochemicals). The
recombinant plasmid, pMJ26, was introduced into the .[.lac-i-Q.].
.Iadd.laciq .Iaddend.bearing E. coli strain, JMTO3, to evaluate
expression of .alpha.-ECGF.
In pMJ26, expression of .alpha.-ECGF, under control of the hybrid
tac promoter, is inducible with IPTG. To measure .alpha.-ECGF
production, logarithmically grown bacterial cultures containing
pMJ26 at A.sub.550 of 0.2 were induced with 1 mM IPTG and grown for
2-4 hours at 37.degree. C. prior to harvesting, lysis and growth
factor isolation. Control extracts were prepared from uninduced
cultures of pMJ26 and from induced and uninduced bacterial cultures
not containing the ECGF gene. All extracts were fractionated by
SDS-PAGE, and the protein visualized by staining with Coomassie
brilliant blue. As shown in FIG. 11, lane b, a prominant band at
approximately 16 .[.kd.]. .Iadd.Kd .Iaddend.is observed in induced
.[.cultrues.]. .Iadd.cultures .Iaddend.of pMJ26. The band is
observed at low levels when pMJ26 is not induced, lane a, (this
reflects the leakiness of the tac promoter) and, as expected, is
absent in either induced or control cultures of bacterial which do
not contain the .alpha.-ECGF gene.
The ability to induce a polypeptide of the expected size,
specifically, in bacteria containing the .alpha.-ECGF gene,
suggests the successful expression of the human .alpha.-ECGF. The
protein was purified by a two-step procedure involving
.[.heparin-Sepharose.]. .Iadd.heparin-SEPHAROSE .Iaddend.column
chromatography followed by reversed phase HPLC analysis. (Burgess,
W. H., Mehlman, T., Friesel, R., Johnson, W. V., and Maciag, T.
(1985) .[.J. Biol. Chem..]. .Iadd.J. Biol. Chem. .Iaddend.260,
11389-11392.) Protein evaluated by this method is essentially pure
and amino terminal and amino acid sequence analyses demonstrate the
predicted amino acid sequence of .alpha.-ECGF of MNYKKPKLLYCSNG.
Data .[.suggests.]. .Iadd.suggest .Iaddend.(FIG. 11) pMJ26 can
express .alpha.-ECGF to approximately 10% of the total protein of
E. coli and remain soluble in this bacteria allowing .[.his.].
.Iadd.this .Iaddend.rapid two-step purification. To establish that
this protein is biologically active, it was compared to bovine ECGF
in several established assays.
In these assays, the functional activities of recombinant human
.alpha.-ECGF were examined. The success of the
.[.heparin-Sepharose.]. .Iadd.heparin-SEPHAROSE .Iaddend.affinity
based purification demonstrates that recombinant .alpha.-ECGF (FIG.
12B). Together these data indicate that the heparin binding
properties of the recombinant material are similar to those of
bovine brain-derived ECGF.
The results of cellular receptor assays (Friesel, R., Burgess, W.
H., Mehlman, T., and Maciag, T. (1986) .[.J. Biol. Chem..].
.Iadd.J. Biol. Chem. .Iaddend.261, 7581-7584: Schreiber, A. B.,
Kenney, J., Kawalski, J., Firesel, R., Mehlman, T., and Maciag, T.
(1985) .[.Proc. Natl. Acad. Sci. U.S.A..]. .Iadd.Proc. Natl. Acad.
Sci. U.S.A. .Iaddend.82, 6138-6143) indicate that the receptor
binding activity of recombinant human .alpha.-ECGF also is similar
to bovine brain-derived ECGF. Radioiodinated bovine .alpha.-ECGF
was incubated with murine endothelial cells at 4.degree. C. in the
presence of increasing quantities of either bovine or recombinant
human .alpha.-ECGF. After 30 minutes, the cell monolayer was washed
and the cell-associated radioactivity determined. As shown in FIG.
12a, the displacement curves for both bovine and human recombinant
.alpha.-ECGF are very similar. The receptor-binding activity of the
recombinant protein was abolished after reduction and alkylation
(FIG. 12A).
The mitogenic activities of native and recombinant
.[..alpha.-ECGR.]. .Iadd..alpha.-ECGF .Iaddend.were in two separate
assays. In the first assay DNA synthesis was monitored by
incorporation of .[.[.sup.3 H]-thymidine.]. (.Iadd..sup.3
H)-thymidine .Iaddend.into TCA-precipitable material as a function
of increasing quantities of .alpha.-ECGF (FIG. 12B). The second
assay compared the stimulation of both preparations of ECGF upon
the proliferation of HUVEC (FIG. 12C). In the [.sup.3 H]-thymidine
incorporation assay (FIG. 12B), the maximal response observed with
bovine brain-derived ECGF, while the dose for each which gave
half-maximal stimulation was similar (EC.sub.50 of bovine
.alpha.-ECGF=1.75 ng/ml; EC.sub.50 of recombinant human
.alpha.-ECGF=0.5 ng/ml). In the HUVEC assay (FIG. 12C), the maximal
stimulation observed with bovine and recombinant human ECGF were
similar, as were the concentrations giving half-maximal stimulation
(EC.sub.50 of bovine .alpha.-ECGF=0.6 ng/ml; .[.EC50.].
.Iadd.EC.sub.50 .Iaddend. of recombinant human .alpha.-ECGF=0.45
ng/ml). Heparin (5 U/ml) was found to potentiate the mitogenic
effect of both bovine and recombinant human .alpha.-ECGF 5-10 fold.
These data demonstrate that human recombinant .alpha.-ECGF has
biological properties similar to bovine ECGF.
Thus, this example describes experimental procedures which provide
human endothelial cell growth factor essentially free of other
proteins of human origin.
UTILITY
ECGF has utility in the growth and amplification of endothelial
cells in culture. Currently, ECGF for cell culture use is extracted
from bovine brain by the protocol of Maciag, et al., .[.[Proc.
Natl. Acad. Sci., 76:11, 5674-5678 (1978)].]. .Iadd.Proc. Natl.
Acad. Sci. U.S.A., 76:11, 5674-5678 (1978).Iaddend.. This crude
bovine ECGF is mitogenic for human umbilical vein endothelial cells
[Maciag, et al., J. Biol. Chem. 257:5333-5336 (1982)] and
endothelial cells from other species. Utilization of heparin with
ECGF and fibronectin matrix permits the establishment of stable
endothelial cell clones. The recommended concentration of this
crude bovine ECGF for use .Iadd.as .Iaddend.a mitogen in vitro is
150 micrograms per milliliter of growth medium.
Recombinant DNA-derived human ECGF has utility, therefore, as an
improved substitute for crude bovine ECGF in the in vitro culturing
of human endothelial cells and other mesenchymal cells for research
use. The activity of human ECGF is expected to be the same as or
better than bovine ECGF in the potentiation of endothelial cell
growth due to the high degree of homology in the amino acid
sequences of both proteins. The expected effective dose range for
potentiating cell division and growth in vitro is 5-10 ng of
purified ECGF per milliliter of culture medium. Production of the
ECGF via recombinant-DNA technologies as outlined in this patent
application and subsequent purification as described by Burgess, et
al., [J. Biol. Chem. 260:11389-11392 (1985)] will provide large
quantities of a pure product of human origin (heretofore
unavailable in any quantity or purity) with which to develop models
of human homeostatis and angiogenesis.
Recombinant DNA-derived human ECGF also has utility in the
potentiation of cell growth on a prosthetic device, rather than a
tissue culture flask or bottle. This device may or may not be
coated with other molecules which would facilitate the attachment
of endothelial cells to the device. These facilitating molecules
may include extracellular matrix components, human serum albumin,
or inert organic molecules.
The extracellular matrix is comprised of several fibrous proteins
imbedded in a gel comprised of glycosaminoglycan polysaccharides.
The glycosaminoglycans are usually linked to a protein core to form
proteoglycans (Alberts et al. in "Molecular Biology of the Cell"
Garland Publishing, Inc. (1983) pp. 692-715; the contents of which
are incorporated herein by reference). Among the protein components
of the extracellular matrix are collagen, elastin, laminin and
fibronectin. Collagen has a stiff, triple-stranded helical
structure and exists in at least 5 major forms (Types 1-V). Types
I-III are predominent in connective tissue, while Type IV is found
in the basal lamina. Type V is widespread in different tissues,
although in relatively small amounts. Fibronectin is a glycoprotein
that promotes cell adhesion and exists as large aggregates in the
extracellular space. Laminin is a component of the basal
lamina.
Glycosaminoglycans are long, unbranched polysaccharide chains
composed of repeating disaccharide units. They are highly
negatively charged and capable of attracting large amounts of
water, thereby forming hydrated gels even at low concentrations.
The glycosaminoglycans include hyaluronic acid, chondroitin
4-sulfate, chondroitin 6-sulfate, dermatan sulfate, .[.heparan.].
.Iadd.heparin .Iaddend.sulfate, heparin and keratan sulfate.
Hyaluronic acid is the only glycosaminoglycan that does not form a
proteoglycan structure.
For potentiation of cell growth, such as on the surface of a
prosthetic device, endothelial cells would be cultured in the
presence of effective doses of ECGF, and optionally one or more
extracellular matrix components. This device would then provide a
non-thrombogenic surface on the prosthetic device, thus reducing
the risk of potentially life-threatening thrombogenic events
subsequent to implantation of the prosthetic device.
ECGF has utility in diagnostic applications. Schreiber, et al.,
[Proc. Natl. Acad. Sci. 82:6138 (1985)] developed a double antibody
immunoassay for bovine ECGF. In this assay, 96-well polyvinyl
chloride plates were coated with rabbit anti-ECGF and the remaining
binding sites subsequently blocked with 10% normal rabbit serum.
Samples of ECGF were then added to the wells and incubated. After
washing, murine monoclonal anti-ECGF was added. After incubation
and several washes, rabbit anti-mouse IgG coupled with peroxidase
was added. The reaction product was quantitated
spectrophotometrically after conversion of O-phenylenediamine in
the presence of hydrogen peroxide. A similarly constructed
immunoassay may be useful for monitoring human ECGF levels in
disease states affecting endothelial cell growth. Purified
recombinant-DNA derived ECGF would be useful as a standard reagent
in quantifying unknown ECGF samples.
ECGF also may have potential in the treatment of damaged or in the
regeneration of blood vessels and other endothelial cell-lined
structures.
It should be appreciated that the present invention is not to be
construed as being limited by the illustrative embodiment. It is
possible to produce still other embodiment. It is possible to
produce still other embodiments without departing from the
inventive concepts herein disclosed. Such embodiments are within
the ability of those skilled in the art. Deposit of Strains Useful
in Practicing the Invention
Biologically pure cultures of strains for practicing this invention
are available at the offices of Rorer Biotechnology Inc.
Access to said cultures will be available during pendency of the
patent application to one determined by the Commissioner to be
entitled thereto under 37 C.F.R. Section 1.14 and 35 U.S.C. Section
122.
At a date prior to issuance a deposit of biologically pure cultures
of the strains within the allowed claims will be made with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., the accession number assigned after successful viability
testing will be indicated by amendment below, and the requisite
fees will be paid.
All restriction on availability of said culture to the public will
be irrevocably removed upon the granting of a patent based upon the
application and said culture will remain permanently available for
a term of at least five years after the most recent request for the
furnishing of a sample and in any case for a period of at least 30
years after the date of the deposit. Should the culture become
nonviable or be inadvertently destroyed, it will be replaced with a
viable culture (s) of the same taxonomic description.
Strain/Plasmid ATCC No. Deposit Date pDH 15 .[.53366.].
.Iadd.53336.Iaddend. Nov. 25, 1985 pDH 14 .[.53365.].
.Iadd.53335.Iaddend. Nov. 25, 1985 pMJ 26 67857 Nov. 23, 1988.
* * * * *