IE83398B1 - DNA encoding a growth factor specific for epithelial cells - Google Patents
DNA encoding a growth factor specific for epithelial cellsInfo
- Publication number
- IE83398B1 IE83398B1 IE1990/0358A IE35890A IE83398B1 IE 83398 B1 IE83398 B1 IE 83398B1 IE 1990/0358 A IE1990/0358 A IE 1990/0358A IE 35890 A IE35890 A IE 35890A IE 83398 B1 IE83398 B1 IE 83398B1
- Authority
- IE
- Ireland
- Prior art keywords
- kgf
- protein
- amino acid
- cells
- acid sequence
- Prior art date
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Description
PATENTS ACT,
358[9O
DNA ENCODING A GROWTH FACTOR SPECIFIC FOR EPITHELIAL
CELLS
Jeffrey S. RUBIN, Paul W. FINCH and Stuart A. AARONSON
FIELD OF THE INVENTION
The present invention relates to growth
factors, particularly to isolation of a
polypeptide growth factor similar to a family of
factors including known fibroblast growth factors
(FGFS). This invention also relates to
construction of complementary DNA (cDNA) segments
from messenger RNA (mRNA) encoding the novel
growth factor. Further, this inyention pertains
to synthesis of products of such DNA segments by
recombinant cells, and to the manufacture and use
of certain other novel products enabled by the
identification and cloning of DNAS encoding this
growth factor.
ABBREVIATIONS USED IN THIS APPLICATION
aFGF
bFGF
EGF
HSAC
KGF
Naoodso,/PAGE
RP-HPLC
TGFG
acidic fibroblast growth factor
basic fibroblast growth factor
epidermal growth factor
heparin-Sepharose affinity
chromatography
kilobases
kilodaltons
keratinocyte growth factor
Sodium dodecylsulphate (SDS)/
polyacrylamide gel electrophoresis
reversed-phase high performance
liquid chromatography
transforming growth factor a
EACKGROUND OE Iflfl INVENTIOE
Growth factors are important mediators
of intercellular communication. These potent
molecules are generally released by one cell type
and act to influence proliferation of other cell
types (see reference I-1 in Experimental Section
I, below). Interest in growth factors has been
heightened by evidence of their potential
involvement in neoplasia (reference II-2 in
Experimental Section II, below). The v-us
transforming gene of simian sarcoma virus encodes
a protein that is homologous to the 8 chain of
platelet-derived growth factor (I-1, I-2).
Moreover, a number of oncogenes are homologues of
genes encoding growth factor receptors (I-1).
Thus, increased understanding of growth factors
and their receptor-mediated signal transduction
pathways is likely to provide insights into
mechanisms of both normal and malignant cell
growth.
one known family of growth factors
affecting connective tissue cells includes acidic
fibroblast growth factor (aFGF), basic fibroblast
growth factor (bPBF), and the related products of
the hst, FGF—5 and int—2 oncogenes.
Further, it is known that some growth
factors, including the following, have heparin-
binding properties: aFGF (I-20, I-21): bFGF (I-
19, I-20): granulocyte/macrophage colony
stimulating factor (I-1); and interleukin 3 (I-
1). Each of these polypeptide factors is
produced by stromal cells (I-1, I-2, I-25). Such
factors appear to be deposited in the
extracellular matrix, or on proteoglycans coating
the stromal cell surface (I-1, I-25). It has
been postulated that their storage, release and
contact with specific target cells are regulated
by this interaction (I-25, I-28).
It is widely recognized, however, that
the vast majority of human malignancies are
derived from epithelial tissues (I-S). Effectors
of epithelial cell proliferation derived from
mesenchymal cells have been described (I-1, I-
, I-3), however, their molecular identities and
structures have not been elucidated.
In light of this dearth of knowledge
about such mesenchymal growth factors affecting
epithelial cells, it is apparent that there has
been a need for methods and compositions and
bioassays which would provide an improved
knowledge and analysis of mechanisms of
regulation of epithelial cell proliferation, and,
ultimately, a need for novel diagnostics and
therapies based on the factors involved therein.
This invention contemplates the
application of methods of protein isolation and
recombinant DNA technologies to fulfill such
needs and to develop means for producing protein
factors of mesenchymal origin, which appear to be
related to epithelial cell proliferation
processes and which could not be produced
otherwise. This invention also contemplates the
application of the molecular mechanisms of these
factors related to epithelial cell growth
processes .
SUMMER! OE EH3 INVENTLQE
The present invention relates to
developments of protein isolation and recombinant
DNA technologies, which include production of
novel growth factor proteins affecting epithelial
cells, free of other peptide factors. Novel DNA
segments and bioassay methods are also included.
The present invention in particular
relates to a novel protein having structural
and/or functional characteristics of a known
family of growth factors which includes acidic
fibroblast growth factor (aFGF), basic fibroblast
growth factor (bFBF) and the related products of
the hst, FGF—5 and int—2 oncogenes. This new member of the
FGF polypeptide family retains the heparin-
binding properties of the FGFs but has evolved a
unique target cell specificity. This growth
factor appears to be specific for epithelial
cells and is particularly active“on
keratinocytes. Therefore, this novel factor has
been designated "keratinocyte growth factor"
(KGF). Notwithstanding its lack of activity on
fibroblasts, since it is the sixth known member
of the FGF polypeptide family, KG? may also be
referred to as FGP-6.
According to the present invention the term "KGF or KGF-like proteins“ means an
isolated glycosylated or unglycosylated keratinocyte growth factor (KGF) protein
comprising: (a) the following amino acid sequence, (b) a portion of the following amino
acid sequence without the N-terminal 31 amino acids, or (c) an amino acid sequence that
differs by the addition, deletion, or substitution of one or more amino acids from the
following amino acid sequence:
MHKWILTWILPTLLYRSCFHIICL
VGTISLACNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYN|ME|RTVAVGlVAlKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAlT
wherein said protein is capable of stimulating DNA synthesis in quiescent BALB/MK
epidermal keratinocytes by more than 500-fold while lacking mitogenic activity on
fibroblasts and endothelial cells.
In a preferred embodiment 5 nM of said protein exhibits less than one-fold stimulation
over background in NIH/3T3 fibroblasts.
in a still preferred embodiment the KGF protein has a specific activity of at least about
3.4 x 104 units per milligram of protein, where one unit of activity is defined as that
amount which causes half of the maximal possible stimulation of DNA synthesis in
BALB/MK keratinocyte cells.
In a still preferred embodiment the KGF protein is glycosylated.
in a still preferred embodiment the KGF protein is not glycosylated.
In a still preferred embodiment the KGF protein comprises the amino acid sequence:
CNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYNIMElRTV'AVG|VAlKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAIT.
in a still preferred embodiment the KGF protein comprises an amino acid sequence
differing by the addition, deletion, or substitution of one or more amino acids from the
following amino acid sequence:
CNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYNIMEIRTVAVGIVAIKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAIT
In a still preferred embodiment the KGF protein comprises an amino acid sequence
differing by the addition, deletion or substitution of one or more amino acids from the
following amino acid sequence:
MHKWILTWILPTLLYRSCFHHCL
VGTISLACNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYNIMEIRTVAVGIVAIKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGlPVRGKKTKKEQKTA
HFLPMA|T.
In a still preferred embodiment the KGF protein comprises a segment of amino acids of
Figure ll-1 B which comprises amino acids 32 to 78 of Figure ll-1 B to confer on the KGF
protein preferential mitogenic activity on an epithelial cell.
In a still preferred embodiment the KGF protein comprises a segment of the following
amino acid sequence:
MHKWILTWILPTLLYRSCFHIICL
VGTISLACNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYNIMEIRTVAVGIVAIKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAIT.
in a still preferred embodiment the KGF protein consists of a segment of the following
amino acid sequence:
MHKWILTWILPTLLYRSCFHHCL
VGTISLACNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRIDKRGKVKGTQEMKN
NYNIMEIRTVAVGIVAIKGVESEF
YLAMNKEGKLYAKKECNEDCNFK
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAIT.
in a still preferred embodiment the isolated glycosylated or unglycosylated keratinocyte
growth factor (KGF) protein consists of a segment of the following amino acid sequence:
CNDMTPEQMATNVNCS
SPERHTRSYDYMEGGDIRVRRLF
CRTQWYLRlDKRGKVKGTQEMKN
NYN|MElRTVAVG|VAl‘KGVESEF
YLAMNKEGKLYAKKECNEDCNF-"K
ELILENHYNTYASAKWTHNGGEM
FVALNQKGIPVRGKKTKKEQKTA
HFLPMAlT,
wherein said segment has mitogenic activity for a keratinocyte cell, and wherein said
segment may further include methionine at the amino terminus.
In a still preferred embodiment the KGF protein has a specific activity of at least about
3.4 x 10“ units per milligram of protein, where one unit of activity is defined as that
amount which causes half of the maximal possible stimulation of DNA synthesis in
BALB/MK keratinocyte cells.
In a still preferred embodiment the KGF protein comprises Met at the amino terminus.
Accordingly, this-invention relates, in part, to purified KGF or KGF-like proteins and
methods for preparing these proteins. Such
purified factors may be made by cultivation of
human cells which naturally secrete these
proteins and application of isolation methods
according to the practice of this invention.
These proteins can be used for biochemical and
biological studies leading, for example, to
isolation of DNA segments encoding KGF or KG?-
like polypeptides.
The present invention also relates to
such DNA segments which encode KGF or KGF-like
proteins. In a principal embodiment, the present
invention relates to DNA segments, which encode
KGF-related products, consisting of: human cDNA
clones 32 or 49, derived from polyadenylated RNA
extracted from the human embryonic lung
fibroblast cell line M426: recombinants and
mutants of these clones; and related DNA segments
which can be detected by hybridization to any of
the above human DNA segments, which related
segments encode KGF-like proteins or portions
thereof.
In the practice of one embodiment of
this invention, the DNA segments of the invention
are capable of being expressed in suitable host
cells, thereby producing KGF or KG?-like
proteins. The invention also relates to mRNAs
produced as the result of transcription of the
sense strands of the DNA segments of this
invention.
In another embodiment, the invention
relates to a recombinant DNA molecule comprising
a vector and a DNA of the present invention.
These recombinant molecules are exemplified by
molecules comprising a KGF CDNA and any of the
following vector DNAs: a bacteriophage A cloning
vector (exemplified by ApCEV9); a DNA sequencing
plasmid vector (e.g., a pUc variant); a bacterial
gene expression vector (e.g., pKK233-2): or a
mammalian gene expression vector (such as pMMT).
I In still another embodiment, the
invention comprises a cell, preferably a
mammalian cell, comprising a vector with a DNA of the
invention. Further, the invention comprises
cells, including insect cells, yeast cells and
bacterial cells such as those of Escherichia coli and
B. subtilis, comprising a vector with a DNAs of the invention.
According to another embodiment of this aspect of
the invention, the transforming DNA is capable of
being expressed in the cell, thereby increasing
in the cell the amount of KGF or KGF-like protein
encoded by this DNA.
The primary KGF translation product
predicted from its cDNA sequence contains an
N-terminal hydrophobic region which likely serves
as a signal sequence for secretion and which is
not present in the mature KGF molecule. In a
most preferred embodiment of the gene expression
aspect of the invention, the cell transformed by
the DNA of the invention secretes the protein
encoded by that DNA in the (truncated) form that
is secreted by human embryonic lung fibroblast
cells.
Still further, this invention
contemplates KGF or KGF-like proteins produced by
expression of a DNA of the invention, or by
translation of an RNA of the invention.
Preferably, these proteins will be of the
secreted form (i.e., lacking an apparent signal
sequence). These protein factors can be used for
functional studies, and can be purified for
additional structural and functional analyses,
such as qualitative and quantitative receptor
binding assays.
Moreover, the ability to produce large
quantities of this novel growth factor by
recombinant techniques will allow testing of its
clinical applicability in situations where
specific stimulation of growth of epithelial
cells is of particular importance. Accordingly,
this invention includes pharmaceutical
compositions comprising KGF or KGF-like
polypeptides for use in the treatment of such
conditions, including, for example, healing of
wounds due to burns or stimulation of
transplanted corneal tissue.
According to this embodiment of the
invention, the novel KGF-like proteins will be
protein products of "unmodified" DNAs and mRNAs
of the invention, or will be modified or
genetically engineered protein products. As a
result of engineered mutations in the DNA
sequences, modified KGF-like proteins will have
one or more differences in amino acid sequence
from the corresponding naturally occurring "wild-
type" proteins. According to one embodiment of
this aspect of this invention, the modified KGF-
like proteins will include "chimeric" molecules
comprising a KGF and at least one other member of
the FGF peptide family.
Ultimately, given results of analogous
successful approaches with other peptide factors
having similar properties, development of such
chimeric KGF-like polypeptides should lead to
superior, "second generation" forms of KGF-like
peptides for clinical purposes. These modified
KGF-like products might be smaller, more stable,
more potent, and/or easier or less expensive to
produce, for example.
According to another embodiment an invitro method of stimulating epithelial cells is
provided comprising adding an amount of the composition comprising KGF protein
or chimeric protein sufficient to stimulate epithelial cell growth.
This invention further comprises novel
bioassay methods for determining expression in
human cells of the mRNAs and proteins produced
from the genes related to DNA segments of the
invention. According to one such embodiment,
DNAs of this invention may be used as probes to
determine steady state levels or kinetics of
induction of related mRNAs. The availability of
the KGF-related CDNA clones makes it possible to
determine whether abnormal expression of this
growth factor is involved in clinical conditions
characterized by excessive epithelial cell
growth, including dysplasia and neoplasia (e.g.,
psoriasis, or malignant or benign epithelial
tumors).
This invention also contemplates novel
antibodies made against a peptide encoded by a
DNA segment of the invention. In this embodiment
of the invention, the antibodies are monoclonal
or polyclonal in origin, and are generated using
KGF-related polypeptides from natural,
recombinant or synthetic chemistry sources.
The antibodies of this invention bind
specifically to KGF or a KGF-like protein which
includes the sequence of such peptide, preferably
when that protein is in its native (biologically
These antibodies can be
active) conformation.
used for detection or purification of the KGF or
KG? or KGF-like protein
factors. In a most preferred embodiment of this
aspect of the invention, the antibodies will
neutralize the growth promoting activity of KGF,
thereby enabling mechanistic studies and,
ultimately, therapy for clinical conditions
involving excessive levels of KGF.
BRIEF DESCRIPTION or THE DRAWINGS
Fig. 1-1 depicts results of heparin-
Sepharose affinity chromatography of conditioned
medium from M426 human embryonic fibroblasts
showing that greater than‘90% of the mitogenic
activity for mouse keratinocytes (BALE/MK) eluted
with O_6 M Nacl. Approximately 150
ml of ultrafiltration retentate derived from
liters of H426 conditioned medium were loaded
onto a heparin-Sepharose column (6 ml bed
volume) in 1 hr. After washing the column with
150 ml of the equilibration buffer, 20 mM
Tris—HCl, pH 7.5/0.3! Nacl, the retained protein
(<5% of the total protein in the retentate) was
eluted with a modified linear gradient of
increasing Nacl concentration. Fraction size
was 3.8 ml and flow rate during gradient elutiom
was 108 ml/hr. Two pl of the indicated
fractions were transferred to microtiter wells
containing a final volume of 0.2 ml for assay of
3H—thymidine incorporation in BALE/MK cells as
described in the Hethods.
U.)
C
Fig. I-2 illustrates results of further
purification of the mitogen from human
fibroblasts using HPLC with an adsorptive
matrix. Panel (A) shows the profile on reversed-
phase‘(Cg,HPLC of BALE/MK mitogenic activity.
Panel (B) presents electrophoretic (Nabodsop/PAGE)
analysis of selected fractions from the C‘
chromatography shown in panel A, demonstrating
that the:peak HPLC fractions contained a single
band on the silver stained gel- Panel (C) is a‘
bar graph of DNA synthesis in BALE/MK cells
triggered by theéfractions analyzed in Panel B,
showihg that the relative mitogenic activity
correlated well with the.intensity of the protein
band across the activity profile.
(A) Reversed-phasetqHPLC of BALE/MK
mitogenic activity. Active fractions eluted
from heparin-Sepharose with O—6H Nacl were
processed with the Centricon -10 and loaded
directly onto a C‘ Vydac column (4.6 x 250 mm)
which had been equilibrated in 0.1%
trifludroacetic acid/20% acetonitrile (ACN).
After washing the column with 4 ml of
equilibration buffer, the sample was eluted with
a modified linear gradient of increasing % ACN.
Fraction size was 0.2 ml and flow rate was 0.5
ml/min. Aliquots for the assay ot'3H-thymidine
incorporation in BALE/MK cells were promptly
diluted loefold with 50 pg/ml bovine serum
albumin/20 mh Tris—HCl, pH 7.5, and tested at a
final dilution of 200-fold. (B) NaDodSOb/PAGE
analysis or selected fractions from the C‘
chromatography shown in panel A. Half of each
fraction was dried, redissolved in NaDodSOb/2-
mercaptoethanol, heat denatured and
electrophoresed in a 14% polyacrylamide gel
which was subsequently stained with silver, The
position of each molecular weight marker (mass
in kDa) is indicated by an arrow. (C) DNA
synthesis in BALE/MK cells triggered by the
fractions analyzed in Panel 8. Activity is
expressed as the fold stimulation over
background which was 100 cpm.
Fig. I-3 presents an alternative
purification step to RP-HPLC, using sieving
kchromatography with a (TSK G3000SW GlasPac)
column run in aqueous solution near physiologic
pH, which resulted in a major peak of mitogenic
activity in the _aALB/MK bibassay.
Holecular sieging HPLC (TSK sooosw)
chromatography of the BALE/MK mitogenic
activityl Approximately 50 pl of a Centricon—
processed, 0.6M Nacl pool from HSAC were loaded
onto a LKB GiasPac TSK GJOOOSW column (8 x 300
mm), previously equilibrated in 20 mM Tris—HCl,
pa 6.8/0.5M Nacl, and eluted as 0.2 ml fractions
at a flow rate of 0.4 ml/min. Aliquots of 2 pl
were transferred to microtiter wells containing
a final volume of 0.2 ml for assay of 3H-
thymidine incorporation in BALE/MK cells. The
elution positions of molecular weight markers
(mass in kDa) were as indicated by the arrows.
. , 2()
Fig. TF4 illustrates a comparis n of
BALE/MK DNA synthesis in response to TSK-purified
mitogen and other growth factors
C°mpari5°“ Of BALE/MK DNA synthesis
in response to TSK—purified mitogen and othe»
growth factors. Incorporation of 3H—thymidine
into trichloracetic acid—insoluble DNA
expressed as fold stimulation over background,
was measured as a function of the concentration
of the indicated growth factors. Background
values with no sample added were 150 cpm_ The
results represent mean values of two independent
experiments. Replicates in each experiment were
within 10% of mean values. TSK—purified
—mitogen, - -; Bar, A
A; aFGF, -
bFGF, o---o.
Fig. I-5 shows comparisons of growth of
BALE/MK cells in a chemically defined.medium in
response to different combinations of growth
factors;
Comparative growth of BALE/MK cells
in a chemically defined medium in response to
different combinations of growth factors_
Cultures were plated at a density of 2.5xl0‘
cells per dish on 35 mm Petri dishes precoated
with poly—D-lysine/fibronectin in a 1:1 mixture
of Eagle‘s minimal essential medium and Hamvs
F12 medium supplemented with transferrin,
Nazseds, ethanolamine and the growth factors
indicated below. After 10 days, the plates were
fixed and stained with Giemsa. Key; a) no
growth factor: b) EGF alone: c) insulin alone;
}
d) KGF alone; e) £6? + insulin. Final
concentrations of the growth factors were as
and
follows: EGF, 20 ng/ml: insulin, 10 pg/ml;
KGF, 40 ng/ml.
Table I-1 summarizes the results from
various purification steps, documenting that
sieving chromatography provided a far better
recovery of activity than the adsorptive RP—HPLC
approach.
Recoveries were calculated by assuming that'all
of the mitogenic activity in the starting
material was due to the isolated factor.
‘One unit of activity is defined as half of the
maximal stimulation of thymidine incorporation
induced by TSK-purified factor in the BALE/MK
bioassay, in which approximately 3 ng of the
TSK-purified factor stimulated 1 unit of
activity.
‘Protein was estimated by using the Bradford
reagent from BioRad (I-23).
b Protein was estimated by using Aéfl = 140.
Table I-Ztrecapitulates data on the
target‘cell:specificities;ofivarioussgrowth"
factors, demonstrating that;the;newly;isolated
factor exhibited.a strong mitogenic effect on
keratinocytes (BALE/HK) and, in striking
contrast,thad:no:detectable“effects.on
fibroblasts or human saphenous vein endothelial
cells.
:12
Comparison of marimal thymidine incorporation
stimulated by K5? andaother growth
factors in a variety of cell lines, expressed as
fold stimulation over background.
This data represents a summary or four different
experiments.
‘Maximal stimulation by aFGF required the
presence of heparin (Sigma), 20 ug/ml.
n.d. - not determined.
Fig. II:l_presents the nucleotide
sequence and deduced amino acid sequence of KG?
cDNA, as well as identification of RNAS
transcribed from the KGF gene. Panel (A)
outlines a schematic representation of human KGF
CDNA clones. Panel (B) documents the KGF cDNAi
nucleotide and predicted amino acid sequences.
(C) Identification of RNA transcripts of Kc?
genes by Northern blot analysis.
V Nucleotide sequence and deduced
amino acid sequence of KGF CDNA, and
identification of KGF gene transcripts. (A)
Schematic representation of human KG? CDNA
clones. overlapping pCEV9 clones 32 and 49,
used in sequence determination, are shown above
a diagram of the complete structure in which
untranslated regions are depicted by a line and
the coding sequence is boxed. The hatched
region denotes sequences of the signal peptide
and the open region of the mature protein.
Selected restriction sites are indicated. (B)
KG? CDNA nucleotide and predicted amino acid
sequences. Nucleotides are numbered on the
right; amino acids are numbered throughout. The
N-terminal peptide sequence derived from
purified KGF is underlined. The hydrophobic N-
terminal domain is italicized. The potential
asparagine-linked glycosylation site is
overlined. The variant polyadenylation signals,
AATTAA and AATACA, close to the 3' end of the
RNA, are boxed. (C) Identification of KG? mRNAs
by Northern blot analysis. Lanes a and c,
poly(A)-selected H426 RNA: lane d, total
cellular H426 RNA. Filters were hybridized with
a VP-labeled 595 hp BamHI/Bdl fragment from
clone 32 (Probe A, Fig. II-lA), lanes a and h,
or a :41 bp Apal/EwRI fragment from cione 49
(Probe B, Fig. II-lA), lanes c and a.
Fig. II-2 illustrates the topological
comparison of the FGF family of related
molecules, including KGF, with emphasis on the
two protein domains that share high homology, the
putative signal peptide sequences, and the two
conserved cysteine residues.
Topological comparison of the FGF
family of related molecules. The two protein
domains that share high homology are shown by
shaded boxes. Hatched boxes indicate putative
signal peptide sequences. The positions of two
conserved cysteine residues (C) are shown.
Fig. II-3 shows (Northern blot) analyses
of expression of KGF—related‘mRNA in selected
normal human cell lines and tissues, revealing
that a single 2.4 kb transcript was present in
RNA from human embryonic lung fibroblasts and
from adult skin fibroblasts; while no transcript
was detected in the (B5/589) epithelial or (HA83)
glial cell lines; or in primary cultures of human
saphenous vein endothelial cells.
Northern blot analysis Of xcy mRNA
in normal human cell lines and tissues, and
comparison with mRNA expression of other growth
factors with known activity on epithelial cells,
Total cellular RNAS were isolated by cesium
trifluoro-acetate gradient centrifugation. l0
pg of RNA were denatured and electrophoresed in
1% formaldehyde gels. Following mild alkali
denaturation (50 mh NaOH for 30?), Rxa was
transferred to nitrocellulose filters using l_M
ammonium acetate as a convectant. Filters were
hybridized to a "2:-iabeiiea CDNA probe
containing the 647bp EwRI fragment from the 5'
end of the KG? coding sequence (A) or similar
probes from the other growth factor DNAs. The
following human cell types were used: squamous
cell carcinomas (A253, A388 and A431); mammary
epithelial cells (56 and R1); keratinocytes
immortalized with Adlz-SV40: primary human
keratinocytes: neonatal foreskin fibroblasts,
AGl523i adult skin fibroblasts (50lT); and
embryonic lung fibroblasts (WI-38 and H426).
Table II-l summarizes a comparison of
the effect of heparin on KGF mitogenic activity
with effects on other growth factors, showing
that thymidine incorporation into DNA by BALB/MK
cells in response to K¢F was inhibited by
heparin, in contrast, to the activities of both
_aFGF and bFGF which were increased by the same
treatment.
Cells were plated in microtiter plates,
grown to confluence in serum containing media
and then placed in a serum—free medium for 24-72
hr prior to sample addition. Mitogenesis assays
were performed as described (see Experimental
Section I, above and II-3). Where indicated,
heparin was included in the culture media at a
final concentration of 20 pg/ml. The
concentration of all the growth factors was 50
ng/ml. The results represent fold stimulation
of 3H-thymidine incorporation in the indicated
assay cell in the presence (+) or absence (-) of
heparin. Each value represents the mean result
from two independent experiments in which each
point, in turn, represents the mean value of
duplicate analyses.
:28
pH, which resulted in a major peak of mitogenic
activity in the BALB/MK bioassay.
Fig. I-4 illustrates a comparison of
BALB/MK DNA synthesis in response to TSK-purified
mitogen and other growth factors.
Fig. I-5 shows comparisons of growth of
BALB/MK cells in a chemically defined medium in
response to different combinations of growth
factors.
Table I-1 summarizes the results from
various purification steps, documenting that
sieving chromatography provided a far better
recovery of activity than the adsorptive RP-HPLC
approach.
Table I-2 recapitulates data on the
target cell specificities of various growth
factors, demonstrating that the newly isolated
factor exhibited a strong mitogenic effect on
keratinocytes (BALB/MK) and, in striking
contrast, had no detectable effects on
fibroblasts or human saphenous vein endothelial
cells.
Fig. II-1 presents the nucleotide
sequence and deduced amino acid sequence of KGF
CDNA, as well as identification of RNAs
transcribed from the KGF gene. Panel (A)
outlines a schematic representation of human KGP
cDNA clones. Panel (8) documents the KGF CDNA
nucleotide and predicted amino acid sequences.
(C) Identification of RNA transcripts of KGF
genes by Northern blot analysis.
Fig. II-2 illustrates the topological
comparison of the FGF family of related
molecules, including KGF, with emphasis on the
two protein domains that share high homology, the
putative signal peptide sequences, and the two
conserved cysteine residues.
Pig. II-3 shows (Northern blot) analyses
of expression of KGF—related mRNA in selected
normal human cell lines and tissues, revealing
that the predominant 2.4 kb transcript was
present in RNA from stromal fibroblasts
but was not detected in epithelial cell
lines.
Table II-1 summarizes a comparison of
the effect of heparin on KGF mitogenic activity
with effects on other growth factors, showing
- that thymidine incorporation into DNA by BALE/MK
cells in response to KGF was inhibited by
heparin, in contrast, to the activities of both
aFGF and bFGF which were increased by the same
treatment.
DESCRIPTION OF SPECIFIQ EMBODIMENTS
This invention relates, in part, to
purified KGF or KGF-like proteins as defined above and methods for
preparing these proteins. A principal embodiment
of this aspect of this invention relates to
homogeneous KGF characterized by an apparent
molecular weight of about 28 kDa based on
migration in NaDodS0,/PAGE, movement as a single
peak on reversed-phase high performance liquid
chromatography, and a specific activity of at
least about 3.4 x 10‘\ufits per milligram, and
preferably at least about 3.2 x 105 units.per
milligram, where one unit of activity is defined
as that amount which causes half of the maximal
possible stimulation of DNA synthesis in certain
epithelial (keratinocyte) cells under standard
assay conditions outlined below.
To identify novel growth factors
specific for epithelial cell types, a clonal
BALB/c mouse keratinocyte cell line, designated
BALE/MK (I-6) was employed as an indicator cell
to detect such factors. These cells are
dependent for their growth upon an exogenous
source of an epithelial cell mitogen even in
medium containing serum (I-6). The development
of chemically defined medium for these cells has
made it possible to demonstrate that two major
mitogenic pathways are required for BALB/MK
proliferation. One involves insulin-like growth
factor I (or insulin at high concentration) and
the other is satisfied by epidermal growth factor
(EGF), transforming growth factor a (TGFa),
acidic fibroblast growth factor (aFGF) or basic
fibroblast growth factor (bFBF) (I-7).
By using BALE/MK as the prototypical
epithelial cell line and NIH/3T3 as its
fibroblast counterpart, conditioned media from
various human cell lines were assayed for new
epithelial cell-specific mitogens. These
bioassays of this invention enabled the
purification to homogeneity of one such novel
growth factor, released by a human embryonic lung
fibroblast line, and designated herein as
keratinocyte growth factor (KGF).
In brief, the bioassay for KGF-like
activity under standard conditions comprises the
following steps:
(i) Mouse keratinocytes (BALE/MK cells) are
grown in culture to confluency and then
maintained for 24-72 hr in serum-free medium;
(ii) Following addition of test samples,
stimulation of DNA synthesis is determined by
incorporation of Rbthymidine into acid-
precipitable DNA.
To determine the target cell specificity
of a mitogenic growth factor, the DNA synthesis
stimulation, expressed as ratio of stimulated
synthesis over background incorporation of
thymidine in the absence of added test sample,
can be compared to analogous stimulation observed
in cells other than keratinocytes under the same
assay conditions. In such comparisons, KGF
mitogenic activity will exhibit marked
specificity for the keratinocytes as opposed to
fibroblasts (at least about 500-fold greater
stimulation) and lesser but significant (at least
about sq-fold) greater activity on keratinocytes
than on other exemplary epithelial cell types
(see Table I-2 for further data, and Materials
and Methods in Experimental Section I for details
of the standard conditions of the bioassay).
By employing a method of KGF production
involving culturing cells and isolating mitogenic
activity, which method comprises ultrafiltration,
heparin-sepharose affinity chromatography (HSAC)
and adsorptive reversed-phase high performance
liquid chromatography (RP-HPLC) or,
alternatively, molecular sieving HPLC (TSK-HPLC),
according to the present invention, a quantity
was isolated sufficient to permit detailed
characterization of the physical and biological
properties of this molecule.
To summarize, the method for production
of KGF from producing cells such as M426 human
embryonic fibroblasts (I-8), for example,
comprises the following steps:
(i) Preparation of conditioned media (e.g.,
liters) using monolayer cultures cycled from
serum-containing to serum-free medium and storing
the serum-free harvest at -70'C until further
use:
(ii) Concentration by ultrafiltration using
membranes having a 10 kDa molecular weight cutoff
in several successive steps with intervening
dilution in buffer (to facilitate removal of low
molecular weight materials), followed by optional
storage at -70’C;
(iii) Affinity chromatography on heparin
attached to a polymeric support (e.g., sepharose)
with elution by a gradient of increasing Nacl
concentration:
(iv) Concentration by a factor of at least
ten- to twenty-fold with small scale
ultrafiltration devices with a 10 kDa molecular
weight cutoff (e.g., a Centricon-10
microconcentrator from Amicon) and storage at
-70'C.
The next step of the purification
process comprises either step (v) or,
alternatively, step (vi), as follows:
(v) Reversed-phase HPLC of active fractions
(0.6 M Nacl pool) from the previous HSAC step in
organic solvent systems;
or,
(vi) Molecular sieve HPLC (e.g, on a TSK—
csooosw Glas-Pac Column from LKB) in aqueous
buffer at near physiological pH (e.g., Tris-Hcl,
pH 6.8/0.5M Nacl) followed by storage at -70‘C.
A preparation made by the TSK step (vi)
was almost as pure as one obtained from RP-HPLC,
as judged by silver-stained Nanodsob/PAGE (data
not shown); but the TSK approach provided a far
better recovery of activity (Table I-1).
Further, the TSK-purified material had a higher
specific activity than the RP-HPLC material. KGF
prepared by the TSK procedure above stimulated
DNA synthesis in epithelial cells at sub-
nanomolar concentrations, but failed to induce
any thymidine incorporation into DNA of
fibroblasts or endothelial cells at comparable or
higher concentrations (up to 5 nu). The activity
was sensitive to acid, heat and solvents used in
the RP-HPLC step. (See Experimental Section I for
data on sensitivities and further details of the
production method.)
Using standard methodology well known in
the art, an unambiguous amino acid sequence was
determined for positions 2-13 from the amino
terminus of the purified KGF, as follows: Ash-
Asp-Met—Thr—Pro-G1u-Gln-Met-Ala—Thr—Asn-Val (see
Experimental Section I).
The present invention also includes DNA
segments encoding KGF and KGF-like polypeptides.
The DNAs of this invention are exemplified by
DNAs referred to herein as: human cDNA clones 32
and 49 derived from polyadenylated RNA extracted
from the human embryonic lung fibroblast cell
line M426} recombinants and mutants of these
clones; and related DNA segments which can be
detected by hybridization to these DNA segments.
As described in Experimental Section II,
to search for cDNA clones corresponding to the
known portion of the KGF amino acid sequence, two
pools of oligonucleotide probes were generated
based upon all possible nucleotide sequences
encoding the nine-amino acid sequence,
Asn-Asp-Met-Thr-Pro-Glu-G1n-Met-Ala. A cDNA
library was constructed in a CDNA cloning vector,
ApCEV9, using polyadenylated RNA extracted from
the human embryonic lung fibroblast cell line
M426 which was the initial source of the growth
factor. Screening of the library (9 x 105
plaques) with the “P-labelled oligonucleotides
identified 88 plaques which hybridized to both
probes.
Of 10 plaque-purified clones that were
analyzed, one, designated clone 49, had a cDNA
insert of 3.5 kb, while the rest had inserts
ranging from 1.8 kb to 2.1 kb. Analysis of the
smaller clones revealed several common
restriction sites, and sequencing of a
representative smaller clone, designated clone
32, along with clone 49, demonstrated that they
were overlapping cDNAs (Fig II-1A). Alignment of
the two cDNAs established a continuous sequence
of 3.85 kb containing the complete KGF coding
sequence. The sense strand DNA nucleotide
sequence, and the predicted primary protein
sequence encoded, are shown for the full-length
composite KGF CDNA sequence in Fig. II-1B.
These DNAs, cDNA clones 32 and 49, as
well as recombinant forms of these segments
comprising the complete KGF coding sequence, are
most preferred DNAS of this invention.
From the CDNA sequence, it is apparent
that the primary KGF, FGF—5 and hst translation products
contain hydrophobic N-terminal regions which
likely serve as signal sequences, based on
similarity to such sequences in a variety of
other proteins. Accordingly, this N-terminal
domain is not present in the purified mature KGF
molecule which is secreted by human embryonic
fibroblasts.
Furthermore, KG? shares with all other
members of the FGF family two major regions of
homology, spanning amino acids 65-156 and 162-189
in the predicted KGF sequence, which are
separated by short, nonhomologous series of amino
acids of various lengths in the different family
members. The sequence of the purified form of
KGF contains five cysteine residues, two of which
are conserved throughout the family of FGF
related proteins. Five pairs of basic‘residues
occur throughout the KGF sequence. This same
pattern has been observed in other FGP family
members.
It should be obvious to one skilled in
the art that, by using the DNAs and RNAs of this
invention in hybridization methods (such as
Southern blot analyses of genomic human DNAs),
especially the most preferred DNAs listed herein
above, without undue experimentation, it is
possible to screen genomic or CDNA libraries to
find other KGF-like DNAs which fall within the
scope of this invention. Furthermore, by so
using DNAS of this invention, genetic markers
associated with the KGF gene, such as restriction
fragment length polymorphisms (RFLPs), may be
identified and associated with inherited clinical
conditions involving this or other nearby genes.
This invention also includes modified
forms of KGF DNAS. According to a chief
embodiment of this aspect of the invention, such
modified DNAS KGF—like
encode proteins
comprising a KGF protein and at
least one other member of the FGF peptide family.
Thus, for example, since there is no significant
N-terminal homology between the secreted form of
KGF and analogous positions in other FGF-related
proteins, polypeptides with novel structural and
functional properties may be created by grafting
DNA segments encoding the distinct N-terminal
segments of another polypeptide in the FGF family
onto a KGF DNA segment in place of its usual N-
terminal sequence. .
The polypeptide chimeras produced by
such modified DNAs are useful for determining
whether the KGF NH:-terminal domain is sufficient
to account for its unique target cell
specificity. Studies on chimeras should also
provide insights into which domains contribute
the different effects of heparin on their
biologic activities.
Indeed, the utility of this approach has
already been confirmed by the successful
engineering and expression of a chimeric molecule
in which 46 amino acids from the N-
terminus of the secreted form of KG? (beginning
with the amino terminal cys residue of the mature
KGF form, numbered 32 in Fig II-1, and ending at
KGF residue 78, arg) is linked to 116 amino
acids of the C—terminal core of aFGF (beginning
at residue 39, arg; and continuing to the C-
terminal end of the aFGF coding sequence. This
chimeric product has a target cell preference for
keratinocytes, like KGF, but its activity is enhanced
by heparin, a characteristic which parallels that
of aFGF rather than KGF. This noyel XGF—like
growth factor may have advantages in clinical
applications where administration of an
‘epithelial-specific growth factor is desirable in
the presence of heparin, a commonly used
anticoagulant. Further details of the
construction of this chimeric molecule and the
properties of the polypeptide are described in
Experimental Section II.
other DNAs of this invention include the
following recombinant DNA molecules comprising a
KGF cDNA and any of the following exemplary
vector DNAS: a bacteriophage A cloning vector
(ApCEV9); a DNA sequencing plasmid vector (a puc
variant); a bacterial expression vector (pKK233-
); or a mammalian expression vector (pMmT/neo).
Such recombinant DNAs are exemplified by
constructs described in detail in the
Experimental Sections.
Most preferred recombinant molecules
include the following: molecules comprising the
coding sequence for the secreted form of KGF and
a bacterial expression vector (e.g., pKK233-2) or
a cDNA encoding the entire primary translation
product (including the NH:-terminal signal
peptide) and a mammalian expression vector
(exemplified by pMMT) capable of expressing
inserted DNAs in mammalian (e.g., NIH/3T3) cells.
Construction of recombinant DNAs
containing KGF DNA and a bacterial expression
vector is described in Experimental Section II.
In brief, KGF cDNA was expressed to produce
polypeptide in E.coli by placing its coding
sequence under control of the hybrid :23 promoter
in the plasmid expression vector pKK233-2 (II-
31).
Construction of recombinant DNAs
comprising KGF DNA and a mammalian vector capable
of expressing inserted DNA: in cultured human or
animal cells, can be carried out by standard gene
expression technology using methods well known in
the art for expression of such a relatively
simple polypeptide. one specific embodiment of a
recombinant DNA of this aspect of the present
invention, involving the mammalian vector pMMT,
is described further below in this section under
recombinant cells of this invention.
DNAs and sense strand RNAs of this
invention can be employed, in conjunction with
protein production methods of this invention, to
make large quantities of substantially pure KGF
or KGF-like proteins. Substantially pure KG?
protein thus produced can be employed, using
well-known techniques, in diagnostic assays to
determine the presence of receptors for this
protein in various body fluids and tissue
samples.
Accordingly, this invention also
comprises a cell, preferably a bacterial or
mammalian cell, comprising a vector with a
DNA of the invention, wherein the DNA is
capable of being expressed. In a preferred
embodiment of this aspect of the invention, the
cell transformed by the DNA of the invention
produces KGP protein in a fully mitogenic form.
Most preferably, these proteins will be of a
secreted form (i.e., lacking an apparent signal
sequence). These protein factors can be used for
functional studies, and can be purified for
additional biochemical and functional analyses,
such as qualitative and quantitative receptor
binding assays.
Recombinant Eumu cells have been
constructed in a bacterial expression vector,
pKK233-2, for production of KGF, as detailed in
Experimental section II. In summary, several
recombinant bacterial clones were tested for
protein production by the usual small scale
methods. All recombinants tested synthesized a
protein that was recognized by antibodies raised
against an amino—terminal KGF peptide (see
below). one recombinant was grown up in a one
liter culture which produced recombinant KGF that
efficiently stimulated thymidine incorporation
into DNA of BALE/MK keratinocyte cells, but was
only marginally active on NIH/3T3 fibroblasts.
Half-maximal stimulation of the BALB/MK cells in
the standard keratinocyte bioassay was achieved
with a concentration of between 2 to 5 ng/ml,
compared to a concentration of 10 to 15 ng/ml for
XGF purified from M426 cells.
one liter of bacterial cells yielded
approximately 50 pg of Mono-S purified
recombinant KGF. It will be apparent to those
skilled in the art of gene expression that this
initial yield can be improved substantially
without undue experimentation by application of a
variety known recombinant DNA technologies.
Recombinant mammalian (NIH/3T3 mouse)
cells have also been constructed using the entire
KG? CDNA coding sequence (including the NR2-
terminal signal peptide) and the vector pMMT/neo,
which carries mouse metallothionine (MT)
promoter and the selective marker gene for
neomycin resistance. The cells are being
evaluated for KGF production, particularly for
secretion of the mature form (lacking signal
peptide) produced by human fibroblasts, using
bioassays of the present invention. This same
vector and host cell combination has been used
successfully to express several other similar
recombinant polypeptides, including high levels
of Platelet-Derived Growth Factor (PDGF) A and B
chains (II-32). Accordingly, it will be
recognized by those skilled in the art that high
yields of recombinant KGF can
be achieved in this manner, using the
aforementioned recombinant DNAs and transformed
cells or this invention.
Ultimately, large-scale production can
be used to enable clinical testing in conditions
requiring specific stimulation of epithelial cell
growth. Materials and methods for preparing
pharmaceutical compositions for administration of
polypeptides topically (to skin or to the cornea
of the eye, for example) or systemically are well
known in the art and can be adapted readily for
administration of KGF and RGF-like peptides
without undue experimentation.
This invention also comprises novel
antibodies made against a peptide encoded by a
DNA segment of the invention. This embodiment of
the invention is exemplified by several kinds of
antibodies which recognize KGF. These have been
prepared using standard methodologies well known
in the art of experimental immunology, as
outlined in Experimental section II. These
antibodies include: monoclonal antibodies raised
in mice against intact, purified protein from
human fibroblasts; polyclonal antibodies raised
in rabbits against synthetic peptides with
sequences based on amino acid sequences predicted
from the KGF CDNA sequence [exemplified by a peptide with the
sequence of KGF residues 32-45 plus an R at its C—terminus,
namely, NDMTPEQMATNVR (using standard one-letter
code for amino acid sequences; see Fig. II-1)];
polyclonal antibodies raised in rabbits against
both naturally secreted KGF from human
fibroblasts and recombinant KGF produced in Enmfi
(see above).
All tested antibodies recognize the
recombinant as well as the naturally occurring
KGF, either in a solid-phase (ELISA) assay and/or
Some exemplary antibodies,
in a Western blot.
which are preferred antibodies of this invention,
appear to neutralize mitogenic activity of KGF in
the BALB/MK bioassay.
Fragments of antibodies of this
invention, such as Fab or F(ab)' fragments, which
retain antigen binding activity and can be
prepared by methods well known in the art, also
fall within the scope of the present invention.
Further, this invention comprises pharmaceutical
compositions of the antibodies of this invention,
or active fragments thereof, which can be
prepared using materials and methods for
preparing pharmaceutical compositions for
administration of polypeptides that are well
known in the art and can be adapted readily for
administration of KGF and KGF-like peptides
without undue experimentation.
These antibodies, and active fragments
thereof, can be used, for example, for detection
of KGF in bioassays or for purification of the
protein factors. They may also be used in
approaches well known in the art, for isolation
of the receptor for KGF, which, as described in
Experimental section II, appears to be distinct
from those of all other known growth factors.
Those preferred antibodies, and
fragments and pharmaceutical compositions
thereof, which neutralize mitogenic activity of
KGF for epithelial cells, as indicated by the
'BALB/MK assay, for instance, may be used in the
treatment of clinical conditions characterized by
excessive epithelial cell growth, including
dysplasia and neoplasia (e.g., psoriasis, or
malignant or benign epithelial tumors).
This invention further comprises novel
bioassay methods for detecting the expression of
genes related to DNAS of the invention. In some
exemplary embodiments, DNAs of this invention
were used as probes to determine steady state
levels of related mRNAs. Methods for these
bioassays of the invention, using KGF DNAS, and
standard Northern blotting techniques, are
described in detail in Experimental section II.
one skilled in the art will recognize
that, without undue experimentation, such methods
may be readily applied to analysis of gene
expression for KGF-like proteins, either in
isolated cells or various tissues. such
bioassays may be useful, for example, for
identification or various classes of tumor cells
or genetic defects in the epithelial growth
processes.
Without further elaboration, it is
believed that one of ordinary skill in the art,
using the preceding description, and following
the methods of the Experimental Sections below,
can utilize the present invention to its fullest
extent. The material disclosed in the
Experimental Sections, unless otherwise
indicated, is disclosed for illustrative purposes
’and therefore should not be construed as being
limitive in any way of the appended claims.
IDENTIFICATION AND CHARACTBRIZATIOR OF A NOVEL
GROWTH FACTOR BPECIPIC FOR EPITHBLIAL CELL8
This section describes experimental work
leading to identification of a growth factor
specific for epithelial cells in conditioned
medium of a human embryonic lung fibroblast ce11
line. The factor, provisionally termed
keratinocyte growth factor (KGF) because of its
predominant activity on this cell type, was
purified to homogeneity by a combination of
ultrafiltration, heparin-Sepharose affinity
chromatography and hydrophobic chromatography on
a C‘ reversed-phase HPLC column, according to
methods of this invention. KGF was found to be
both acid and heat labile, and consisted of a
single polypeptide chain with an apparent
molecular weight of approximately 28,000 daltons.
Purified KGP was a potent mitogen for epithelial
cells, capable of stimulating DNA synthesis in
quiescent BALB/MK epidermal keratinocytes by more
than 500-fold with activity detectable at 0.1 nu
and maximal at 1.0 nu. Lack of mitogenic
activity on either fibroblasts or endothelial
cells indicated that KGF possessed a target cell
specificity distinct from any previously
characterized growth factor. Microsequencing
revealed an amino-terminal sequence containing no
significant homology to any known protein. The
release of this novel growth factor by human
embryonic fibroblasts indicates that KGF plays a
role in mesenchymal stimulation of normal
epithelial cell proliferation.
early passage of M426 human embryonic
fibroblasts (I-8) was plated onto 175 cm2'r-flasks
and grown to confluence over 10-14 days in
Dulbecco's modified Eagle's medium (DMEM; GIBCO)
supplemented with 10% calf serum (GIBCO). Once
confluent, the monolayers were cycled weekly from
serum-containing to serum-free medium, the latter
consisting of DMEM alone. The cells were washed
twice with 5 ml of phosphate buffered saline
prior to addition of 20 ml of DMM. After 72
hrs, culture fluids were collected and replaced
with 35 ml of serum-containing medium. The
conditioned medium was stored at -7o°c until
further use.
-i
liters of conditioned medium were thawed,
Approximately ten
prefiltered through a 0.50 micron filter
(Millipore HAWP 142 50) and concentrated to 248
ml using the Pellicon cassette system (Hillipore
XX42 00K 60) and a cassette having a 10 kDa
molecular weight cutoff (Millipore PTGC 000 05).
After concentration, the sample was subjected to
two successive rounds of dilution with one liter
of 20 mM Tris-HCl, pH 7.5/0.3M Nacl, each
followed by another step of ultrafiltration with
the Pellicon system. Activity recovered in the
retentate was either immediately applied to
heparin-Sepharose resin or stored at -70'C.
fieparin-Sepharose gfginity
C o to a S . The retentate from
ultrafiltration was loaded onto heparin-Sepharose
resin (Pharmacia) which had been equilibrated in
mm Tris-Hcl, pH 7.5/0.3 M NaCl. The resin was
washed extensively until the optical density had
returned to baseline and then subjected to a
1inear—step gradient of increasing Nacl
concentration. After removing aliquots from the
fractions for the thymidine incorporation
bioassay, selected fractions were concentrated
ten- to twenty-fold with a Centricon-10
microconcentrator (Amicon) and stored at -70'C.
v - - . Active
fractions (0.6 M Nacl pool) from the HSAC were
thawed, pooled and further concentrated with the
centricon-10 to a final volume of S200 pl. The
sample was loaded onto a Vydac C‘ HPLC column (The
Separations Group, Hesperia, CA) which had been
equilibrated in 0.1% trifluoroacetic acid (TFA,
Fluka)/20% acetonitrile (Baker, HPLC grade) and
eluted with a linear gradient of increasing
acetonitrile. Aliquots for the bioassay were
immediately diluted in a 10-fold excess or 50
pg/ml BSA (Fraction V, Sigma)/20 mu Tris-Hcl, pH
7.5. The remainder of the sample was dried in a
speed-Vac (Savant) in preparation for structural
analysis.
molecular Sieve HPLC. Approximately 50
pl of the twice concentrated heparin—Sepharose
fractions were loaded onto a TsK—G3000SW Glas—Pac
Column (LKB) which had been equilibrated in 20 mM
Tris-Hcl, pH 6.8/0.5M Nacl. The sample was
eluted in this buffer at a flow rate of 0.4
ml/min. After removing aliquots for the
bioassay, the fractions were stored at -70'C.
-
gels were prepared with Naoodso‘ according to the
Polyacrylamide
procedure of Laemmli (I-9). Samples were boiled
for 3 min in the presence of 2.5%
-mercaptoethanol (vol/vol). The gels were fixed
and stained with silver (I-10) using the reagents
and protocol from BioRad. Molecular weight
markers were from Pharmacia.
pfih Synthesis Stimulation. Ninety-six
well microtiter plates (Falcon No. 3596) were
precoated with human fibronectin (Collaborative
Research) at 1 pg/cmz prior to seeding with
BALB/MK cells. Once confluent, the cells were
maintained for 24-72 hr in serum-free medium
containing 5 pg/ml transferrin (Collaborative
Research) and 30 nM Na1Seo3 (Baker).
Incorporation of 31-I-thymidine (S uci/ml final
concentration, NEN) into DNA was measured during
a 6 hr period beginning at 16 hrs following
addition of samples. The assay was terminated by
washing the cells once with ice cold phosphate-
buffered saline and twice with 5% trichloroacetic
acid. The precipitate was redissolved in 0.25 H
Naqfi, transferred into liquid scintillation fluid
(Biofluor, NEN) and counted.
Stimulation of DNA synthesis was
monitored as described above for BALB/MK cells on
a variety of other cell lines. NIH/3T3
fibroblasts (I-11) were available from the
National Institutes of Health, while CCL208
Rhesus monkey bronchial epithelial cells (I-12)
were obtained from the American Type Culture
Collection. The B5/589 human mammary epithelial
cell line, prepared as described in (I-13), was
obtained from Martha Stampfer (Lawrence Berkeley
Laboratory, Berkeley). The mammary cells were
grown in RPMI 1640 supplemented with 10% fetal
calf serum and 4 ng/ml EGF. when maintained in
serum-free conditions, the basal medium was DMEM.
Primary cultures of human saphenous vein
endothelial cells were prepared and maintained as
described elsewhere (I-14). Epidermal growth
factor and insulin were from Collaborative
Research. Acidic FGF and bFGF were obtained from
California Biotechnology, Inc. Recombinant TGFa
was obtained from Genentech, Inc. Media and
serum were either from GIBCO, Biofluids, Inc. or
the NIH media unit.
Erolifigragion Assay. Thirty-five mm
culture dishes were precoated sequentially with
poly-D-lysine (20 ug/cmf) (sigma) and human
fibronectin, and then seeded with approximately
2.5 x 10‘ BALB/MK cells. The basic medium was a
1:1 mixture of Eagle's low Ca” minimal essential
medium and Ham's F-12 medium, supplemented with 5
pg/ml transferrin, 30 nu Na2seo3 and 0.2 mM
ethanolamine (Sigma). Medium was changed every 2
or 3 days. After 10 days, the cells were fixed
in formalin (Fisher Scientific Co.) and stained
with Giemsa (Fisher Scientific Co.).
pg (-150 pmol) of protein from the active
Approximately
fractions of the q‘column were redissolved in 50%
TFA and loaded onto an Applied Biosystems gas-
phase protein sequenator. Twenty rounds of Edman
degradation were carried out and identifications
of amino acid derivatives were made with an
automated on-line HPLC (Model 120A, Applied
Biosystems).
t t c .
Preliminary screening of conditioned media from
various cell lines indicated that media from some
fibroblast lines contained mitogenic activities
detectable on both BALE/MK and NIH/3T3 cells.
Whereas boiling destroyed the activity on
BALB/MK, mitogenic activity on NIH/3T3 remained
intact. Based on the known heat stability of EGF
(I-15) and TGFa (I-16), it was reasoned that the
BALB/MK mitogenic activity might be due to an
agent different from these known epithelial
growth factors.
H426, a human embryonic lung fibroblast
line, was selected as the most productive source
of this activity for purification of the putative
growth factor(s). Ultratiltration with the
Pellicon system provided a convenient way of
reducing the sample volume to a suitable level
Various
for subsequent chromatography.
combinations of sieving, ion exchange and
isoelectric focusing chromatography were tried
during the development of a purification scheme,
but all resulted in unacceptably low yields.
On the other hand, heparin-Sepharose affinity
chromatography (HSAC), which has been employed in
the purification of other growth factors (I-_
I-22), proved to be useful as an early
purification step in the present invention.
While estimates of recovered specific activity
were uncertain at this stage because of the
likely presence of other factors, the apparent
yield-of activity was 50-70% with a corresponding
enrichment of approximately 1000 fold.
As shown in Fig. I-1, greater than 90%
of the BALB/MK mitogenic activity eluted from the
HSAC column with 0.6M Nacl. This peak of
activity was not associated with any activity on
NIH/3T3 cells (data not shown). A much smaller
peak of BALB/MK mitogenic activity consistently
emerged with 0.8 - 1.2M Nacl.
Due to the reproducibility of the HSAC
pattern, active fractions could be identified
presumptively on the basis of the gradient and
optical density profile. Prompt concentration of
-20 fold with the Centricon-10 was found to be
essential for stability, which could be
maintained subsequently at -7o'c for several
months.
Final purification was achieved by RP—HPLC
with a c"Vydac column, a preparative method
suitable for amino acid sequence analysis. While
the yield of activity from the C, step was usually
only a few percent, this loss could be attributed
to the solvents employed. In other experiments,
exposure to 0.1% TFA/50% acetonitrile for 1 hr at
room temperature reduced the mitogenic activity
of the preparation by 98%. Nonetheless, as shown
in Fig. I-2, a single peak of BALB/MK stimulatory
activity was obtained, coinciding with a distinct
peak in the optical density profile. The peak
fractions produced a single band upon
Naoodsob/PAGE and silver staining of the gel (Fig.
I-28), and the relative mitogenic activity of
each tested traction (Fig. I-2C) correlated well
with the intensity of the bands across the
activity profile.
An alternative purification step to the
HPLC technique described above, using sieving
chromatography with a TSK GBOOOSW GlasPac column
run in aqueous solution near physiologic pH,
resulted in a major peak of activity in the
BALE/M bioassay (Fig. I-3). This preparation
was almost as pure as the one obtained from RP-
IZIPLC as judged by silver-stained Naoodso,/PAGE
(data not shown) but provided a far better
recovery of activity (Table I-1). The TSK-
purified material was used routinely for
biological studies as it had a higher specific
activity.
In both types of purified preparations
(i.e., purified by HPLC or molecular sieving),
the profile of mitogenic activity was associated
’with a distinct band on Nabodsoy/PAGE which
appeared to be indistinguishable in the two
preparations.
Physical and Biological Characterization
Q: the Growth Eactor, The purified factor had an
estimated molecular weight of about 28 kDa based
on NaDodSo,/PAGE under reducing (Fig. I-2) and
non-reducing conditions (data not shown). This
value was in good agreement with its elution
position on two different sizing columns run in
solvents expected to maintain native conformation
(TSK-G3ooo-SW, Fig. I-3, and superose-12, data
not shown). From these data, the mitogen appears
to consist of a single polypeptide chain with a
molecular weight of 25-30 kDa.
The heat and acid lability of the
mitogenic activity were demonstrated using the
BALE/Hm mitogenesis bioassay. While activity was
unaffected by a 10 min incubation at 50'c, it was
reduced by 68% after 10 min at 60‘c and was
Exposure to
undetectable after 3 min at 100'C.
.5M acetic acid for 60 min at room temperature
resulted in a decline in activity to 14% of the
control. In comparison, the mitogenic activity
of the known growth factor, EGF, was not
diminished by any of these treatments.
The dose response curve for the purified
growth factor depicted in Fig. 1-4 illustrates
that as little as 0.1 nM led to a detectable
stimulation of DNA synthesis. Thus, the activity
range was comparable to that of the other growth
factors analyzed to date. A linear relationship
was observed in the concentration range 0.1 - 1.0
nM with maximal stimulation of 600 fold observed
at 1.0 nM. The novel factor consistently induced
a higher level of maximal thymidine incorporation
than EGF, aFGF, or bFGF in the BALB/MK
keratinocytes (Fig. 1-4).
The distinctive target cell specificity
of this factor was demonstrated by comparing its
.activities on a variety of cell types with those
of other growth factors known to possess
epithelial cell mitogenic activity. As shown in
Table I-2, the newly isolated factor exhibited a
strong mitogenic effect on BALB/MK but also
induced demonstrable incorporation of thymidine
into DNA of the other epithelial cells tested.
In striking contrast, the factor had no
detectable mitogenic effects on mouse (or human,
data not shown) fibroblasts or human saphenous
vein endothelial cells.
By comparison, none of the other known
growth factors appeared to preferentially
stimulate keratinocytes. TGFa and EGF showed
potent activity on fibroblasts, while the FGFs
were mitogenic for endothelial cells as well as
fibroblasts (Table I-2). Because of its
specificity of epithelial cells and the
sensitivity of keratinocytes in particular, the
novel mitogen was provisionally designated as
keratinocyte growth factor (KGF).
To establish that KGF not only would
stimulate DNA synthesis but would also support
sustained cell growth, the ability of BALB/MK
cells to grow in a fully-defined, serum-free
medium supplemented with this growth factor was
assessed. As shown in Fig. I-5, KGF served as an
excellent substitute for EGF but not insulin (or
insulin-like growth factor I) in this chemically
defined medium. Thus, KGF appears to act through
the major signalling pathway shared by EGF, aFGF
and bFGF for proliferation of BALB/MK cells.
c e eve e -te
Amino Acid geggencg of Kgz.
characterize the growth factor, approximately 150
To further
pmol of C‘-purified material were subjected to
amino acid sequence analysis. A single sequence
was detected with unambiguous assignments made
for cycles 2-13, as follows: X-Asn-Asp-Met-Thr-
Pro-Glu-G1n-Met-Ala-Thr-Asn-Va1. High background
noise precluded an assignment for the first
position which is, therefore, indicated by an X.
A computer search using the FAST?
program (I-24) revealed that the N-terminal amino
acid sequence of KGF showed no significant
homology to any protein in the National
Biomedical Research Foundation data bank, thus
supporting the novelty of this epithelial growth
factor.
The studies described in this
Experimental Section identified a human growth
factor which has a unique specificity for
epithelial cells. By employing ultrafiltration,
HSAC and RP-HPLC or TSK sieving chromatography
according to the present invention, a quantity
sufficient to permit detailed characterization of
V the physical and biological properties of this
molecule was isolated.
A single silver-stained band
corresponding to a molecular weight of about
28,000 daltons was detected in the active
fractions from RP-HPLC, and the intensity of the
band was proportional to the level of mitogenic
activity in these fractions. A band
indistinguishable from that obtained by RP-HPLC
was seen in the active fractions from TSK
chromatography. The purified protein stimulated
DNA synthesis in epithelial cells at sub-
nanomolar concentrations, but failed to induce
any thymidine incorporation in fibroblasts or
endothelial cells at comparable or higher
concentrations (up to 5 nM). This distinctive
target cell specificity combined with the single
novel N-terminal amino acid sequence determined
from the purified molecule lead to the conclusion
that KGF represents a new growth factor.
In a chemically defined medium the
purified factor was able to complement the
insulin-like growth factor I/insulin growth
requirement of BALB/MK cells and therefore must
act through a signal transduction pathway shared
with EGF, TGFa and the FGFs. Moreover, the new
factor was more potent than any of the known
epithelial cell mitogens in stimulating thymidine
incorporation in BALB/MK cells. Preliminary
evidence indicates that this factor is also
capable of supporting proliferation of secondary
cultures of human keratinocytes (data not shown).
Handling and storage of KG? were
problematical during its purification. Besides
()
its inherent lability to acid and heat, it was
unstable to lyophilization or dialysis. After
HSAC, complete loss of activity occurred within
24 hr despite the use of carrier proteins,
heparin, protease inhibitors, siliconized tubes
or storage at either 4' or -20‘C. only
concentrating the sample at this stage could
preserve its activity.
Furthermore, in order to transfer the
dried, purified factor it was necessary to
utilize either strong acid or detergent,
consistent with an adsorptive tendency or
insolubility. Thus, for preservation of
activity, the purified factor was maintained in
solution at high concentrations at -7o'c where it
remained stable for several months.
The ability of KGF to bind heparin may
signify a fundamental property of this factor
that has a bearing on its function hxfinu Growth
factors with heparin-binding properties include
aFGF (I-20 — I-22), bFGF (I-19, I-22), granulocyte/macrophage
colony stimulating factor (Roberts, R., et al., 1988, Nature
332, 376-378) and interleukin 3. (I-25) Each of these is
produced by stromal cells (I-I-27). Such
factors appear to be deposited in the
extracellular matrix, or on proteoglycans coating
the stromal cell surface (I-25, I-28). It has
been postulated that their storage, release and
contact with specific target cells are regulated
by this interaction (I-25, I-28). While
mesenchymal-derived effectors of epithelial cell
proliferation have also been described (I-I-
31), their identities have not been elucidated.
Its heparin-binding properties, release by human
embryonic fibroblast stromal cells, and
epithelial cell tropism provide KGF with all of
the properties expected of such a paracrine
mediator of normal epithelial cell growth.
The partial amino acid sequence
determined for this new growth factor has enabled
molecular cloning of its coding sequence and
determination of its structural relationship to
known families or growth factors, as described in
Experimental section II, below.
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N., strooband, P., Johnson, A.,
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Hunter, T. and Cooper, J.A. (1985) Annm
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Wright, N. and Allison, H. (1984) The
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3-5.
Weissman, B.E. and Aaronson, s.A. (1983)
Cell 32, 599-606.
Falco, J.P., Taylor, W.G., DiFiore,
P.P., Weissman, B.E., and Aaronson, S.A.
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Herril, C.R., Goldman, D., Sedman, S.A.
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1562.
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Shing, Y., Folkman, J., Sullivan, R.,
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H., Baird, A. and Bohlen, P. (1984)
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Maciag, T., Mehlman, T., Priesel, R. and
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Lipman, D.J. and Pearson, R.W. (1985)
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EXPERIMENTAL SBCTION :1
cDNA SEQUENCE OF
A NOVEL EPITHBLIAL CELL BPECIPIC GROWTH FACTOR
DEFINE8 A NEW MEMBER OF THE PG? FAMILY
Work in the previous Experimental
Section I identified and purified a novel
heparin-binding growth factor, designated
keratinocyte growth factor (XGF), which is
particularly active on keratinocytes and appears
to be specific for epithelial cells. This second
Experimental Section describes the isolation and
characterization of cDNA clones encoding KGF,
using synthetic oligonucleotides, based upon the
experimentally determined NR5-terminal amino acid '
sequence, as hybridization probes. Nucleotide
sequence analysis identified a 582-bp open
reading frame which would code for a 194-amino
acid polypeptide that is between 41% and 33%
identical to the heparin-binding acidic and basic
fibroblast growth factors (FGFs), and the related
products of the hst, FGF~S and int—2 oncogenes. The KGF
gene RNA transcript is expressed in normal
fibroblasts of both embryonic and adult origin,
but not in epithelial, endothelial or glial
cells. Thus, KGF appears to be normally
expressed by the mesenchyme, indicating a role in
the regulation of epithelial cell proliferation.
purification and N—terminal sequencing of KGF has
been previously described (see Experimental
Section I, above and II-3). Pools (50 pmole) of
deoxyoligonucleotides described under Results
were 5' end-labelled using 83 pmole of T-uP-ATP
(3000 Ci/mmole, Amersham) and 10 units of T4
polynucleotide kinase. The recombinant phage
carrying cDNA clones were replica plated onto
nitrocellulose filters and hybridized with
"P-labelled deoxyoligonucleotides in 20%
formamide, 10% dextran sulphate, 10 mM Tris-HCl
(pH 7.5), 8 X SSC, 5x Denhardt's and 50 pg/ml
denatured salmon sperm DNA, overnight at 42'c.
Filters were washed in 0.5 x SSC, 0.1% SDS at
50'C and exposed to Kodak X-omat AR film.
se e . The nucleotide sequence
of the KGF cDNA was determined by the dideoxy
chain termination method (II-26), of overlapping
restriction fragments, subcloned into pUc vectors
(II-27)
st 5
mature, secreted form or the polypeptide was
KGF cDNA encoding the
placed under control of the hybrid :13 promoter
in the plasmid expression vector pKK233-2 (II-
31), as follows. To accomplish this, a specific
length of KGF CDNA that contained the information
to code for the mature KGF molecule (i.e.,
without its signal peptide) was amplified using
the polymerase chain reaction (PCR) technique
(II-32). The fragment was directionally inserted
between two sites in the vector, namely the AkoI
site, made blunt ended by S1 nuclease digestion,
and the Hmdlll site, using standard recombinant
DNA methodology. The ends of the KGF cDNA
produced by the PCR method were as follows: the
' end was blunt and began with an ATG codon,
followed by the codon TGC for cys residue, number
33, which is the amino terminal residue of the
mature form of KG? (see Fig. II-1), and then the
entire KGF coding sequence. The stop codon, TAA,
and the four bases immediately following, TTGC,
were also included on the 3' end of the cDNA.
The primer used in the PCR method to direct DNA
synthesis to the desired position on the 3' end
of the cDNA included a HmdIII site for insertion
of the amplified cDNA into the vector DNA.
b es
raised in mice against intact, purified protein
from human fibroblasts using 5 or more
subcutaneous injections. Test bleeds were
screened with a solid-phase (ELISA) assay using
highly purified KGF from the conditioned medium of human
fibroblast cells as antigen. Hybridomas were prepared by
routine methods and supernatants were screened with the
ELISA assay to detect KGF-reactive antibodies.
Positive clones were serially subcloned by the
usual methods, and selected subclones were grown
as ascites tumors in mice for production of large
amounts of antibodies. Antibodies were purified
from ascites fluids employing standard techniques
(e.g., hydroxyapatite or immunoaffinity resins).
Polyclonal antibodies against a
synthetic peptide were raised in rabbits by
standard methods, as follows. The peptides were
made by solid phase technology and coupled to
thyroglobulin by reaction with glutaraldehyde.
Serial subcutaneous injections were made and test
bleeds were screened by ELISA as well as other
techniques, including western blot analysis and
mitogenesis bioassay. IgG immunoglobulins were
isolated by affinity chromatography using
immobilized protein G.
Polyclonal antibodies were raised in
rabbits against both naturally secreted KGF from
human fibroblasts and recombinant KGF produced in
E.afli (see next section), using the following
protocol:
) Initial injection and first boost were
administered in the inguinal lymph
nodes:
ii) subsequent boosts were made
intramuscularly.
Screening of test bleeds included ELISA as well
as Western blot analysis and mitogenesis
bioassay, and IgG was purified as for antibodies
against synthetic peptides, above.
Isolation of CQNA clones encoding the
ove owth factor. To search for CDNA clones
corresponding to the KGF coding sequence, two
pools of oligonucleotides with lengths of 26
bases were generated based upon a nine-amino acid
sequence, Asn-Asp-Met-Thr-Pro-Glu-G1n-Met-Ala, as
determined by microsequencing of purified KGF
(see Experimental Section I, above and reference
II-3). one oligonucleotide pool contained a
mixture of all 256 possible coding sequences for
the nine amino acids, while the other contained
inosine residues at the degenerate third position
of the codons for Thr and Pro.
This latter design reduced the number of
possible coding sequences in the pool to 16.
Inosine in a tRNA anticodon can form hydrogen
bonds with A, C or U (II-4), and oligonucleotides
that contain deoxyinosine have been shown to
hybridize efficiently with the corresponding cDNA
(II-5).
A CDNA library was constructed in a cDNA
cloning vector, pcEV9 (II-6) using
polyadenylated RNA extracted from the human
embryonic lung fibroblast cell line M426 (II-7),
the initial source of the growth factor.
screening of the library (9 x 10’ plaques) with
the “P-labelled 26-mer
oligonucleotides
identified 88 plaques which hybridized to both
pools of oligonucleotide probes.
C a c e ' at o se
se ted c .
clones that were analyz
e ci
or 10 plaque-purified
ed, one, designated clone
, had a cDNA insert of 3.5 kb, while the rest
had inserts ranging from 1.8 kb to 2.1 kb.
Analysis of the smaller clones revealed
several common restriction sites.
Sequencing of '
a representative smaller clone, designated clone
, along with clone 49
were overlapping cDNAs
c ' t o
, demonstrated that they
(Fig II-1A).
S ecee e
(clones 32 and 49) established a continuous
sequence of 3.85 kb containing the complete KGF
coding sequence (Fig. II-13).
be an initiation codon
An ATG likely to
was located at nucleotide
position 446, establishing a 582-base pair open
reading frame that ended at a TAA termination
codon at position 1030.
This open reading frame
would encode a 194—amino acid polypeptide with a
calculated molecular weight of 22,512 daltons.
The sequence flanking the ATG codon did
not conform to the proposed GcC(G/A)CcATGG
consensus for optimal initiation by eukaryotic
ribosomes (II-8), however, there was an A three
nucleotides upstream of the ATG codon. An A at
this position is the most highly conserved
nucleotide in the consensus. This ATG codon was
preceded 85 nucleotides upstream by a TGA stop
codon in the same reading frame.
A 19-amino acid sequence that was
homologous to the experimentally determined
NH:-terminus of purified KGF began 32 amino acids
downstream of the proposed initiation codon.
There was complete agreement between the
predicted and experimentally determined amino
acid sequences, where unambiguous assignments
could be made.
To search for homology between KGF and
any known protein, a computer search of the
National Biomedical Research Foundation data base
using the FASTP program of Lipman and Pearson was
conducted (II-9). By this approach, a striking
degree of relatedness between the predicted '
primary structure of KGF and those of acidic and
«J
u
basic FGF, as well as the related hst, FGF—5 and
inuz-encoded proteins was revealed.
Expression of mRNA transcripts of the
ene uma e s. In preliminary attempts
to examine expression of KGF mRNA in human cells,
a probe spanning the majority of the KGF coding
sequence (Probe A, Figure II-1A) detected a
single 2.4 kb transcript by Northern blot”
analysis of total M426 RNA (Figure II-1C). This
was considerably shorter than the length of the
composite CDNA sequence, 3.85 kb.
However, on screening poly(A)-selected
M426 RNA, an additional transcript of
approximately 5 kb was detected. Furthermore, a
probe derived from the untranslated region of
clone 49, 3' to the end of clone 32 (Probe 3,
Figure II-1A), hybridized only to the larger
message (Figure II-1C). Thus, it appears that
the KGF gene is transcribed as to alternate RNAs.
Two other members of the FGF gene family, bFGF
(II-29) and mu: (II-30), also express multiple
RNAS, the significance of which remains to be
determined.
To investigate the normal functional
role of KGF, the expression of its transcript in
a variety of human cell lines and tissues was
examined. As shown in Figure II-3, the
predominant 2.4 kb KGF transcript was detected in
each of several stromal fibroblast lines derived
from epithelial tissues of embryonic, neonatal
and adult sources, but not from epithelial cell
lines of normal origin. The transcript was also
detected in RNA extracted from normal adult
kidneys and organs of the gastrointestinal tract,
but not from lung or brain. The striking
specificity of KGF RNA expression in stromal
cells from epithelial tissues indicated that this‘
factor plays a normal role in mesenchymal
stimulation of epithelial cell growth.
For comparison, the mRNAs of other
growth factors with known activity on epithelial
cells were also analyzed in the same tissues as
listed above. Among the epithelial and stromal
cell lines analyzed, there was no consistent
pattern of expression of aFGF or bFGF transcripts
(Fig. II-3). The EGP transcript was not
expressed in any of the same cell lines, and was
only observed in kidney, among the various
tissues. Finally, the TGPa message was not
detected in any or the stromal fibroblast lines
and was expressed at varying levels in each or
the epithelial cell lines. It was also detected
at low levels in kidney among the tissues
examined (Fig. II-3).
?’5
b’t'o o G m to e a t v‘
e ar . Heparin has been shown to substantially
increase the mitogenic activity of aFGF for a
variety of target cells in culture, and to
stabilize it from heat inactivation (II-21, II-
22). Despite binding tightly to bFGF, heparin
had minimal effects on its mitogenic activity
(II-22). In view of the relatedness of KGF to
the FGFs, the effect of heparin on KGF mitogenic
activity was examined. As shown in Table II-1,
thymidine incorporation by BALB/MK cells in
response to KGF was inhibited 16 fold when
heparin was included in the culture medium. In
contrast, the activities of both aFGF and bFGF
were increased by the same treatment.
o u t o - b .
Several kinds of antibodies which recognize KGF
or KGF-like polypeptides have been prepared using
standard methodologies well known in the art of
experimental immunology and summarized in the
Methods section, above. These include:
monoclonal antibodies raised in mice against
intact, purified protein from human fibroblasts;
polyclonal antibodies raised in rabbits against
synthetic peptides with sequences based on amino
acid sequences predicted from the KGF cDNA
polyclonal antibodies raised in
sequence:
rabbits against both naturally secreted KGF from
human fibroblasts and recombinant KGF produced in
E4mN (see next section).
Monoclonal antibodies from three
different hybridomas have been purified. All
three recognize the recombinant as well as the
naturally occurring KGF in a solid-phase (ELISA)
assay. None cross-reacts with KGF under
denaturing conditions (in a Western blot), and
none neutralizes mitogenic activity of KGF in the
BALE/MK bioassay.
Polyclonal antibodies were generated
with a synthetic peptide with the amino acid
sequence NDMTPEQMATNVR, corresponding to residues
numbered 32 through 44 in KGF (see Fig. II-1),
plus an R (Arg) residue instead of the actual Asn
residue encoded by the CDNA at position 45. The Asn
residue is probably glycosylated in the
natural KGF polypeptide and, therefore, was not
identified in the amino acid sequencing data
obtained directly from that polypeptide (see
Discussion, below). Polyclonal antibodies
generated with this synthetic peptide recognize
both naturally occurring and recombinant KGF in
ELISA and Western blot analyses at a level of
sensitivity of at least as low as 10 ng protein.
These antibodies, however, do not neutralize
mitogenic activity of KGF in the BALB/MK
bioassay.
Polyclonal antisera against intact
natural KGF protein recognizes KGF in both ELISA
and Western blot assays. Such antibodies also
appear to inhibit mitogenic activity of KGF in
the BALE/MK bioassay.
es o G N '. KGF
CDNA was expressed to produce polypeptide in E.afli
by placing its coding sequence under control of
the hybrid tr; promoter (comprising elements of
ggp and lag promoters), in the plasmid pKK233-2
(II-31). To accomplish this, a specific length
of KGF CDNA that contained the information to
code for the mature KGF molecule (i.e., without
its signal peptide) was amplified using the
polymerase chain reaction technique (II-32). The
fragment was directionally inserted between two
sites in the vector, namely the Akol site, made
blunt ended by S1 nuclease digestion, and the
iHndIII site, using standard recombinant DNA
methodology. Selected recombinants were
sequenced at their cDNA 5' ends to ensure correct
alignment of the ATG initiation codon with the
regulatory elements of the tr; promoter.
Several recombinants were tested for
protein production by the usual small scale
In brief, the clones were grown to mid-
methods.
exponential phase (oo,,, '0.5) , treated with 1 mm
isopropyl 5-D-thiogalactopyranoside (IPTG) for 90
minutes, and cell extracts were run on SD8-
polyacrylamide gels for Western blot analysis.
All recombinants tested synthesized a protein
that was recognized by antibodies raised against
an amino-terminal KG? peptide. one recombinant
was selected which showed the greatest induction
from IPTG, for further protein analyses.
one liter of bacteria was grown up in
NZY broth containing 50 pg/ml ampicillin and 12.5
pg/ml tetracycline, to Obfis '0.5, and treated for
90 min. with IPTG. The cells were collected by
centrifugation, resuspended in 50 mm sodium
phosphate (pH 7.3), 0.2 M NaCl, and lysed by
sonication. Cell debris was removed by
centrifugation, and lysate applied directly to a
heparin-sepharose affinity column.
As determined by Western blot analysis
and mitogenic activity in keratinocytes,
recombinant KGF was eluted in 0.5-0.6 M Nacl.
Subsequent purification of the HSAC material with
a Mono-S (FPLC) column (Pharmacia) yielded a
preparation of KGF estimated to be 290% pure, as
judged by electrophoretic analysis using SD8-
polyacrylamide gels and silver-staining.
Recombinant KGF efficiently stimulated
thymidine incorporation into BALB/MK keratinocyte
cells, but was only marginally active on NIH/3T3
fibroblasts. Half-maximal stimulation of the
BALB/MK cells in the standard keratinocyte
bioassay was achieved with a concentration of
between 2 to 5 ng/ml, compared to a concentration
of 10 to 15 ng/ml for KGF purified from M426
cells. one liter of bacterial cells yielded
approximately 50 pg of Mono—s purified_
recombinant KGF.
Construction o; a chimera containing KGE
and aFGF seggences. The studies above indicated
that KGF possessed two distinctive
characteristics which might be encoded by
distinct portions or domains of the polypeptide
sequence, as is well known to occur in coding
sequences of other multifunctional polypeptides.
To test this possibility, a chimeric DNA segment
encoding the NH:-terminal sequence of KGF grafted
onto the C-terminal core of aFGF was constructed,
as follows. A Saul restriction enzyme site
(CCTGAGG) in the 5' end of the KGF CDNA, within
codons for residues 76, 77, and 78 (Tyr, Leu and Arg
respectively; see Fig. II—1) was cut and joined to an
homologous site in the aFGF CDNA within codons for
amino acids 37 (Phe), 38 (Leu) and 39 (Arg). The 3'
and 5' ends of this chimeric DNA were joined to
the vector DNA of the plasmid pKK233-2 by the
same method used for insertion of the KGF.cDNA
()
encoding the secreted form of polypeptide (see
Methods, above).
when recombinant E4mH cells were
constructed using the vector carrying the
chimera, and expression tests were conducted as
described for mature KGF, above, a novel product
with properties of both KGF and aPGF was
produced. The peptide was enriched by heparin-
sepharose chromatography and found to have a
target cell preference for keratinocytes, like
KGF, with minimal activity on fibroblasts
(NIH/3T3). The mitogenic activity of this
chimeric polypeptide lacks, however,
susceptibility to inhibition by heparin, a
characteristic which parallels that of aFGF
rather than KGF} In fact, the mitogenic activity
on keratinocytes is actually enhanced by heparin,
as is the case for aFGP. Thus the peptide
domains responsible for target cell specificity
and heparin sensitivity are clearly distinct and
readily separable in KGP, according to the
practice of the present invention.
The experiments described in this
section illustrate the practice of several
principal embodiments of the present invention.
These include isolation of cDNAs encoding KGF,
expression of such cDNAs in recombinant cells,
production of various antibodies reactive with
KGF, and construction and expression of a
chimeric CDNA encoding a novel growth factor with
amino acid sequences and related functionalities
of both KGF and aFGF. The following points
related to these embodiments may also be noted to
enhance the understanding of the present
invention.
The sequence predicted from the KGF cDNA
agreed with the amino acid sequence determined
from the purified KGF form secreted by human
fibroblasts. Moreover, the sequence offered
potential explanations for positions where
definitive amino acid assignments could not be
made by direct amino acid sequencing. Residues
32 and 46 are predicted from the cDNA sequence to
be cysteines, and hydrolyzed derivatives of
unmodified cysteine residues are not detectable
following Edman degradation. The-predicted KGF
amino acid sequence also contained one potential
N-linked glycosylation site (Asn-X-Ser/Thr) from
residues 45 through 47. If Asn 45 were
glycosylated, it would not be detected by the
amino acid sequencing methods employed here. In
fact, KGF migrates as a broad band on
NaDodSo@/PAGE at a higher molecular weight than
predicted for the purified protein. This may be
accounted for by glycosylation.
The FGFS are heparin-binding mitogens
with broad target cell specificities (II-10).
FGF—5 and hst are transforming genes originally detected
by DNA-mediated gene transfer.
The product of the nu-2 gene is expressed
normally during mouse embryogenesis (II-14) and
iaberrantly after proviral integration of mouse
mammary tumor virus (II-15).
KGF is the sixth member of the
fibroblast growth factor family to be identified
(II-28). While the name FGF-6 does not seem
suitable because KG? is devoid of activity on
fibroblasts, this nomenclature may also be used
for this growth factor, to denote its structural
relationship to the FGF family. As all
previously characterized growth factors either
exclude epithelial cells as targets or include
them among a number of sensitive target cells,
the highly specific nature of KGF mitogenic
activity for epithelial cells, and the
sensitivity of keratinocytes in particular, make
it unique.
In studies to date, expression of the
KGF transcript appears to be specific for stromal
cells derived from epithelial tissues, suggesting
its function in normal epithelial cell
proliferation. The availability of the KGF cDNA
clone will make it possible to determine whether
abnormal expression of this growth factor can be
implicated in clinical conditions characterized
by epithelial cell dysplasia and/or neoplasia.
Moreover, the ability to produce large quantities
of this novel growth factor by recombinant
techniques should allow testing of its clinical
applicability in situations where specific growth
of epithelial cells is of particular importance.
Alignment of the KGF sequence with the
five other proteins of the FGF family revealed
two major regions of homology, spanning amino
acids 65-156 and 162-189 in the predicted KGF
sequence, which were separated by a short,
nonhomologous series of amino acids with varying
lengths in different members of the family (Fig.
II-2). In the case of Mb), the length of this
sequence was 17 residues, while in hu, the two
homologous regions were contiguous. In KGF the
intervening sequence consisted of five amino
acids.
In the aligned regions, the KGF amino acids sequence
was about 44%, identical to int—2 (mouse), 41% identical
to FGF—5 (human), 39% identical to bFGF (human), 37%
identical to aFGF (human) and 33% identical to
hu (human). In this same region, all six
proteins were identical at 19% or the residues,
and allowing for conservative substitutions, they
showed 28% homology. i
As shown in Fig. II-2, the amino termini
of these related proteins are nonhomologous and:
of variable length. The primary KGF, FGF—S and hst
translation products contain hydrophobic
N-terminal regions which likely serve as signal
sequences (II-16). The fact that this N-terminal
domain is not present in the mature KGF molecule
(Fig. II-1B) further supports this conclusion.
In contrast, the FGFS are synthesized apparently
without signal peptides (II-10). The mu) protein
contains an atypically short region of N-terminal
hydrophobic residues (II-17), but it is not known
if the protein is secreted. Moreover, the mu:
protein contains a long C- terminal extension
compared to the other family members.
Purified KGF contains five cysteine
residues, two of which are conserved throughout
the family of FGF related proteins (Fig. II-2).
Also of note are the five pairs of basic residues
throughout the KGF sequence. This same pattern
has been observed in other FGF family members and
may be involved in their interaction with heparin
(II-18). Dibasic sites are also common targets
for proteolytic processing and such processing
might account for the microheterogeneity observed
in some KGF preparations (unpublished data).
The KGF CDNA sequence was AT rich
throughout its length, but particularly so in the
3' untranslated region where the AT content was
70% as compared to 60% in the putative coding
sequence and 63% in the 5' untranslated region.
The 3' untranslated region contained a large
number of ATTTA sequences, which have been
proposed to be involved in the selected .
degradation of transiently expressed, unstable
RNAs (II-19). There was no classical AATAAA
polyadenylation signal but two variant sequences,
AATTAA and AATACA (II-20), were detected 24 and
19 nucleotides, respectively, upstream of the
poly(A) sequence at the 3' end of the cDNA.
It has been suggested that the heparin
effect on acidic F6? is either due to
stabilization of the active conformation of the
growth factor or to formation of a tertiary
complex with acidic FGF and its receptor (II-21,
II-22). If so, heparin may stabilize a
conformation of KGF that is not as active as the
free molecule, or form a tight complex that is
unable to efficiently interact with its receptor.
While its ability to bind heparin
reflects the structural similarities of KGF with
the FGF's, the differences in target cell
specificities between these related mitogens is
remarkable. The FGF's induce division of most
nonterminally differentiated cells of both
embryonic mesodermal and neuroectodermal origin.
In addition to fibroblasts and vascular
endothelial tissues, mesodermally derived targets
in culture include myoblasts, chondrocytes and
osteoblasts (II-23). FGF's are also mitogenic
for glial astrocytes and neuroblasts (II-24).
The product of the oncogene isolated from
Kaposi's sarcoma, which is identical to ha, also
stimulates proliferation of NIH/3T3 and capillary
endothelial cells (II-25). To date, KGF induced
mitogenesis has only been observed in epithelial
cells, and the absence of any detectable activity
in fibroblasts or endothelial cells has also been
demonstrated (see Experimental Section I, above
and II-3). It seems likely, therefore, that KGF
acts through a different cell surface receptor
than the FGFs.
There is no significant N-terminal
homology between KGF and other FGF-related
proteins. Thus, the construction of chimeric
molecules between KGF and a prototype PC? was
undertaken to determine whether the KGF
N-terminal domain is sufficient to account for
its unique target cell specificity. The results
on the first such recombinant polypeptide
sequence indicate that the N-terminal domain of
KGF essentially encodes the cell preference for
keratinocytes, while the susceptibility of KGF to
heparin is encoded somewhere in the C-terminal
core region which was replaced by sequences of
aFGF. This novel KGF-like growth factor may have
advantages in clinical applications where
administration of an epithelial-specific growth
factor is desirable in the presence of heparin, a
commonly used anticoagulant. Additional studies
on chimeras should also provide insights into
which specific domains in the C-terminal core
contribute the different effects of heparin on
their biologic activities.
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For purposes of completing the
background description and present disclosure,
each of the published articles, patents and
patent applications heretofore identified in this
specification is hereby incorporated by
reference into the specification.
The foregoing invention has been
described in some detail for purposes of clarity
and understanding. It will also be obvious that
various combinations in form and detail can be
made without departing from the scope of the
invention.
Table I-1. Growth Factor Purification
Purification Protein tout specific
stop activity . activity
(no) (min) (mlu/ma)
Conditioned medium 1.5 x 103' 2.5 x 10‘ 1.3 x 10‘
(10 liters)
Ultraffltration 1.3 x 103' 3.2 x 10‘ 2.5 x 10‘
(retenute)
nsAc 0.73" 1.5 x 10‘ 2.2 x 10‘
0.6 It not pool
tsx-csooo su 8.6 x 1o'3” 2.7 x 103 3.2 x 105
c‘-nPLc 6.1 x 1o‘3b 2.1 x 102 3.4 x so‘
Recoveries were calculated by assuming that all
of the mitogenic activity in the starting
material was due to the isolated factor.»
'One unit of activity is defined as half of the
maximal stimulation of thymidine incorporation
induced by TSK-purified factor in the BALB/MK
bioassay, in which approximately 3 ng of the
TSK-purified factor stimulated 1 unit of
activity.
‘Protein was estimated by using the Bradford
reagent from BioRad (I-23).
“Protein was estimated by using Ag: - 140.
Table I-2. Target Cell Specificity of Growth
Factors
Growth Factor figithel in; Fibrglngg findothel ial
IALI/NK as/559 CCLZOB uxn/313$ Human saphenous
vein
rat 500-1000 2-3 5-10 <1 <1
ear 100-200 20-60 10-30 10-20 n.d.
ran 150-300 n.d. n.d. 10-20 n.d.
aFGF' 300-500 2-: 5-10 50-70 5
bFGF 100-200 2-3 2-5 50-70 5
Comparison of manimal thymidine incorporation
stimulated by KGF and other growth
factors in a variety of cell lines, expressed as
fold stimulation over background.
This data represents a summary of four different
experiments.
'Maxima1 stimulation by aFGF required the
presence of heparin (Sigma), 20 ug/ml.
n.d. = not determined.
TABLE II—l. Effect of Heparin on KG? Mitogenic
Activity.
£:s.vL':_F.:ms !4_I-Ia! Hum}
9 9
any 150 9.5 <1 <1
eF6F 1os 259 10.6 as
been so 124 45.7 1n
Cells were plated in microtiter plates,
grown to confluence in serum containing media
and then placed in a serum—free medium for 24-72
hr prior to sample addition. Mitogenesis assays
were performed as described (see Experimental
Section 1, above and II-3). Where indicated,
heparin was included in the culture media at a
final concentration of 20 ng/ml. The
concentration of all the growth factors was so
ng/ml. The results represent fold stimulation
of 3H-thymidine incorporation in the indicated
assay cell in the presence (+) or absence (-) of
heparin. Each value represents the mean result
from two independent experiments in which each
point, in turn, represents the mean value of
duplicate analyses.
Claims (1)
- CLAIMS An isolated glycosylated or unglycosylated keratinocyte growth factor (KGF) protein comprising: (a) the following amino acid sequence, (b) a portion of the following amino acid sequence without the N-terminal 31 amino acids, or (c) an amino acid sequence that differs by the addition, deletion, or substitution of one or more amino acids from the following amino acid sequence: MHK-WILTWILPTLLYRSCFHIICL VGTISLACNDMTPEQMATNVNCS SPERI-lTRSYDYMEGGDlRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNIMEIRTVAVGIVAIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVALNQKGlPVRGKKTKKEQKTA HFLPMAIT wherein said protein is capable of stimulating DNA synthesis in quiescent _ BALB/MK epidermal keratinocytes by more than 500-fold while lacking mitogenic activity on fibroblasts and endothelial cells. The KGF protein of claim 1, wherein 5 nM of said protein exhibits less than one- fold stimulation over background in NIH/3T3 fibroblasts. The KGF protein of any of claims 1 to 2 which has a specific activity of at least about 3.4 X10‘ units per milligram of protein, where one unit of activity is defined as that amount which causes half of the maximal possible stimulation of DNA synthesis in BALB/MK keratinocyte cells. The KFG protein of any of claims 1 to 3 which is glycosylated. The KGF protein of any of claims 1 to 3 which is not glycosylated. The KGF protein of any of claims 1 to 5 comprising the amino acid sequence: CNDMTPEQMATNVNCS SPERHTRSYDYMEGGDlRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNlMElRTVAVGlV/XIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVA__LNQKGlPVRGKKTKKEQKTA HFLPMAIT. The KGF protein of any of claims 1 to 5, comprising an amino acid sequence differing by the addition, deletion, or substitution of one or more amino acids from the following amino acid sequence: CNDMTPEQMATNVNCS SPERHTRSYDYMEGGDIRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNIMEIRTVAVGIVAIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELlLENHYNTY/XSAKWTHNGGEM FVALNQKGIPVRGKKTKKEQKTA HFLPMAIT The KGF protein of any of claims 1 to 5, comprising an amino acid sequence differing by the addition, deletion or substitution of one or more amino acids from the following amino acid sequence: Mt-lKWlLTWlLPTLLYRSCFHllCL VGTISLACNDMTPEQMATNVNCS SPERHTRSYDYMEGGDIRVRRLF C_RTQWYLRlDKRGKVKGTQEMKN NYNIMEIRTVAVGIVAIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVALNQKGIPVRGKKTKKEQKTA HFLPMAIT. The KGF protein of any of claims 1 to 5, comprising a segment of amino acids of Figure ll-1 B which comprises amino acids 32 to 78 of Figure ll—1B to confer on the KGF protein preferential mitogenic activity on an epithelial cell. The KGF protein of any of claims 1 to 5, comprising a segment of the following amino acid sequence: MHKWILTWILPTLLYRSCFHIICL VGTISLACNDMTPEQMATNVNCS SPERHTRSYDYMEGGDIRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNIMEIRTVAVGIVAIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVALNQKGIPVRGKKTKKEQKTA HFLPMAIT. The KGF protein of claim 10, consisting of a segment of the following amino acid sequence: MHKWILTWILPTLLYRSCFHIICL VGTISLACNDMTPEQMATNVNCS SPERHTRSYDYMEGGDIRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNIMEIRTVAVGIVAIKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVALNQKGIPVRGKKTKKEQKTA HFLPMAIT. An isolated glycosylated or unglycosylated keratinocyte growth factor (KGF) protein according to any of claims 1 to 5 consisting of a segment of the following amino acid sequence: CNDMTPEQMATNVNCS SPERHTRSYDYMEGGDIRVRRLF CRTQWYLRIDKRGKVKGTQEMKN NYNlMElRTVAVGlVAlKGVESEF YLAMNKEGKLYAKKECNEDCNFK ELILENHYNTYASAKWTHNGGEM FVA__LNQKGlPVRGKKTKKEQKTA HFL—PMA|T, wherein said segment has mitogenic activity for a keratinocyte cell, and wherein said segment may further include methionine at the amino terminus. The KGF protein of claim 12 which has a specific activity of at least about 3.4 x 10“ units per milligram of protein, where one unit of activity is defined as that amount which causes half of the maximal possible stimulation of DNA synthesis in BALB/MK keratinocyte cells. The KGF protein of any of claims 1 to 13, wherein said KGF protein comprises’ Met at the amino terminus. A pharmaceutical composition comprising at least one KGF protein according to any of claims ‘1 to 14. A pharmaceutical composition comprising at least one KGF protein according to any of claims 1 to 14 for stimulating epithelial cells. Use of the KGF protein according to any of claims 1 to 14 for preparing a medicament for stimulating epithelial cells. Use of the KGF protein according to any of claims 1 to 14 for preparing a medicament, wherein the medicament is to be topically applied to the skin or eye. Use according to claim 18, wherein the medicament is to be topically applied to the skin. A chimeric protein which comprises within a single polypeptide molecule a KGF protein of any of claims 1 to 14 and at least one other polypeptide portion of the fibroblast growth factor family. The chimeric protein of claim 20, comprising amino acids 32 to 78 of
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IE35890A IE900358L (en) | 1990-01-31 | 1990-01-31 | Dna encoding a growth factor |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
USUNITEDSTATESOFAMERICA31/01/19893 | |||
IE35890A IE900358L (en) | 1990-01-31 | 1990-01-31 | Dna encoding a growth factor |
Publications (2)
Publication Number | Publication Date |
---|---|
IE83398B1 true IE83398B1 (en) | |
IE900358L IE900358L (en) | 1990-07-31 |
Family
ID=11011129
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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IE35890A IE900358L (en) | 1990-01-31 | 1990-01-31 | Dna encoding a growth factor |
Country Status (1)
Country | Link |
---|---|
IE (1) | IE900358L (en) |
-
1990
- 1990-01-31 IE IE35890A patent/IE900358L/en not_active IP Right Cessation
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