Note: Descriptions are shown in the official language in which they were submitted.
CA 02089143 2001-03-15
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GROWTH FACTOR COMPOSITIONS, PREPARATION AND USE
FIELD OF THE INVENTION
This invention relates to novel growth factor compositions of matter which
have anti-ulcer properties. The compositions are protein-like polypeptide
hormones
or peptides (amino acids in a chain) which are newly found species of the
epidermal
growth factor (EGF) family. More particularly, the compositions comprise the
novel
nicked (i.e., broken chain) and non-nicked (intact chain) human EGF: hEGF1-48,
and its congeners hEGF1-47, and hEGF1-49. The invention includes pure hEGF1-
48, hEGF1-48 in dosage form, treatment methods using hEGF1-48 and methods of
preparing pure hEGF1-48.
BACKGROUND OF THE INVENTION
Human EGF is a polypeptide of 53 amino acids with a molecular weight of
approximately 6,000 daltons. The amino acid sequence is known. The known
hEGF1-53 has a variety of biological/pharmacological effects including
stimulation of
RNA, DNA and protein synthesis; stimulation of cell growth, and inhibition of
gastric
acid secretion. EGF has been found to be homologous with another polypeptide
hormone urogastrone. The literature occasionally identifies this peptide as
EGF-
urogastrone, which is abstracted as Abstract 3492, The Merck Index, 11th Ed.,
552
(1989).
Patents relating to EGF, urogastrone and fragments thereof described as
EGF1-47, EGF1-48 and EGF1-51
2089143
WO 92/02246 PCT/US91/05396 4..
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include those of Gregory et al. U.S. Patent Nos. 3,883,497;
4,032,633; 4,035,485 and 4,820,690, and the patent of
Camble et al U.S. Patent No. 3,917,824.
Bioloctical Activity
The known information about the biological
activity of EGF has led to a consensus that human EGF1-53
is the most potent of the EGF-like moieties, with other
compounds, including hEGFl-48, being less active.
EGF was initially described by Stanley Cohen and
his coworkers. They observed that extracts of salivary
glands from rats induced precocious eyelid opening and
tooth eruption when administered to newborn rat pups.
Subsequently, the peptide EGF was purified from these
extracts and characterized. EGF was shown to be a potent
mitogen (i.e., an agent causing or inducing mitosis or cell
transformation) for a variety of cell types. EGF has both
mitogenic and acid suppressive activities in the GI tract.
As indicated, EGF was isolated from salivary
glands from which it is secreted into the gastrointestinal
lumen (i.e., cavity or channel). It is also secreted into
the GI tract from a variety of other sources. This has led
to numerous attempts to characterize the activities of EGF
in the GI tract.
Reports show that EGF produced a dose-dependent
suppression of gastric acid secretion in dogs. Other work
has confirmed this acid suppressive activity in several
animal species including humans. EGF is less effective as
a suppressor of gastric acid secretion than well known acid
~....WO 92/02246 ~ ~ ~! (~ 1 ~ ~ PCT/US91/05396
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suppressive therapies such as histamine-H2-antagonist or
proton pump inhibitors. This suppression of gastric acid
secretion follows when EGF is administered by injection
(i.e., by parenteral administration) but does not follow
when EGF is taken by mouth even at very high doses.
EGF administered into the gastrointestinal lumen
(i.e., the stomach or other segment of the GI cavity or
channel) does have trophic effects. Thus, increases in
tissue mass and DNA synthesis have been reported following
l0 intragastric administration and intralumenal infusions of
EGF.
The oral administration of EGF has also been
shown to promote the healing of experimentally induced
gastric and duodenal ulcers which have been induced by any
of a variety of agents including ace~'c acid, laser
treatment, cysteamine and indomethacin. ~ .~e recently, we
have found that the mechanism of action i~r this activity
is related to the ability of EGF to accelerate the re-
epithelialization (i.e., new repair growth) rate of the
induced lesions (FIGURE 1, described below). This is in
contrast to acid suppressive therapies which appear to
affect portions of the healing process that precede the re-
epithelialization phase (FIGURE 2).
Structure,/Activitv
As indicated above, human EGF is a 53 amino acid
peptide which is derived as a result of cleavage from a
much larger protein. EGF contains six cysteine residues
which form three covalent disulfide bonds.
W0 92/02246 ~ p 8 914 3 , PCT/US91/05396
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The structure activity relationship of EGF has
been the subject of investigation by a number of
laboratories. Molecular forms of EGF-like moieties include
EGF1-52, EGF1-51, EGF1-49, EGF1-48, and EGF1-47, as well as
a variety of chemically cleaved molecules and molecules
with numerous amino acid substitutions. These molecular
forms of EGF are reported to be less active than EGF1-53
with respect to mitogenic activity and receptor cell
binding activity. With the exception of EGF1-52 none of
the fragments of EGF has been evaluated for its in vivo
activity, probably owing in each case to the prevailing
opinion that it would be less efficacious than EGF1-53.
Although these molecular forms have been published as
having been isolated, none with the possible exception of
EGF1-52 has been purified to homogeneity and characterized
for homogeneity and identity.
Summary of the Invention
The present invention is based on the discovery
of the pure human EGF (hEGF) species, especially as species
purified to homogeneity: non-nicked (or intact chain)
polypeptides EGF1-47, EGF1-48, and EGF1-49; and the pure
human EGF (hEGF) species: nicked (or broken chain)
polypeptides EGF1-47, EGF1-48, and EGF1-49.
The invention is thus based on the discovery that
conventional means of obtaining EGF1-48 and its congeners
EGF1-47 and EGF1-49 (sometimes herein referred to as EGF
congeners) -- such as by chemical synthesis, limited
r..WO 92!02246 ~ ~ ~ ~ ~ ~ ~ pCT/US91/05396
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proteolysis of intact EGF, and recombinant microbial
techniques -- do not produce the EGF species alone but
rather produce the mixed species of the hitherto unnoticed
comigrant nicked and non-nicked mixture which by
conventional methods including chromatographic techniques
have prior to the present invention never been isolated as
the separate pure nicked and pure non-nicked species
(FIGURE 3).
The invention thus provides the nicked and non
to nicked forms of human EGF1-48 and its adjacent EGF1-47 and
EGF1-49 congeners also in nicked and non-nicked forms (each
as a discrete and separate novel molecular entity). These
compounds have unexpected therapeutic utility for the
treatment of gastrointestinal lesions. Thus, EGF1-48 and
its congeners in pure nicked or pure non-nicked form, each
possess unique and unexpected activity.
It is also found that pure EGF1-48 and its pure
congeners each in either nicked or non-nicked form have
structural stability and resistance to enzymatic
2o degradation (gastric juice and trypsin) which stability and
resistance result in unexpected utility for treating
gastrointestinal lesions.
Brief Description of the Drawincrs
FIGURE 1 is a graph showing the dose response of
EGF expressed as contracture rate in treatment of canine
gastric ulcer; ulcers were induced via laser therapy of the
antrum. The ulcer healing process was followed by repeated
endoscopic examination during which images of the lesion
WO 92/02246 2 d 8 914 ~ , . PCT/US91/05396
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.. _
were obtained. For each animal, lesion sizes were measured
and the rate of reepithelialization was determined as the
rate constant for a least squares fit of the lesion sizes
over the time course of the experiment;
FIGURE 2 is a schematic of the ulcer phases
showing the variation in ulcer size following the
initiating event and during pre-contracture, contracture
and healing;
FIGURE 3 is a plot of the chromatographic
separation profile over time of the hEGF1-48 eluting first
as the non-nicked (intact) species and then as the nicked
species; the EGF1-48 fraction was isolated and purified
from the broth of a fermentation during which expression
was induced. This representative chromatogram demonstrated
an additional chromatographic separation of the purified
hEGF1-48 into the nicked and intact species using an ion
exchange resin as described within:
FIGURE 4 is a graph of the dose effect of EGF,
relative to the dose effect of hEGF1-48, on indomethacin-
induced gastric lesions in the rat, expressed as percent
improvement compared to the saline group; rats received an
injurious dose of indomethacin (orally) and either saline
or the indicated dose (orally) of EGF or hEGFl-48. At 12
hours post dosing, animals were sacrificed and the extent
of gastric damage assessed:
FIGURE 5 is a 3-graph set comparing the % AUC for
EGF1-49, EGF1-48, and EGF, respectively; samples of each
parent peptide, dissolved in human gastric juice were
incubated at 37°C. At the indicated times aliquots were
,.~ wo 9a/oaa46 ~r ~ ~ ~ i ~
PGT/US91/OS~96
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withdrawn and t~ amount of intact parent pept~~e was
determined as the area under the curve of the A214
(absorbance at 214 nanometers) chromatogram;
FIGURE 6 is a composite graph showing over time
the relative percentages of unhydrolyzed EGF (hEGFi-48 and
EGF1-53) in 1% and 3% trypsin; the experiment is the same
as described in Figure 5 except trypsin is the enzyme
source;
FIGURE 7 is a gene design diagram for hEGFl-48;
showing the nucleotide and corresponding amino acid
sequence for the EGF coding region of the EGF expression
cassette inserted in the P. Dastoris strains; for the
strain containing only the hEGFi-48 coding region, the
nucleotides coding for residues 49-53 were not included in
the cassette;
FIGURE 8 is a graph showing the mitogenesis and
competition binding analysis of EGF and hEGFl-48 in Balb
3T3 cells; and
FIGURE 9 is a graph showing the mitogenic effect
of EGF and hEGFl-48 in IRK cells.
Detailed Description and Preferred Embodiments
The invention in one aspect comprises a
polypeptide selected from hEGFl-48 or its adjacent congener
or a pharmaceutically acceptable salt thereof, which
polypeptide is pure non-nicked or pure nicked, preferably
a polypeptide which is non-nicked, and preferably EGF1-48
of the formula I:
WO 92/02246 ~ ~ ~ ~ ~ ~ ~ . PCT/US9.1/05396 ._
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H.Asn.Ser.Asp.Ser.Glu.Cys.Pro.Leu.Ser.His.Asp.Gly.Tyr
Tyr.Met.Cys.Val.Gly.Asp.His.Leu.Cys
25 26
~--~ Ile. _Glu.Ala.Leu.Asp.Lys.Tyr.Ala.Cys I
Tyr.Gly.Val.Val.Cys.Asn
Ile.Gly.Glu.Arg.Cys.Gln.Tyr
HO.Lys.Leu.Asp.Arg
A preferred salt is the trifluoroacetate or the acetate
salt.
Also preferred is a polypeptide of the formula I
which is nicked EGF1-48 or its nicked adjacent congener or
a pharmaceutically acceptable salt thereof, preferably
nicked between the 25-26 position of the polypeptide chain.
We have found that hEGFl-48 is surprisingly more
effective than hEGF1-53 in the treatment of experimentally
induced lesions in the gastrointestinal tract (FIGURE 4).
A typical result, for example, in the treatment of
indomethacin-induced lesions in the rat was an apparent
modest reduction (16%) in lesion size (read after 12 hours)
in the use of hEGFl-53 at a zero time oral dose of 1.0
nanomoles per Kg. (but without reduction at higher dosage).
The size reduction observed was however not statistically
different from controls. A typical result in the
comparable use of intact hEGFl-48 at various oral doses
(0.5, 1.0, 5.0, and 10.0 nmol per Kg) varied from 37 to 46%
improvement in lesion size reduction read at 12 hours
versus controls, a result that was found to be significant
based on t-test analysis. This unique therapeutic utility
is enhanced by the unexpected and heretofore unappreciated
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PCT/US91/05396
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structural stability and rf~..:.stance, as indicated, to
enzymatic degradation of nicked and non-nicked EGF1-48 and
its congeners. The time course of degradation of pure
intact (non-nicked) hEGF1-48, yr example, in gastric fluid
was found in the typical case to be only slight at one
hour, slight to moderate at four hours, and marginally more
degraded at 19 hours (FIGURE 5). By contrast, the
comparable time course of hEGFi-53 was approaching almost
complete degradation at one hour. Similarly, the time
course of degradation of intact hEGF1-48 in 1% or 3%
trypsin (w/w) was ca. 10% and ca. 25% respectively after
four hours. By contrast, the comparable time course of
degradation of hEGFl-53 in 1% and 3% trypsin was ca. 50%
after one hour and 90-100% after one hour, respectively
(FIGURE 6).
As used herein, the term "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not
impart any undesired toxicological effects. Examples of
such salts are (a) acid addition salts formed with
inorganic acids, for example hydrochloric acid, hydrobromic
acid, sulfuric acid, phosphoric acid, nitric acid and the
like; and salts formed with organic acids such as, for
example, acetic acid, oxalic acid, tartaric acid, succinic
acid, malefic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
pamoic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acids, naphthalenedisulfonic acids,
polygalacturonic acid; (b) salts with polyvalent metal
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cations such as zinc, calcium, bismuth, barium, magnesium,
aluminum, copper, cobalt, nickel, cadmium, and the like; or
(c) salts formed with an organic cation formed from N,N'-
dibenzylethylenediamine or ethylenediamine; or (d)
combinations of (a) and (b) or (c), e.g., a zinc tannate
salt: and the like. The preferred acid addition salts are
the trifluoroacetate salt and the acetate salt.
As used herein in reference to the EGF species,
the term "non-nicked" means the intact polypeptide in which
the three disulfide bonds are intact or unbroken and the
polypeptide chain is intact. The term "nicked" means that
the three disulfide bonds are intact and the polypeptide
chain is nicked or broken between at least one pair of
adjacent residues of the polypeptide chain such as the 25
26 pair of residues.
The invention in another aspect comprises
pharmaceutical compositions in dosage form, preferably for
oral administration, containing an effective amount of the
described poiypeptide (which may be the nicked or non-
nicked EGF1-48 or its nicked or non-nicked adjacent cogener
EGF1-47 or EGF1-49) and a pharmaceutically acceptable
diluent or carrier, for the prevention or management or
treatment of diseases of the gastrointestinal mucosa such
as erosive or inflammatory diseases in a subject. One uses
an amount of the polypeptide that is effective to prevent
or manage the disease in the subject or to promote the
management or healing thereof. For human treatment, the
non-nicked EGF1-48 is to be administered in a dosage
regimen, preferably oral, in pharmacologic amounts between
CA 02089143 2001-03-15
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about 0.001 nanomoles/kg and at least about 100 nanomoles/kg per day in
pharmaceutically acceptable dosage form. The regimen for nicked EGF1-48 or
congener requires a higher dosage in an amount ranging from about 0.01
nanomoles/kg to at least about 10 micromoles/kg per day. Treatment of GI
disease
conditions may be achieved by the oral route without inhibiting gastric acid
secretion
in the subject. The invention contemplates pharmaceutical compositions for the
prevention or management of or treatment for erosive or inflammatory disease
conditions such as erosive or ulcerative esophagitis; inflamed, erosive or
atrophic
(i.e., being atrophied) conditions of the small or large intestine including
but not
limited to atrophic gastritis, duodenal ulcers, gastric ulcers, duodenitis,
inflammatory
bowel disease, ulcerative colitis; and the like. In a preferred embodiment of
an oral
dosage form and use thereof, an appropriate amount of nicked or non-nicked
EGF1-
48 or an adjacent congener (as the case may be) is dissolved in aqueous
solution,
which may include a water soluble cellulose stabilizer such as described in
U.S. Pat.
No. 4,717,717, and administered orally. Other oral dosage forms described
herein
can also be used.
Nicked or non-nicked EGF1-48 or an adjacent congener may be administered
therapeutically as part of a common oral formulation which includes a known
anti-
ulcer agent.
Examples of such anti-ulcer agents known in the art are: the so-called
histamine H-2 receptor antagonists,
CA 02089143 2001-03-15
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e.g. cimetidine, ranitidine, and famotidine; gastric specific anti-cholinergic
agents
such as pirenzepine; prostaglandin E2 analogues such as misoprostol or
arboprostil;
agents such as sucralfate or carbenoxolone; H+/K+ ATPase inhibitors such as
omeprazole; and antacids such as aluminum hydroxide/magnesium hydroxide
mixtures. For a layman's description of these and other drugs, see Joe
Graedon's
The New People's Pharmacy, Chapter 5, 134-163, 1985. Bantam Books, Inc., New
York.
The known anti-ulcer agent may be present in the composition in an amount
consistent with its known therapeutic activity. Thus, for example, an oral
composition
containing cimetidine may contain between 100 and 1000 mg. of cimetidine.
The oral pharmaceutical composition may be formulated by means known to
the art in the form of, for example, aqueous or oily solutions or suspensions,
emulsions, tablets, capsules, lozenges, chewing gums or dispersable powders.
In another aspect, the invention comprises a method for prevention or
management or treatment of diseases of the gastrointestinal mucosa including
erosive or inflammatory diseases in a subject which comprises administering to
the
subject an amount of nicked or non-nicked EGF1-48 or its adjacent congener, or
a
pharmaceutically acceptable salt thereof that is effective to prevent or
manage the
disease in the subject or to promote the management or healing thereof. For
human
~Q8~I~3
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treatment, the non-nicked EGF1-48 is to be administered in
a dosage regimen, preferably oral, in pharmacologic amounts
between about 0.001 nanomoles/kg and at least about 100
nanomoles/kg per day in pharmaceutically acceptable dosage
form. The regimen for nicked EGFl-48 or congener requires
a higher dosage in an amount ranging from about 0.01
nanomoles/kg to at least about 10 micromoles/kg per day.
Treatment of GI disease conditions may be achieved by the
oral route without inhibiting gastric acid secretion in the
subject. The invention contemplates treatment of disease
conditions such as erosive or ulcerative esophagitis;
inflamed, erosive or atrophic (i.e., being atrophied)
conditions of the small or large intestine including but
not limited to atrophic gastritis, duodenal ulcers, gastric
ulcers, duodenitis, inflammatory bowel disease, ulcerative
colitis: and the like.
In still another aspect the invention concerns a
method of making non-nicked hEGFl-48 comprising the steps
of
A. growing a human EGF expression strain of the
methylotrophic yeast P, pastoris in a fermentation growth
medium having a methanol feed, at acid pH, preferably pH 5,
for a methanol-sustained growth period resulting in the
selectively induced formation of a mature growth medium
broth containing yeast expressed non-nicked or intact
hEGFl-48 and excluding nicked hEGFl-48, and
B. separating the hEGFl-48 from the broth by
means excluding other proteins, especially EGF species
other than hEGF1-48, which means excluding other proteins
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preferably may include~first treating the mature broth with
trypsin to selectively degrade the other proteins while
leaving intact the hEGFl-48, employing, for example, 1 to
3% trypsin in the broth for one hour at 37 ~, followed by
HPLC chromatography.
The fermentation is carried out at an acid pH,
preferably at pH 5. The methanol feed portion of the
fermentation (as described elsewhere herein) is maintained
for about 24 to about 40 hours, more preferably for optimum
production, about 36 hours. Under these conditions of
relatively short methanol induction, we have found
surprisingly that the sole EGF product expressed in the
broth is the desired non-nicked hEGFl-48. We have found,
however, that when the methanol feed portion of the
fermentation is carried out for substantially longer
periods, i.e., a long methanol induction period (e. g., more
than 40 hours), a mixture of both products is obtained:
non-nicked hEGFl-48 and nicked hEGFi-48. This result is
exemplified by the following methanol-sustained incubation
runs, which are typical:
TABLE I
Incubation
Time
Broth No. in MEOH % Nicked
477 110 hrs 50.3
490 40 hrs 2.7
The method employing the short methanol induction
is preferred because it facilitates the workup and
purification of the desired non-nicked hEGFl-48 in non-salt
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or salt form. The separation of non-nicked hEGF from the
broth may be carried out by art-recognized means.
In still another aspect, the invention concerns
a method of making hEGF comprising the steps of:
A. growing a human EGF expression strain of the
methylotrophic yeast P. nastoris in a fermentation growth
medium having a methanol feed for a methanol-sustained
growth period resulting in the formation of a mature growth
medium broth containing a mixture of yeast expressed non-
to nicked hEGF and nicked hEGF,
B. isolating the hEGF mixture from the broth by
steps comprising subjecting the hEGF to column
chromatography comprising adsorption on and elution from a
strong cation exchange resin under acid conditions to cause
the non-nicked hEGF and the nicked hEGF to be eluted as
separate eluates respectively, and
C. isolating the non-nicked hEGF and the nicked
EGF from the respective eluates. Preferably, the period of
methanol-sustained growth is substantially longer than 40
hours, up to 100 hours or longer, as desired, to produce a
sufficient quantity of nicked hEGF in the broth mixture.
A preferred resin for column chromatography is a
sulfoethylaspartamide resin. We have found surprisingly
that while intact and nicked hEGFl-48 have the same
retention time on reversed phase HPLC columns, they can be
separated on the strong cation exchange column under acidic
conditions (FIGURE 3). Under these conditions, nicked
EGF1-48 has one extra positive charge as compared with
intact EGF1-48. The preferred column is a column of
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suitable dimension preferably measuring 4.6 x 200 mm., 300
Angstrom units, 5 microns, employing sulfoethylaspartamide-
SCX (available from the Nest Group). For elution, the
preferred mobile phases are mobile phase A comprising five
millimole phosphoric acid titrated to pH 3 with KOH and
containing 25% acetonitrile and mobile phase B comprising
mobile phase A that contains 0.3 molar KC1. The preferred
elution conditions comprise a linear gradient from phase A
to 70% phase B over 45 minutes at 1 ml. per minute. The
nicked EGF eluates after the non-nicked EGF and the eluate
containing it is lyophilized in pure form. It is nicked
between residues 25 and 26 and was found by protein
sequencing to be totally nicked. The non-nicked EGF is
obtained separately in pure form by lyophilizing the eluate
containing it. The invention contemplates the production
of any of the species: EGF1-48, EGF1-47, and EGF1-49 by
appropriate selection of the producing yeast strain which
strain is known or is available by art-recognized means.
Another aspect of this invention is the
production of hEGFl-49. The present preferred method
includes the steps comprising:
A. Enzymatically treating EGF1-53 by suitable
means such as treating with human gastric juice,
carboxypeptidase or the like, preferably using human
gastric juice as the source of enzyme, for a time
sufficient to convert most of the starting material to
EGF1-49, about two hours at 37°C in gastric juice; and
B. Separating, preferably by chromatographic
procedures, the EGF1-49 from other materials in the
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reaction mixture. The enzyme reaction may be stopped or
quenched by a variety of means including but not limited
to: addition of alcohol or other organic solvent,
adjustment of pH above 3.0, or immersion of the reaction
vessel in an ice cold bath to reduce the temperature. In
the currently preferred embodiment, the chromatographic
procedures described for the isolation of EGF1-48 also have
been used effectively to purify EGF1-49.
As indicated, the nicked and non-nicked EGF1-48
and congeners are for purposes of the invention preferably
derived recombinantly by microbial methods, i.e., by rDNA
techniques.
EGF Products By Recombinant Technoloav
The knowledge of the amino acid sequence of
urogastrone allowed design and construction of synthetic
genes encoding this peptide, which in turn allowed
development of recombinant expression systems. By 1982,
the first recombinant expression system for hEGF was
reported, utilizing the bacterium E. co i to produce hEGF
and yielding 2.3 mg/1 of biologically active material.
Later, the use of the S. cerevisiae a-mating factor leader
sequence to direct secretion of hEGF from ,5~. cerevisiae
increased the expression level of a (1-52) form of hEGF to
5 mg/1. More recently, an improved Bacillus expression
host has been reported to secrete 240 mg/1 of hEGF with no
appreciable degradation. With the exception of the
Bacillus system, for which no published information on
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productivity during scale-up is available, expression
levels of hEGF in these recombinant systems are low.
The methylotrophic (requiring methyl alcohol as
a nutrient) yeast, Pichia pastoris, has been developed as
an improved host for production of recombinant products.
Recombinant Pichia pastoris strains advantageously can
secrete recombinant proteins in the gram per liter range,
can adapt to fed batch or continuous cultivation, have an
extremely stable recombinant phenotype (i.e., physical,
biochemical and physiological makeup of the yeast), and can
maintain high yields over several orders of fermentation
scale-up.
What follows is a description of the development
and scale-up including the best mode according to the
invention to a pilot-plant scale of a process for
production and purification of bioactive hEGF, for
illustrative purposes usually as the EGF1-48 species that
is secreted into the growth medium of a recombinant strain
of ~ pastoris.
A. Expression and Biochemical Analysis of hEGF Secreted
by Pichia pastoris
The alcohol oxidase (AOX1) promoter used to drive
heterologous (i.e., different species) peptide synthesis in
P. pastoris expression systems is derived from the primary
alcohol oxidase gene. Alcohol oxidase catalyzes the
oxidation of methanol to formaldehyde and hydrogen peroxide
as the first step in methanol metabolism. Development of
*..:.WO 92/02246 2 0 8 914 ~J pCT~US91105396
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a fermentation protocol which induces expression of AO~C1
regulated heterologous genes has been previously described.
Briefly, the fermentation consists of three
distinct stages. First, the cells are grown on glycerol to
accumulate cell biomass while repressing heterologous gene
expression. Second, glycerol is fed at a rate which keeps
yeast cell growth carbon-limited; the cell mass increases
further during this stage but the carbon limitation allows
derepression of the methanol metabolic pathway so that the
cells begin to adapt to growth on methanol. In the third
stage, full expression of the heterologous peptide is
induced by introduction of a methanol feed. This protocol
was used to induce expression of hEGF from three
recombinant strains.
Two P. ~astoris strains are designated G+EGF817S1
and G+EGF819S4. They contain two and four copies,
respectively, of an hEGF expression cassette coding for
EGF1-53 integrated into the AOX1 locus of the host strain
GS115. A third strain G+EGF206S10 contains six copies of
an hEGF cassette coding for EGFl-48 integrated at the H1S4
locus of the host strain GS115. Each expression cassette
contains the P. pastoris alcohol oxidase (AOX1) promoter
and regulatory sequences, DNA sequences coding for the S.
cerevisiae a-mating factor prepro leader sequence fused to
a synthetic gene encoding respectively for hEGFl-53, hEGFl-
53, and hEGFl-48 (FIGURE 7); and the AOX1 transcription
termination sequence. The transforming DNA also includes
the P. pastoris HIS4 sequence.
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HPLC was routinely used to quantitate the various
hEGF species present, in cell-free broth from fermentations
of strain G+EGF819S4. At the smaller fermentation scales,
a series of HPLC profiles typically was taken over 36 hours
following methanol induction of EGF expression. At the
earliest times a single peptide peak appeared on the
chromatogram. By seven or eight hours into the methanol
induction phase a second peak was evident and represented
the major species present. After 36 hours of methanol
to induction, a single major peak, which eluted appreciably
earlier in the gradient than the two peaks seen previously,
was evident. Mass spectral analysis and amino acid
analysis identified the three peaks as EGF1-52, EGF1-51,
and EGF1-48, respectively. Thus, the EGF was being
converted to a progressively shorter peptide over time. We
found that the conversion of hEGF from the EGF1-52 to the
EGF1-48 form was pH dependent. The conversion pattern
described above occurred when the fermentation preferably
was conducted at pH 5.
Only one hEGF species, non-nicked hEGF1-47, was
produced by strain G+EGF206S10. This strain contains six
copies of a DNA sequence encoding hEGFl-48.
B. Biological Activity of hEGFl-48
The recombinant human EGF1-48 produced by the
yeast was compared with human EGF1-53 in terms of mitogenic
activity in normal rat kidney fibroblasts (NRK-49F) and two
murine cell lines. The mitogenic response to EGF is cell
line dependent. Maximal stimulation in the rat cell line
~WO 92/02246 ~ ~<g 9:1 ~..~ . PCT/US91/05396
- 21 -
is reached at about 10 ~~ M and remains unchanged up to about
108 M for EGF1-53. For EGF1-48, the effect is observed
around 10 ~~ M. The mitogenic response to EGF1-53 in the
murine lines occurs at a slightly different concentration
and remains near maximal over a much more narrow
concentration range (FIGURE 8). The response to EGF1-48 in
murine cell lines is about equivalent to the response to
EGF1-53 except that the maximal effect occurs at slightly
higher concentration and this peak effect is not diminished
l0 by increasing the EGF1-48 concentration further in contrast
to the response to EGFl-53 (FIGURE 8). Mitogenic
evaluation using another murine cell line, CH310T1/2, was
substantially similar to the Balb 3T3 data except that
EGF1-48 appeared slightly more potent. In all cell lines
tested, the maximal response to EGF1-48 is at least as
great as that of EGF1-53 (FIGURE 8, FIGURE 9).
C. Fermentation Scale-Up and hEGF Production at 250-Liter
S a a
A general consideration in scale-up of
fermentation processes is the relatively lower oxygen
transfer capacity of larger fermentors compared to
laboratory models. This consideration is especially
relevant to recombinant P' i pastoris expression strains.
Heterologous gene expression is induced in these strains by
the introduction of a methanol feed which is also used for
both cell growth and metabolic energy production. Due to
the highly reduced state of carbon in methanol relative to
carbohydrates, methanol metabolism requires more oxygen per
W0 92/02246 2 ~.g ~ ~ PCT/US91/05396 .
22
mole of carbon than do~s,<.Carbohydrate metabolism. The high
,, .
oxygen requirement'for methanol metabolism is often the
factor that limits the rate of methanol feed, and thus
limits the growth and productivity.
15-Liter Fermentation
Preliminary fermentation investigations showed,
and it is a highly significant feature of the invention,
that nearly quantitative conversion of hEGF to the EGF1-48
form occurred after 36 hours of methanol feed. The
protocol developed for a 15-liter fermentation allowed time
for complete conversion of the EGF1-52 form to hEGFl-48 by
using a feed rate of 100 ml/h of methanol which filled the
fermentor to 11 liters in 42 hours. A typical oxygen
utilization of 33 moles 02/g methanol measured in these fed
batch recombinant fermentations is slightly higher than the
30 moles OZ/g methanol reported for continuous fermentations
of wild-type P. pastoris. Thus, a feed rate of 100 ml/h
methanol at an 8-liter volume would require an oxygen
transfer rate of 330 moles 02 1 'h ~ . The fermentation can
be adapted to fermentors with lower oxygen transfer
capacity by reducing the methanol feed rate. To examine
the effect of adapting to fermentors with significantly
lower oxygen transfer capacity, production of hEGFl-48 was
determined at methanol feed rates of 50 ml/h and 25 ml/h in
the 15-liter fermentor. These reduced feed rates gave no
significant differences in the amount of hEGFi-48 produced,
up to about 5 grams per run. However, the time required to
produce the 5 grams was 5 days longer at 25 ml/h than at
TWO 92/02246 ~ o ~ o i 4 ~ , PCT/US91/0539b
23
100 ml/h. Thus, the fermentation process can be readily
adapted to any fermentor without loss of yield, although
the productivity would be lower in fermentors with less
efficient oxygen transfer.
250-Liter Fermentation
EGF production in P. pastoris was scaled to a
250-liter pilot plant fermentor (New Brunswick Scientific,
Edison, NJ). A proportional scale-up of the methanol feed
would be 1.7 1/h; however, as anticipated, the oxygen
transfer capacity initially limited the methanol feed rate
to half this rate. Therefore, the operating pressure was
increased from 5 to 10 psig and the air sparge was enrich
with oxygen to increase oxygen transfer and allow a higher
methanol feed rate. These changes allowed an increase in
the methanol feed rate to 1.2 1/h. Based on the laboratory
studies, the volumetric yield can be maintained at this
lower feed rate by running the fermentor 18 hours longer.
From inoculation of the fermentor to harvest, the
250-liter fermentations ran 80 hours. These fermentations
consumed 45 liters of methanol and allowed reproducible
recovery by centrifugation of clarified broth containing 50
~ 3 grams hEGFl-48. The hEGFl-48 production per liter of
methanol feed was the same at the 250-liter scale as that
at the laboratory scale.
D. Pilot Scale Purification
At the pilot scale (250-liter fermentor),
recovery and purification of hEGF were monitored by a rapid
WO 92/02246 ~, ~ ~ 1 ~ PCT/US91/05396
- 24 -
isocratic HPLC assay for hE6F1-48. The purification was
greatly simplified by the fact that hEGFl-48 is by far the
predominant peptide , in-. t3ie broth. The HPLC profile of a
sample of broth from the end of one of the 250-liter runs
showed only one major peptide peak. In the initial
recovery step the peptide was removed from 200 liters of
clarified broth by adsorption on a reverse phase resin.
The adsorption was performed stepwise in a batch mode.
After greater than 90% of the EGF was bound to
reverse phase resin, the broth and resin were pumped
through a column where the resin was retained by a 10 ~,
mesh screen. The resin was washed with 0.05 M acetic acid,
and the EGF was then eluted from the resin with four to
eight liters of eluent to effect a volume reduction from
the original broth of almost two orders of magnitude. This
rapid volume reduction reduces liquid handling in the later
steps. After an adsorption-desorption step on a cation
exchange resin to remove colored contaminants, hEGFl-48
comprised more than 85% of the total peptides as determined
by analytical HPLC. The hEGFl-48 was then chromatographed
by preparative HPLC, the fractions were analyzed by
analytical HPLC, and the selected fractions were pooled.
The HPLC was loaded with an aliquot containing 6.7 g EGF.
The recovery of EGF in the fractions was 100% ; the later
fractions had higher purity. If the purity criteria were
set much higher, for example above 99%, it is likely that
the loading of the HPLC would have to be reduced in order
to avoid appreciable losses in fractions which could not be
pooled.
wo ~aioaa~ 2 (~ $ ~ i ~ PCT/US91/05396
- 25 -
The acetonitril: introduced into the sample
during the HPLC step was removed by binding the EGF to a
cation exchange resin and washing with 0.05 M acetic acid.
This step also removed most of the trifluoroacetic acid
(TFA). TFA was less than 0.1% of the final product which
was lyophilized as an acetate salt. The final product
obtained was the purified acetate salt of non-nicked hEGFl-
48. Before lyophilization, the EGF was sterilized by
filtration through a 0.2 ~, membrane.
The cation exchange adsorption-desorption
procedure used for acetonitrile removal is the same as that
which resulted in complete recovery at the decolorization
step. On the basis of overall experience with this
procedure, a recovery of better than 95% is normal. Thus,
in routine operation at the 250-liter scale, the process
described is expected to produce batches of more than 30 g
of purified EGF.
EXPERIMENTAL PROCEDURES
A. EGF Production Strains
Three different recombinant strains of P.
pastoris were tested for the production of hEGF. Two
strains, as indicated, contained respectively two and four,
respectively, copies of an hEGF expression cassette coding
for EGF1-53 integrated into the AOX1 locus of the host
strain GS115. A third strain G+EGF206S10 contains six
copies of an hEGF cassette coding for EGFl-48 integrated at
the H1S4 locus of the host strain GS115. Each expression
cassette contains the P. pastoris alcohol oxidase AO 1
CA 02089143 2001-03-15
-26-
promoter, and regulatory sequences, DNA sequences coding for the S. cerevisiae
a-
mating factor prepro leader sequence fused to a synthetic gene encoding
respectively for hEGF1-53, hEGF1-53 and EGF1-48 (FIG. 7), and the AOX1
transcription termination sequence. The transforming DNA also includes the P.
astoris H1S4 sequence.
Recombinant hEGF-producing strains of P. pastoris were developed by
transformation of the auxotrophic His-Pichia host strain GS115 with vectors
containing two, five or six hEGF expression cassettes. The expression vector
comprised of two hEGF1-53 expression cassettes, as indicated, is pA0817, and
that
having five hEGF1-53 cassettes is pEGF819. An expression vector comprised of
six
hEGF1-48 cassettes is called pEGF206.
Pichia pastoris strain GS115 was the host for transformation with these
vectors.
Deposit of Cultures
Viable cultures of the P. pastoris strain GS115 were deposited, under the
terms of the Budapest Treaty at the American Type Culture Collection,
Rockville,
Maryland USA ("ATCC") on Aug. 15, 1987 and were assigned ATCC Accession No.
20864, as documented by PCT Patent Publication No. WO 90/03431, Pub. Date, 5
Apr. 1990. Undigested vectors pA0817 and pEGF819 and linearized vector
pEGF206 were transformed into GS115 by the spheroplast method [Cregg et al.,
Mol. Cell. Biol. 5, 3376-3385 (1985)]. After selection and analysis by
CA 02089143 2001-03-15
-27-
Southern hybridization, the following strains were identified: strain
G+EGF817S1
contains two copies of the hEGF1-53-encoding cassette integrated at the AOX1
locus; strain G+EGF819S4 contains four copies of the hEGF1-53-encoding
cassette
(one copy was lost from the five-copy plasmid vector by recombination during
transformation) integrated at the AOX1 locus, and strain G+EGF206S10 contains
six
copies of the hEGF1-48-encoding cassette integrated at the HIS4 locus.
B. Fermentation Protocols
Fifteen-liter fermentations (in a 15-liter Biolafitte fermentor) were started
in a
six-liter volume containing four liters of basal salts [52 ml/I 85% phosphoric
acid, 1.8
g/I calcium sulfate~2H20, 28.6 g/I potassium sulfate, 23.4 g/I magnesium
sulfate~7H20, 6.5 g/I potassium hydroxide] and 400 g of glycerol. After
sterilization,
25 ml of PTM, trace salts solution [6.0 g/I cupric sulfate~5H 20, 0.08 g/I
sodium
iodide, 3.0 g/I manganese sulfate~HZO, 0.2 g/I sodium molybdate~2H20, 20 g/I
zinc
chloride, 0.02 g/I boric acid, 0.5 g/I cobalt chloride, 65.0 g/I ferrous
sulfate~7H20, 0.2
g/I biotin and 5.0 ml/I sulfuric acid (conc)] were added, and the pH was
adjusted and
subsequently maintained at 5.0 by the addition of ammonia gas throughout the
fermentation. Excessive foaming was controlled by the addition of 5%
STRUKTOLT"~
J673 antifoam (STRUKTOLT"~ is a trade-mark of Schill & Silacher GmbH & Co.).
The
fermentor was inoculated with a volume of 500 ml of an overnight culture (OD
600=1
to 4) of the EGF-expressing strain in Yeast Nitrogen Base (YNB) , 2%
CA 02089143 2001-03-15
-28-
glycerol, 0.1 M potassium phosphate, pH 6. The dissolved oxygen was maintained
above 20% by increasing the air flow rate up to 20 liter/minute, the agitation
up to
1000 rpm and/or the pressure of the fermentor up to 1.5 bar during the
fermentation.
After exhaustion of the initial glycerol charge, a 50% glycerol feed,
containing
12 ml/I PTM , trace salts, was initiated at a rate of 120 ml/h; the glycerol
feed
continued for 6 hours, at which time the methanol feed, 100% methanol plus 12
ml/1 PTM ~ trace salts, was started at a rate of 20 ml/h. The methanol feed
was
increased by 10% each half hour until a feed rate of 100 ml/h was reached. The
fermentation was then continued for 25-35 hours.
The conditions for 2-liter and 250-liter fermentors were scaled
proportionately
from the 15-liter fermentor, except that the final methanol feed rate was
limited to the
highest rate at which the dissolved oxygen concentration could be maintained
above
20% air saturation. In the 2-liter and 250-liter fermentors, the pH was
controlled with
NH4 OH rather than NH 3, and in the 250-liter fermentor, the air sparge was
supplemented with 02 in some runs.
C. Analytical HPLC
Broth samples to be assayed by HPLC were treated by centrifugation for
three minutes in a microcentrifuge to remove cells. Reverse phase HPLC was
performed on a BONDAPAKT"" C18 (0.25 x 30 cm) column with a C18 guard column
(BONDAPAKT"" is a trade-mark of Waters Investments Corporation, of Delaware.)
Mobile Phase A consisted of 0.1 % by weight TFA in deionized
WO 92/02246 ~ ~ ~ ~r PCT/US91/05396
- 29 -
water, and Mobile Phase B was 95% acetonitrile/5% H20 with
0.1% TFA. The column was equilibrated with a mixture of
80% A and 20% B at a flow rate of 1 ml/min. for 20 minutes
before each run.
Each analytical run was 50 minutes. The first
five minutes were isocratic at 80% A, 20% B; then the
concentration of B was increased linearly over the next 25
minutes to 30% B; and, finally the concentration of B was
increased linearly to 55% during the final 20 minutes. UV
absorbance was monitored at 210 nm. The diffPrPnt upr.r
systems used at several sites gave comparable results.
A shorter analytical HPLC procedure was developed
for process control at the pilot scale. The shorter
procedure consisted of a ten minute run at isocratic
conditions of 72% A, 28% B.
D. Analytical Mass Spectometry
Small amounts of EGF1-47, EGF1-48, EGF1-51, and
EGF1-52 were purified from the fermentation broth by
reverse phase HPLC. These purified samples were analyzed
by fast atom bombardment mass spectometry.
E. Bioactivity Assays
Mitogenic (i.e., cell replication) stimulation by
hEGFl-48 was determined in three cell lines by tritiated
thymidine uptake. Murine Balb 3T3 cells, C3H10T1/2 cells,
and normal rat kidney fibroblasts (NRK-49F) cells were
plated into 24 well plates in DMEM (4.5 g/1 glucose),
phenol red-free, containing 5% Colorado calf serum
(Colorado Serum Company). Cells were grown at 37~C in 5%
CA 02089143 2001-03-15
-30-
C02 atmosphere. The medium was changed every three days. Cells reached
confluence in about three to four days and were allowed to remain at
confluence for
24-48 hours before assay. The medium was removed and replaced with DMEM
containing 0.1 % BSA (Sigma) and 10 U/ml penicillin/streptomycin (Gibco). The
cells
were serum starved for 22 hours, after which EGF1-53 and EGF1-48 were added in
the dose range 0.0 to 30.0 nM , for 24 hours, Dilutions were made from a stock
solution of each EGF species whose concentration was determined by amino acid
analysis. For maximal stimulation, cells were incubated with 5% calf serum.
After the
24 hour incubation period, 100,000 cpm/well of [3H]Thymidine (Amersham) were
added, and the plates were incubated at 34°C for 90 minutes. The cells
were then
washed with 1.5 ml cold PBS, followed by a 20 minute incubation at 4 °C
with 1 ml
cold fixative (50% methanol, 10% acetic acid and 40% PBS). Fixative was
aspired
off and replaced with 0.4 ml of 1 % SDS. The plates were placed on an orbital
shaker
for about 15 minutes or until the cells detached. The cell suspension was then
transferred to scintillation vials and 10 ml SCINTIVERSET"" BD scintillation
fluid was
added (SCINTIVERSET"~ is a trade-mark of Fisher Scientific Company L.L.C., of
New Hampshire). Vials were vortexed and placed in a beta counter (LKB 1219,
Rackbeta). By these assays intact hEGF1-48 had mitogenic activity comparable
to
that of hEGF1-53.
F. Purification Protocols at 250-Liter Scale
Human EGF-containing broth was separated from cells by centrifugation at a
3 LPM feed rate and 40 second
CA 02089143 2001-03-15
-31 -
shoot time (i.e., 40 second interval between discharges) in an Alfa-Laval
BTPX205
stacked disc, intermittent discharge, continuous centrifuge at
approximately13,000 x
g. The cell concentrate was diluted with deionized water to its original
volume and
centrifuged as before. The clarified broths from the two separations were
combined
and further clarified by centrifuging again at a 6 LPM feed rate with a 20
minute
shoot time.
Human EGF was removed from the resulting broth by step-wise addition of a
reverse phase resin that had been wetted in two volumes methanol (ml/g). Two
aliquots of 200 g each (300 g in Run 1 ), and subsequent aliquots of 300 g
each of
VYDACT"' 281TPB 15-20 were added to the broth, and the mix was stirred for 15
minutes after each addition (VYDACT"" is a trade-mark of The Separations
Group,
Inc., of California). Subsequent to each resin addition, the amount of unbound
EGF
remaining in the broth was measured by the shorter analytical HPLC procedure.
Additional aliquots of resin were added until less than 10% of the starting
EGF
remained unbound.
The resin was separated from the broth by pumping the resin-broth mixture
through a column (30-cm diameter, Amicon) with a 10,u mesh screen on the
bottom
support; the top screen was removed prior to the procedure. After the broth
was
passed through the column, the top screen was replaced, and the resin was
washed
with 0.05M acetic acid. The EGF was then eluted with two 4-liter aliquots of
38%
ethanol acidified with 3 ml/I glacial acetic acid. The eluate was decolored by
loading
an aliquot containing not more than 25 g EGF into a column containing six
liters of
CA 02089143 2001-03-15
-32-
MACROSORBT"" KAX-CM cation exchange resin equilibrated in 0.06 mM acetic acid
(MACROSORBT"~ is a trade-mark of Sterling Drug Inc., of New York). The EGF was
then eluted from the column with 12 liters of 0.3M ammonium acetate and the
column regenerated, as recommended by the manufacturer, with 1 M sodium
acetate
and 0.1 M sodium hydroxide before decolorizing additional aliquots.
Aliquots of the eluate from the cation exchange column containing not more
than 8 g EGF each were loaded onto a two-inch diameter radial compression
Waters
C18 column for preparative HPLC (Waters Delta prep, Model 3000). The column
was washed with a mixture of nine parts A and one part B (90% A, 10% B, the
same
composition as described for analytical HPLC); EGF was eluted in a 40-minute
linear
gradient, increasing B from 10% to 25%. Samples (40 ml) were collected from 15
minutes to 30 minutes, and EGF purity was assessed by analytical HPLC. Samples
were selected and pooled to give a final purity greater than 95%. To remove
acetonitrile, the pooled fractions were loaded onto a 6-liter cation exchange
column
(Macrosorb KAX-CM resin), and washed with 0.05M acetic acid until the
acetonitrile
concentration in the effluent was below 10 ppm, as determined by gas
chromatography. EGF was eluted with 0.3M ammonium acetate. The eluate was
filtered through a 0.2~ filter and lyophilized to a final moisture content of
8%. The
product obtained was pure non-nicked hEGF1-48.
The description shows in detail means for producing hEGF and especially
hEGF1-48 in pure non-nicked form.
WO 92/02246 ~ ~ PCT/US91/05396
- 33 -
The invention has been described in detail with
respect to particular embodiments thereof, but reasonable
variations and modifications, within the spirit and scope
of the present disclosure, are contemplated by the present
disclosure and the appended claims.
WO 92/02246 2 0 g 914 ~ PCT/US91/05396
- 34 -
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2~ Cohen, S. Epidermal growth factor. J. Invest.
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3~ Cohen, S, and G. Carpenter. Human epidermal growth
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1321 (1975).
4. Gregory, H. Isolation and structure of urogastrone and
its relationship to epidermal growth factor. Nature
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5. Gregory, H. and B.M. Preston. The primary structure of
human urogastrone. Int. J. Peptide Protein Res. 9:
107-118 (1977).
6. Gregory, H. and I.R. Willshire. The isolation of the
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1765-1774 (1975).
7. Carpenter, G. The regulation of cell proliferation:
Advances in the biology of action of epidermal growth
factor. J. Invest. Dermatol. 71: 283-288 (1978).
8. Hollenberg, M.D. Epidermal growth factor-urogastrone:
A polypeptide acquiring hormonal status. Vitamins
Hormones 37: 69-110 (1979).
