U.S. patent application number 11/585862 was filed with the patent office on 2007-11-01 for epithelial cell specific growth factor keratinocyte growth factor (kgf).
This patent application is currently assigned to The Government of U.S.A. as represented by the Secretary, Department of Health and Human Services. Invention is credited to Stuart A. Aaronson, Paul W. Finch, Jeffrey S. Rubin.
Application Number | 20070253963 11/585862 |
Document ID | / |
Family ID | 36127691 |
Filed Date | 2007-11-01 |
United States Patent
Application |
20070253963 |
Kind Code |
A1 |
Rubin; Jeffrey S. ; et
al. |
November 1, 2007 |
Epithelial cell specific growth factor keratinocyte growth factor
(KGF)
Abstract
Discoveries are disclosed that show particular aspects of
recombinant DNA technology can be used successfully to produce
hitherto unknown human keratinocyte growth factor (KGF) protein
free of other polypeptides. These proteins can be produced in
various functional forms from spontaneously secreting cells or from
DNA segments introduced into cells. These forms variously enable
biochemical and functional studies of this novel protein as well as
production of antibodies. Means are described for determining the
level of expression of genes for the KGF protein, for example, by
measuring mRNA levels in cells or by measuring antigen secreted in
extracellular or body fluids.
Inventors: |
Rubin; Jeffrey S.; (Potomac,
MD) ; Finch; Paul W.; (Croton-on-Hudson, NY) ;
Aaronson; Stuart A.; (New York, NY) |
Correspondence
Address: |
OFFICE OF TECHNOLOGY TRANSFER;NATIONAL INSTITUTES OF HEALTH
6011 EXECUTIVE BLVD SUITE 325
MSC 7660
BETHESDA
MD
20892-7660
US
|
Assignee: |
The Government of U.S.A. as
represented by the Secretary, Department of Health and Human
Services
Rockville
MD
|
Family ID: |
36127691 |
Appl. No.: |
11/585862 |
Filed: |
October 25, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11346626 |
Feb 3, 2006 |
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11585862 |
Oct 25, 2006 |
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08477983 |
Jun 7, 1995 |
7026291 |
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11585862 |
Oct 25, 2006 |
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08106775 |
Aug 16, 1993 |
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08477983 |
Jun 7, 1995 |
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07780847 |
Oct 23, 1991 |
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08106775 |
Aug 16, 1993 |
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07304281 |
Jan 31, 1989 |
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07780847 |
Oct 23, 1991 |
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Current U.S.
Class: |
424/158.1 ;
435/320.1; 435/325; 435/6.16; 435/7.1; 435/70.1; 514/9.1; 514/9.2;
514/9.6; 530/350; 530/389.1; 536/23.5 |
Current CPC
Class: |
C07K 16/22 20130101;
C07K 2317/73 20130101; A61K 38/00 20130101; G01N 33/74 20130101;
C07K 14/50 20130101; G01N 2333/4742 20130101 |
Class at
Publication: |
424/158.1 ;
435/320.1; 435/325; 435/006; 435/007.1; 435/070.1; 514/012;
530/350; 530/389.1; 536/023.5 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 39/00 20060101 A61K039/00; C07H 21/04 20060101
C07H021/04; C07K 14/00 20060101 C07K014/00; C07K 16/00 20060101
C07K016/00; C12N 15/00 20060101 C12N015/00; C12N 15/87 20060101
C12N015/87; C12P 21/04 20060101 C12P021/04; G01N 33/53 20060101
G01N033/53 |
Claims
1. A human keratinocyte growth factor (KGF) having an apparent
molecular weight of about 28 kDa as determined by migration in
NaDodSO.sub.4/PAGE, and a specific activity of at least about
3.4.times.10.sup.4 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 under standard assay conditions.
2. Human KGF according to claim 1, wherein said specific activity
is at least about 3.2.times.10.sup.5 units per milligram
protein.
3. A bioassay for KGF-like activity in a test sample which
comprises the following steps: i) growing keratinocytes in culture
to confluence and maintaining said confluent culture in serum-free
medium; ii) adding a test sample to said confluent culture of
keratinocytes; and iii) determining the stimulation of DNA
synthesis in said keratinocytes.
4. A method of producing KGF from cultured cells comprising the
following steps: i) Culturing KGF-producing cells in culture medium
under conditions such that KGF is produced; ii) concentrating said
culture medium so that a first concentrate is formed; iii)
contacting said concentrate with heparin under conditions such that
KGF present in said first concentrate binds to the heparin whereby
a heparin-KGF complex is formed; iv) separating said heparin-KGF
complex from said concentrate; v) treating said heparin-KGF complex
under conditions such that said KGF dissociates from the heparin so
that a solution of free KGF is formed; vi) concentrating said
solution so that a second concentrate is formed; vii) fractionating
said second concentrate so that KGF is separated from the remaining
components.
5. A method of producing KGF from cultured cells, according to
claim 4, wherein said KGF-producing cells are M426 human embryonic
fibroblasts.
6. A DNA segment encoding a human keratinocyte growth factor (KGF)
protein.
7. A DNA segment, according to claim 6, wherein said protein has
the amino acid sequence defined in FIG. II-1.
8. A DNA segment encoding a chimeric KGF-like protein which
comprises within a single polypeptide molecule functional segments
of human KGF and at least one other polypeptide of the fibroblast
growth factor family.
9. A recombinant DNA molecule comprising a DNA segment according to
claim 6 or claim 8 and a vector.
10. A culture of cells transformed with said recombinant DNA
molecule according to claim 9.
11. A method of producing a human KGF protein comprising culturing
said cells according to claim 10 in a culture medium under
conditions such that said protein is produced and isolating said
protein from said cells.
12. A method of producing a human KGF protein comprising culturing
said cells according to claim 10 in a culture medium, wherein said
protein is secreted from said cell, and isolating said protein from
said medium.
13. A human KGF or KGF-like protein having the amino acid sequence
in FIG. II-1B.
14. A human KGF or KGF-like protein, according to claim 13, which
is not glycosylated.
15. An antibody specific for a peptide having the amino acid
sequence of human KGF or KGF-like protein, according to claim
13.
16. The antibody according to claim 15 which neutralizes the
mitogenic activity of human KGF.
17. A bioassay for expression of a gene encoding KGF, comprising
the steps of: i)isolating mRNA from tissues or cells; and ii)
annealing said RNA to a DNA probe encoding a human KGF; iii)
determining the amount of DNA:RNA hybrid containing said DNA
probe.
18. A bioassay for KGF antigen comprising the steps of: i)
extracting polypeptides from body fluids or tissue samples; and ii)
determining the level of human KGF antigen by reaction with an
antibody specific for a peptide having the amino acid sequence of
human KGF or KGF like protein, according to claim 13.
19. A pharmaceutical composition for treatment of conditions
requiring specific stimulation of epithelial cells, comprising KGF
according to claim 1 or claim 13, and an acceptable pharmaceutical
carrier.
20. A pharmaceutical composition for treatment of conditions
requiring specific inhibition of stimulation of epithelial cells by
KGF, comprising antibodies to KGF according to claim 15, and an
acceptable pharmaceutical carrier.
Description
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/346,626 filed Feb. 3, 2006, which is
a continuation application of U.S. patent application Ser. No.
08/477,983, filed Jun. 7, 1995, which is a continuation application
of U.S. patent application Ser. No. 08/106,775, filed Aug. 16,
1993, abandoned, which is a continuation application of U.S. patent
application Ser. No. 07/780,845, filed Oct. 23, 1991, abandoned,
which is a continuation application of U.S. patent application Ser.
No. 07/304,281, filed Jan. 31, 1989, abandoned.
FIELD OF THE INVENTION
[0002] 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 invention 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
[0003] aFGF acidic fibroblast growth factor [0004] bFGF basic
fibroblast growth factor [0005] EGF epidermal growth factor [0006]
HSAC heparin-Sepharose affinity chromatography [0007] kb kilobases
[0008] kDa kilodaltons [0009] KGF keratinocyte growth factor [0010]
NaDodSO.sub.4/PAGE Sodium dodecylsulphate (SDS)/polyacrylamide gel
electrophoresis [0011] RP-HPLC reversed-phase high performance
liquid chromatography [0012] TGF.alpha. transforming growth factor
.alpha.
BACKGROUND OF THE INVENTION
[0013] 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
(James, R. and Bradshaw, R. A. (1984), Ann. Rev. Biochem. 53,
259-292). Interest in growth factors has been heightened by
evidence of their potential involvement in neoplasia (Sporn, M. B.
and Todaro, G. J. (1980), N. Eng. J. Med. 303, 878-880). The v-sis
transforming gene of simian sarcoma virus encodes a protein that is
homologous to the B chain of platelet-derived growth factor (James,
R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292;
Doolittle, R. F., et al. (1983) Science 221, 275-277). Moreover, a
number of oncogenes are homologues of genes encoding growth factor
receptors (James, R. and Bradshaw, R. A. (1984) Ann. Rev. Biochem.
53, 259-292). 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.
[0014] One known family of growth factors affecting connective
tissue cells includes acidic fibroblast growth factor (aFGF), basic
fibroblast growth factor (bFGF), and the related products of the
hst, and int-2 oncogenes.
[0015] Further, it is known that some growth factors, including the
following, have heparin-binding properties: aFGF (Maciag, T.,
Mehlman, T., Friesel, R. and Schreiber, A. B. (1984) Science 225,
932-935; Conn, G. and Hatcher, V. B. (1984) Biochem. Biophys. Res.
Comm. 124, 262-268); bFGF (Gospodarowicz, D., Cheng, J., Lui,
G.-M., Baird, A. and Bohlen, P. (1984) Proc. Natl. Acad. Sci. USA
81, 6963-6967; Maciag, T., Mehlman, T., Friesel, R. and Schreiber,
A. B. (1984) Science 225, 932-935); granulocyte/macrophage colony
stimulating factor (James, R. and Bradshaw, R. A. (1984) Ann. Rev.
Biochem. 53, 259-292); and interleukin 3 (James, R. and Bradshaw,
R. A. (1984) Ann. Rev. Biochem. 53, 259-292). Each of these
polypeptide factors is produced by stromal cells (James, R. and
Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292, Doolittle,
R. F., Hunkapiller, M. W., Hood, L. E., Devare, S. G., Robbins, K.
C., Aaronson, S. A. and Antoniades, M. N. (1983) Science 221,
275-277, Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D.,
Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378). Such
factors appear to be deposited in the extracellular matrix, or on
proteoglycans coating the stromal cell surface (James, R. and
Bradshaw, R. A. (1984) Ann. Rev. Biochem. 53, 259-292, Roberts, R.,
Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and
Dexter, T. M. (1988) Nature 332, 376-378). It has been postulated
that their storage, release and contact with specific target cells
are regulated by this interaction (Roberts, R., Gallagher, J.,
Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)
Nature 332, 376-378, Vlodavsky, I., Folkman, J., Sullivan, R.,
Fridman, R., Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987)
Proc. Natl. Acad. Sci. USA 84, 2292-2296).
[0016] It is widely recognized, however, that the vast majority of
human malignancies are derived from epithelial tissues (Wright, N.
and Allison, M. (1984) The Biology of Epithelial Cell Populations
(oxford University Press, New York) Vol. 1, pp. 3-5). Effectors of
epithelial cell proliferation derived from mesenchymal tissues have
been described (James, R. and Bradshaw, R. A. (1984) Ann. Rev.
Biochem. 53, 259-292, Doolittle, R. F., Hunkapiller, M. W., Hood,
L. B., Devare, S. G., Robbins, K. C., Aaronson, S. A. and
Antoniades, M. N. (1983) Science 221, 275-2772, Waterfield, M. D.,
Scrace, G. J., Whittle, N., Strooband, P., Johnson, A., Wasteton,
A., Westermark, B., Heldin, C.-H., Huang, J. S. and Deuel, T. F.
(1983) Nature 304, 35-39), however, their molecular identities and
structures have not been elucidated.
[0017] 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.
[0018] 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.
SUMMARY OF THE INVENTION
[0019] 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.
[0020] 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 (bFGF) and the
related products of the hst, 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, KGF may also be referred to as FGF-6.
[0021] Accordingly, this invention relates, in part, to purified
KGP 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 KGF-like
polypeptides.
[0022] 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.
[0023] 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 KGF-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.
[0024] 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 .lamda. cloning vector (exemplified by
.lamda.pCEV9); 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).
[0025] In still another embodiment, the invention comprises a cell,
preferably a mammalian cell, transformed 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, transformed with 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.
[0026] 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.
[0027] Still further, this invention contemplates KGF or is
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.
[0028] 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.
[0029] 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 segments of amino acid
sequences of KGF and at least one other member of the FGF peptide
family.
[0030] 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.
[0031] 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).
[0032] 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.
[0033] 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 active)
conformation. These antibodies can be used for detection or
purification of the KGF 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 OF THE DRAWINGS
[0034] FIG. 1 depicts results of Heparin-Sepharose affinity
chromatography of conditioned medium from M426 human embryonic
fibroblasts. Approximately 150 ml of ultrafiltration retentate
derived from five liters of M426 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 7.50/0.3M 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 elution was 108 ml/hr. Two .mu.l of
the indicated fractions were transferred to microtiter wells
containing a final volume of 0.2 ml for assay of .sup.3H-thymidine
incorporation in BALB/MK cells as described in the Methods.
[0035] FIGS. 2A, 2B, and 2C 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
C.sub.4HPLC of BALB/MK mitogenic activity. Active fractions eluted
from heparin-Sepharose with 0.6M NaCl were processed with the
Centricon -10 and loaded directly onto a C.sub.4 Vydac column
(4.6.times.250 mm) which had been equilibrated in 0.1%
trifluoroacetic 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 of
.sup.3H-thymidine incorporation in BALB/MK cells were promptly
diluted 10-fold with 50 .mu.g/ml bovine serum albumin/20 mM
Tris-HCl, pH 7.5, and tested at a final dilution of 200-fold. (B)
NaDodSO.sub.4/PAGE analysis of selected fractions from the C.sub.4
chromatography shown in-panel A. Half of each fraction was dried,
redissolved in NaDodSO.sub.4/2mercaptoethanol, 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
BALB/MK cells triggered by the fractions analyzed in Panel B.
Activity is expressed as the fold stimulation over background which
was 100 cpm.
[0036] FIG. 3 presents an alternative purification step to RP-HPLC,
using molecular sieving HPLC (TSK 3000SW) chromatography of the
BALB/MK mitogenic activity. Approximately 50 .mu.l of a
Centricon-processed, 0.6M NaCl pool from HSAC were loaded onto a
LKB GlasPac TSK G3000SW column (8.times.300 mm), previously
equilibrated in 20 mM Tris-KCl, pH. 6.8/0.5M NaCl, and eluted as
0.2 ml fractions at a flow rate of 0.4 ml/min. Aliquots of 2 .mu.l
Were transferred to microtiter wells containing a final volume of
0.2 ml for assay of .sup.3H-thymidine incorporation in BALB/MK
cells. The elution positions of molecular weight markers (mass in
kDa) were as indicated by the arrows.
[0037] FIG. 4 illustrates a comparison of BALB/MK DNA synthesis in
response to TSK-purified mitogen and other growth factors.
Incorporation of .sup.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, .cndot.______.cndot.; EGF,
.DELTA.______.DELTA.; aFGF, .smallcircle.______.smallcircle.; bFGF,
.largecircle.______.largecircle..
[0038] FIG. 5 shows comparative growth of BALB/MK cells in a
chemically defined medium in response to different combinations of
growth factors. Cultures were plated at a density of
2.5.times.10.sup.4 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 Ham's F12 medium supplemented with
transferring Na.sub.2SeO.sub.3, 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) EGF and dialyzed fetal calf serum (final
concentration, 10%); f) KGF and EGF; g) KGF and insulin; h) EGF and
insulin. Final concentrations of the growth factors were as
follows: EGF, 20 ng/ml; insulin, 10 .mu.g/ml; and KGF, 40
ng/ml.
[0039] FIG. 6 outlines a schematic representation of human KGF 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. Selected restriction sites are indicated.
[0040] FIG. 7 documents the KGF cDNA nucleotide and predicted amino
acid sequences. Nucleotides are numbered on the left; 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.
[0041] FIG. 8 shows identification of KGF mRNAs by Northern blot
analysis. Lanes a and c, poly(A)-selected M426 RNA; lanes b and d,
total cellular M426 RNA. Filters were hybridized with a
.sup.32P-labeled 695 bp BamHI/BclI fragment from clone 32 (Probe A,
FIG. 6), lanes a and b, or a 541 bp ApaI/EcoRI fragment from clone
49 (Probe B, FIG. 6), lanes c and d.
[0042] FIG. 9 illustrates the topological comparison of the FGF
family of related molecules, including KGF, with emphasis on the
two protein domains that share high homology (shaded boxes), the
putative signal peptide sequences (hatched boxes), and the two
conserved cysteine residues (positions labeled with a "C").
[0043] FIG. 10 shows northern blot analysis of KGF 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. 10 .mu.g of RNA were denatured and
electrophoresed in 1% formaldehyde gels. Following milk alkali
denaturation (50 mM NaOH for 30'), RNA was transferred to
nitrocellulose filters using 1 M ammonium acetate as a convectant.
Filters were hybridized to a .sup.32P-labelled cDNA probe
containing the 647 bp EcoRI fragment from the 5' end of the KGF
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
(B5/589); immortalized bronchial epithelial cells (S6 and R1);
keratinocytes immortalized with Ad12-SV40; primary human
keratinocytes; neonatal foreskin fibroblasts, (AG1523); adult skin
fibroblast-s (501T); and embryonic lung fibroblasts (WI-38 and
M426), 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 (HA 83) glial cell lines or in primary cultures of
human saphenous vein endothelial cells.
DESCRIPTION OF SPECIFIC EMBODIMENTS
[0044] This invention relates, in part, to purified KGF or KGF-like
proteins 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 NaDodSO.sub.4/PAGE, movement as a single peak
on reversed-phase high performance liquid chromatography, and a
specific activity of at least about 3.4.times.10.sup.4 units per
milligram, and preferably at least about 3.2.times.10.sup.5 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.
[0045] To identify novel growth factors specific for epithelial
cell types, a clonal BALB/c mouse keratinocyte cell line,
designated BALB/MK (Weissman, B. E. and Aaronson, S. A. (1983) Cell
32, 599-606) 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 (Weissman, B. B. and Aaronson, S. A. (1983) Cell
32, 599-606). 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 .alpha. (TGF.alpha.),
acidic fibroblast growth factor (aFGF) or basic fibroblast growth
factor (bFGF) (Falco, J. P., Taylor, W. G., DiFiore, P. P.,
Weissman, B. E., and Aaronson, S. A. (1988) Oncogene 2,
573-578).
[0046] By using BALB/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).
[0047] In brief, the bioassay for KGF-like activity under standard
conditions comprises the following steps:
[0048] (i) Mouse keratinocytes (BALB/MK cells) are grown in culture
to confluency and then maintained for 24-72 hr in serum-free
medium;
[0049] (ii) Following addition of test samples, stimulation of DNA
synthesis is determined by incorporation of .sup.3H-thymidine into
acid-precipitable DNA.
[0050] 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 50-fold) greater activity on
keratinocytes than on other exemplary epithelial cell types (see
Table 2 for further data, and Materials and Methods in Experimental
Section I for details of the standard conditions of the
bioassay).
[0051] 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 RPLC
(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.
[0052] To summarize, the method for production of KGF from
producing cells such as M426 human embryonic fibroblasts (Aaronson,
S. A. and Todaro, G. J. (1968) Virology 36, 254-261), for example,
comprises the following steps:
[0053] (i) Preparation of conditioned media (e.g., 10 liters) using
monolayer cultures cycled from serum-containing to serum-free
medium and storing the serum-free harvest at -70.degree. C. until
further use;
[0054] (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.degree. C.;
[0055] (iii) Affinity chromatography on heparin attached to a
polymeric support (e.g., Sepharose) with elution by a gradient of
increasing NaCl concentration;
[0056] (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.degree. C.
[0057] The next step of the purification process comprises either
step (v) or, alternatively, step (vi), as follows:
[0058] (v) Reversed-phase HPLC of active fractions (0.6 M NaCl
pool) from the previous HSAC step in organic solvent systems;
or,
[0059] (vi) Molecular sieve HPLC (e.g, on a TSK-G3000SW 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.degree.
C.
[0060] A preparation made by the TSK step (vi) was almost as pure
as one obtained from RP-HPLC, as judged by silver-stained
NaDodSO.sub.4,/PAGE (data not shown); but the TSK approach provided
a far better recovery of activity (Table 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 nM). 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.)
[0061] 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:
Asn-Asp-Met-Thr-Pro-Glu-Gln-Met-Ala-Thr-Asn-Val (see Experimental
Section I).
[0062] 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.
[0063] 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-Gln-Met-Ala. A cDNA library was
constructed in a cDNA cloning vector, XpCEV9, using poly-adenylated
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.times.10.sup.5 plaques) with the
.sup.32P-labelled oligonucleotides identified 88 plaques which
hybridized to both probes.
[0064] 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. 6).
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. 7.
[0065] 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.
[0066] From the cDNA sequence, it is apparent that the primary KGF
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.
[0067] Furthermore, KGF 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 FGF family members.
[0068] 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.
[0069] This invention also includes modified forms of KGF DNAs.
According to a chief embodiment of this aspect of the invention,
such modified DNAs encode. KGF-like proteins comprising segments of
amino acid sequences of KGF 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.
[0070] The polypeptide chimeras produced by such modified DNAs are
useful for determining whether the KGF NH.sub.2-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.
[0071] Indeed, the utility of this approach has already been
confirmed by the successful engineering and expression of a
chimeric molecule in which about 40 amino acids from the NH.sub.2--
terminus of the secreted form of KGF (beginning with the amino
terminal cys residue of the mature KGF form, numbered 32 in FIG. 7,
and ending at KGF residue 78, arg) is linked to about 140 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 lacks susceptibility to heparin, a
characteristic which parallels that of aFGF rather than KGF. 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. Further details of the construction of this chimeric
molecule and the properties of the polypeptide are described in
Experimental Section II.
[0072] 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 .lamda. cloning
vector (.lamda.pCEV9); a DNA sequencing plasmid vector (a pUC
variant); a bacterial expression vector (pKK233-2); or a mammalian
expression vector (pMMT/neo). Such recombinant DNAs are exemplified
by constructs described in detail in the Experimental Sections.
[0073] 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
N-terminal signal peptide) and a mammalian expression vector
(exemplified by pMMT) capable of expressing inserted DNAs in
mammalian (e.g., NIH/3T3) cells.
[0074] 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 trk
promoter in the plasmid expression vector pKK233-2 (Amman, E. and
Brosius, J. (1985) Gene 40, 183).
[0075] Construction of recombinant DNAs comprising KGF DNA and a
mammalian vector capable of expressing inserted DNAs 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.
[0076] 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 KGF 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.
[0077] Accordingly, this invention also comprises a cell,
preferably a bacterial or mammalian cell, transformed with a DNA of
the invention, wherein the transforming 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 KGF 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.
[0078] Recombinant E. coli 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 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.
[0079] One liter of bacterial cells yielded approximately 50 .mu.g
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.
[0080] Recombinant mammalian (NIH/3T3 mouse) cells have also been
constructed using the entire KGF cDNA coding sequence (including
the NH.sub.2-terminal signal peptide) and the vector pMMT/neo,
which carries mouse metallothionine (MMT) 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 (Sakai, R. K.,
Scharf, S., Faloona, F., Mullis, K. B., Norn, G. T., Erlich, H. A.
and Arnheim, N. (1985) Science 230, 1350-1354). 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 of this
invention.
[0081] 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 KGF-like peptides without undue
experimentation.
[0082] 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 namely, NDMTPEQMATNVR (using standard one-letter
code for amino acid sequences; see FIG. 7)]; polyclonal antibodies
raised in rabbits against both naturally secreted KGF from human
fibroblasts and recombinant KGF produced in E. coli (see
above).
[0083] All tested antibodies recognize the recombinant as well as
the naturally occurring KGF, either in a solid-phase (ELISA) assay
and/or in a Western blot. Some exemplary antibodies, which are
preferred antibodies of this invention, appear to neutralize
mitogenic activity of KGF in the BALB/MK bioassay.
[0084] 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.
[0085] 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.
[0086] 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).
[0087] 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.
[0088] 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 of various classes of tumor cells or genetic defects
in the epithelial growth processes.
[0089] 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.
Experimental Section I
Identification and Characterization of a Novel Growth Factor
Specific for Epithelial Cells
[0090] 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 cell 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.sub.4 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
KGF 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
nM and maximal at 1.0 nM. 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.
Methods and Materials
[0091] Preparation of Conditioned Media. an Early Passage of M426
human embryonic fibroblasts (Aaronson, S. A. and Todaro, G. J.
(1968) Virology 36, 254-261) was plated onto 175 cm.sup.2 T-flasks
and grown to confluence over 10-14 days in Dulbeccols 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 DMEM. After 72 hrs,
culture fluids were collected and replaced with 35 ml of
serum-containing medium. The conditioned medium was stored at
-70.degree. C. until further use.
[0092] Ultrafiltration. Approximately ten liters of conditioned
medium were thawed, prefiltered through a 0.50 micron filter
(Millipore HAWP 142 50) and concentrated to 200 ml using the
Pellicon cassette system (Millipore 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 ultra-filtration with the
Pellicon system. Activity recovered in the retentate was either
immediately applied to heparin-Sepharose resin or stored at
-70.degree. C.
[0093] Heparin-Sepharose Affinity Chromatography (HSAC).
[0094] The retentate from ultrafiltration was loaded onto
heparin-Sepharose resin (Pharmacia) which had been equilibrated in
20 nM Tris-HCl, pH 7.5/0.3 M NaCl. The resin washed extensively
until the optical density had returned to baseline and then
subjected to a linear-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.degree. C.
[0095] Reversed-Phase HPLC (RP-HPLC). 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 .ltoreq.200 .mu.l. The
sample was loaded onto a Vydac C.sub.4 HPLC column (The Separations
Group, Hesperia, Calif.) 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 of 50 .mu.g/ml BSA (Fraction V, Sigma)/20 mM
Tris-HCl, pH 7.5. The remainder of the sample was dried in a
Speed-Vac (Savant) in preparation for structural analysis.
[0096] Molecular Sieve HPLC. Approximately 50 .mu.l 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.degree. C.
[0097] NaDodSO.sub.4-Polyacrylamide Gel Electrophoresis
(NaDodSO.sub.4/PAGE). Polyacrylamide gels were prepared with
NaDodSO.sub.4 according to the procedure of Laemmli (Laemmli, U.K.
(1970) Nature 227, 680-685). Samples were boiled for 3 min in the
presence of 2.5% 2-mercaptoethanol (vol/vol). The gels were fixed
and stained with silver (Merril, C. R., Goldman, D., Sedman, S. A.
and Ebert, N. H. (1981) Science 211, 1437-1438) using the reagents
and protocol from BioRad. Molecular weight markers were from
Pharmacia.
[0098] DNA Synthesis Stimulation. Ninety-six well micro-liter
plates (Falcon No. 3596) were precoated with human fibronectin
(Collaborative Research) at 1 .mu.g/cm.sup.2 prior to seeding with
BALB/MK cells. Once confluent, the cells were maintained for 24-72
hr in serum-free medium containing 5 .mu.g/ml transferrin
(Collaborative Research) and 30 nM Na.sub.2SeO.sub.3 (Baker).
Incorporation of .sup.3H-thymidine (5 .mu.Ci/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 M NaOH, transferred into
liquid scintillation fluid (Biofluor, NEN) and counted.
[0099] Stimulation of DNA synthesis was monitored as described
above for BALB/MK cells on a variety of other cell lines. NIH/3T3
fibroblasts (Jainchill, J. L., Aaronson, S. A. and Todaro, G. J.
(1969) J. Virol. 4, 549-553) were available from the National
Institutes of Health, while CCL208 Rhesus monkey bronchial
epithelial cells (Caputo, J. L., Hay, R. J. and Williams, C. D.
(1979) In Vitro 15, 222-223) were obtained from the American Type
Culture Collection. The B5/589 human mammary epithelial cell line,
prepared as described in (Stampfer, M. R. and Bartley, J. C. (1985)
Proc. Nail. Acad. Sci. USA 82, 2394-2398), 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 (Sharefkin, J. B., Fairchild, K. D., Albus, R. A.,
Cruess, D. F. and Rich, N. M. (1986) J. Surgical Res. 41, 463-472).
Epidermal growth factor and insulin were from Collaborative
Research. Acidic FGF and bFGF were obtained from California
Biotechnology, Inc. Recombinant TGF.alpha. was obtained from
Genentech, Inc. Media and serum were either from GIBCO, Biofluids,
Inc. or the NIH media unit.
[0100] Proliferation Assay. Thirty-five mm culture dishes were
precoated sequentially with poly-D-lysine (20 .mu.g/ccm.sup.2)
(Sigma) and human fibronectin, and then seeded with approximately
2.5.times.10.sup.4 BALB/MK cells. The basic medium was a 1:1
mixture of Eagle's low Ca.sup.2+ minimal essential medium and Ham's
F-12 medium, supplemented with 5 .mu.g/ml transferrin, 30 nM
Na.sub.2SeO.sub.3 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.).
[0101] Protein microsequencing. Approximately 4 .mu.g (.about.150
.mu.mol) of protein from the active fractions of the C.sub.4 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).
Results
[0102] Growth Factor Detection and Isolation. Preliminary screening
of conditioned media from various cell lines indicated that media
from some fibroblast lines contained mitogenic activities
detectable on both BALB/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 (Cohen,
S. (1962) J. Biol. Chem. 237, 1555-1562) and TGF.alpha. (DeLarco,
J. E. and Todaro, G. J. (1978) Proc. Natl. Acad. Sci. USA. 75,
4001-4005), it was reasoned that the BALB/MK mitogenic activity
might be due to an agent different from these known epithelial
growth factors.
[0103] M426, a human embryonic lung fibroblast line, was selected
as the most productive source of this activity for purification of
the putative growth factor(s). Ultrafiltration with the Pellicon
system provided a convenient way of reducing the sample volume to a
suitable level for subsequent chromatography. Various 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 (Raines, E. W.
and Ross, R. (1982) J. Biol. Chem. 257, 5154-5160; Shing, Y.,
Folkman, J., Sullivan, R., Butterfield, C., Murray, J. and
Klagsburn, M. (1984) Science 223, 1296-1299; Gospodarowicz, D.,
Cheng, J., Lui, G.-M., Baird, A. and Bohlen, P. (1984) Proc. Natl.
Acad. Sci. USA 81, 6963-6967; Maciag, T., Mehlman, T., Friesel, R.
and Schreiber, A. B. (1984) Science 225, 932-935; Conn, G. and
Hatcher, V. B. (1984) Biochem. Biophys. Res. Comm. 124, 262-268;
Lobb, R. R. and Fett, J. W. (1984) Biochemistry 23, 6295-6299),
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.
[0104] As shown in FIG. 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.
[0105] 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 10-20
fold with the Centricon-10 was found to be essential for stability,
which could be maintained subsequently at -70.degree. C. for
several months.
[0106] Final purification was achieved by RP-HPLC with a C.sub.4
Vydac column, a preparative method suitable for amino acid sequence
analysis. While the yield of activity from the C.sub.4 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.
2A, 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 NaDodSO.sub.4/PAGE and
silver staining of the gel (FIG. 2B), and the relative mitogenic
activity of each tested fraction (FIG. 2C) correlated well with the
intensity of the bands across the activity profile.
[0107] An alternative purification step to the HPLC technique
described above, using sieving chromatography with a TSK G3000SW
GlasPac column run in aqueous solution near physiologic pH,
resulted in a major peak of activity in the BALB/MK bioassay (FIG.
3). This preparation was almost as pure as the one obtained from
RP-HPLC as judged by silver-stained NaDodSO.sub.4/PAGE (data not
shown) but provided a far better recovery of activity (Table 1).
The TSK-purified material was used routinely for biological studies
as it had a higher specific activity.
[0108] 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 NaDodSO.sub.4/PAGE which
appeared to be indistinguishable in the two preparations.
TABLE-US-00001 TABLE 1 Growth Factor Purification Total Specific
Purification Protein activity activity step (mg) (units)*
(units/mg) Conditioned medium 1.4 .times. 10.sup.3a 2.5 .times.
10.sup.4 1.8 .times. 10.sup.1 (10 liters) Ultrafiltration 1.3
.times. 10.sup.3a 3.2 .times. 10.sup.4 2.5 .times. 10.sup.1
(retentate) HSAC 0.73.sup.b 1.6 .times. 10.sup.4 2.2 .times.
10.sup.4 0.6 MM NaCl pool TSK-G3000 SW 8.4 .times. 10.sup.-3b 2.7
.times. 10.sup.3 3.2 .times. 10.sup.5 C.sub.4-HPLC 6.1 .times.
10.sup.-3b 2.1 .times. 10.sup.2 3.4 .times. 10.sup.4 *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. .sup.aProtein was estimated by using
the Bradford reagent from BioRad (Bradford, M. 1976, Anal. Biochem.
72, 248-254). .sup.bProtein was estimated by using A.sub.214.sup.1%
= 140.
[0109] Physical and Biological Characterization of the Growth
Factor. The purified factor had an estimated molecular weight of
about 28 kDa based on NaDodSO.sub.4/PAGE under reducing (FIG.
2A-2C) 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-G3000-SW, FIG. 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.
[0110] The heat and acid lability of the mitogenic activity were
demonstrated using the BALB/MK mitogenesis bioassay. While activity
was unaffected by a 10 min incubation at 50.degree. C., it was
reduced by 68% after 10 min at 60.degree. C. and was undetectable
after 3 min at 100.degree. C. Exposure to 0.5M 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.
[0111] The dose response curve for the purified growth factor
depicted in FIG. 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. 4).
[0112] 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 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.
[0113] By comparison, none of the other known growth factors
appeared to preferentially stimulate keratinocytes. TGF.alpha. and
EGF showed potent activity on fibroblasts, while the FGFs were
mitogenic for endothelial cells as well as fibroblasts (Table 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).
[0114] 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.
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. TABLE-US-00002 TABLE 2 Target Cell Specificity of Growth
Factors Endothelial Epithelial Fibroblast Human saphenous Growth
Factor BALK/MK BS/589 CCL208 NIH/3T3 vein KGF 500-1000 2-3 5-10
<1 <1 EGF 100-200 20-40 10-30 10-20 n.d. TGFa 150-300 n.d.
n.d. 10-20 n.d. aFGF* 300-500 2-3 5-10 50-70 5 bFGF 100-200 2-3 2-5
50-70 5 *Maximal stimulation by aFGF required the presence of
heparin (Sigma), 20 .mu.g/ml. n.d. = not determined.
[0115] Microsequencing Reveals a Unique N-terminal Amino Acid
Sequence of KGF. To further characterize the growth factor,
approximately 150 .mu.mol of C.sub.4-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-Gln-Met-Ala-Thr-Asn-Val. High
background noise precluded an assignment for the first position
which is, therefore, indicated by an X.
[0116] A computer search using the FASTP program (Lipman, D. J. and
Pearson, R. W. (1985) Science 227, 1435-1441) 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.
Discussion
[0117] The studies described in this Experimental Section
identified a human growth factor which has a unique specificity for
epithelial cells. By employing ultra-filtration, HSAC and RP-HPLC
or TSK sieving chromatography according to the present invention, a
quantity sufficient to permit detailed characterization of the
physical and biological properties of this molecule was
isolated.
[0118] 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.
[0119] 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, TGF.alpha. 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).
[0120] Handling and storage of KGF 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.degree. or -20.degree. C. Only concentrating
the sample at this stage could preserve its activity.
[0121] 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 -70.degree. C. where it remained
stable for several months.
[0122] The ability of KGF to bind heparin may signify a fundamental
property of this factor that has a bearing on its function in vivo.
Growth factors with heparin-binding properties include aFGF
(Maciag, T., Mehiman, T., Friesel, R. and Schreiber, A. B. (1984)
Science 225, 932-935; Conn, G. and Hatcher, V. B. (1984) Biochem.
Biophys. Res. Comm. 124, 262-268; Lobb, R. R. and Fett, J. W.
(1984) Biochemistry 23, 6295-6299), bFGF (Gospodarowicz, D., Cheng,
J., Lui, G.-M., Baird, A. and Bohlen, P. (1984) Proc. Natl. Acad.
Sci. USA 81, 6963-6967, Lobb, R. R. and Fett, J. W. (1984)
Biochemistry 23, 6295-6299) granulo-cyte/macrophage colony
stimulating factor (Roberts, R., Gallagher, J., Spooncer, E.,
Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988) Nature 332,
376-378) and interleukin 3 (Roberts, R., Gallagher, J., Spooncer,
E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988) Nature
332, 376-378). Each of these is produced by stromal cells (Roberts,
R., Gallagher, J., Spooncer, E., Allen, T. D., Bloomfield, F. and
Dexter, T. M. (1988) Nature 332, 376-378; Libermann, T. A.,
Friesel, R., Jaye, M., Lyall. R. M., Westermark, B., Drohen, W.,
Schmidt, A., Maciag, T. and Schlessinger, J. (1987) EMBO J., 61
1627-1632; Shipley, G. D., Sternfeld, M. D., Coffey, R. J. and
Pittelkow, M. R. (1988) J. Cell Biochem. Supp 12A, 125, abstr.
C420). Such factors appear to be deposited in the extracellular
matrix, or on proteoglycans coating the stromal cell surface
(Roberts, R., Gallagher, J., Spooncer, E., Allen, T. D.,
Bloomfield, F. and Dexter, T. M. (1988) Nature 332, 376-378,
Vlodavsky; I., Folkman, J., Sullivan, R., Fridman, R.,
Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987) Proc. Natl.
Acad. Sci. USA 84, 2292-2296). It has been postulated that their
storage, release and contact with specific target cells are
regulated by this interaction (Roberts, R., Gallagher, J.,
Spooncer, E., Allen, T. D., Bloomfield, F. and Dexter, T. M. (1988)
Nature 332, 376-378, Vlbdavsky, I., Folkman, J., Sullivan, R.,
Fridman, R., Ishai-Michaeli, R., Sasse, J. and Klagsburn, M. (1987)
Proc. Natl. Acad. Sci. USA 84, 2292-2296). While
mesenchymal-derived effectors of epithelial cell proliferation have
also been described (Gilchrest, B. A., Karassik, R. L., Wilkins, L.
M., Vrabel, M. A. and Maciag, T. (1983) J. Cell Physiol. 117,
2325-240, Chan, K. Y. and Haschke, R. H. (1983) Exp. Eye Res. 36,
231-246, Stiles, A. D., Smith, B. T. and Post, M. (1986) Exp. Lung
Res. 11, 165-177), 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.
[0123] 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
of growth factors, as described in Experimental Section II,
below.
Experimental Section II
cDNA Sequence of A Novel Epithelial Cell Specific Growth Factor
Defines a New Member of the FGF Family
[0124] Work in the previous Experimental Section I identified and
purified a novel heparin-binding growth factor, designated
keratinocyte growth factor (KGF), 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
N-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 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.
Materials and Methods
[0125] Isolation of cDNA clones. The purification and N-terminal
sequencing of KGF has been previously described (see Experimental
Section I, above and Rubin, J. S., Osada, H., Finch, P. W., Taylor,
W. G., Rudikoff, S. and Aaronson, S. A. (1989) Proc. Natl. Acad.
Sci. USA (in press), February, 1989). Pools (50 pmole) of
deoxyoligo-nucleotides described under Results were 5 end-labelled
using 83 pmole of .tau.-.sup.32P-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 .sup.32P-labelled deoxyoligonucleotides
in 20% formamide, 10% dextran sulphate, 10 mM Tris-HCl (pH 7.5),
8.times.SSC, 5.times. Denhardt's and 50 .mu.g/ml denatured salmon
sperm DNA, overnight at 42.degree. C. Filters were washed in
0.5.times.SSC, 0.1% SDS at 50.degree. C. and exposed to Kodak
X-omat AR film.
[0126] DNA sequencing. The nucleotide sequence of the KGF cDNA was
determined by the dideoxy chain termination method (Sanger, F.,
Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA
74, 5463-5467), of overlapping restriction fragments, subcloned
into pUC vectors (Yanisch-Perron, C., Vieira, J. and Messing, J.
(1985) Gene 33, 103-119)
[0127] Construction of a bacterial expression vector for KGF cDNA.
KGF cDNA encoding the mature, secreted form of the polypeptide was
placed under control of the hybrid trk promoter in the plasmid
expression vector pKK233-2 (Amman, E. and Brosius, J. (1985) Gene
40, 183), 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 (Sakai, R. K., Scharf,
S., Faloona, F., Mullis, K. B., Norn, G. T., Erlich, H. A. and
Arnheim, N. (1985) Science 230, 1350-1354). The fragment was
directionally inserted between two sites in the vector, namely the
NcoI site, made blunt ended by S1 nuclease digestion, and the
HindIII site, using standard recombinant DNA methodology. The ends
of the KGF cDNA produced by the PCR method were as follows: the 5'
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 KGF (see FIG. 7), 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
HindIII site for insertion of the amplified cDNA into the vector
DNA.
[0128] Production of antibodies against KGF and KGF-related
peptides. Monoclonal antibodies were 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 KGP from human
epithelial 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).
[0129] 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.
[0130] Polyclonal antibodies were raised in rabbits against both
naturally secreted KGF from human fibroblasts and recombinant KGF
produced in E. coli (see next section), using the following
protocol:
[0131] i) Initial injection and first boost were administered in
the inguinal lymph nodes;
[0132] 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.
Results
[0133] Isolation of cDNA clones encoding the novel growth 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-Gln-Met-Ala, as determined by
microsequencing of purified KGF (see Experimental Section I, above
and reference Rubin et al., Proc. Natl. Acad. Sci. USA 86:802-806
(1989). 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.
[0134] 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 (Crick, F. H. C. (1966) J. Mol. Biol.
19, 548-555), and oligonucleotides that contain deoxyinosine have
been shown to hybridize efficiently with the corresponding cDNA
(Ohtsuka, E., Matsuki, S., Ikehara, M., Takashi, Y. and Matsubara,
K. (1985) J. Biol. Chem. 260, 2605-2608).
[0135] A cDNA library was constructed in a cDNA cloning vector,
pCEV9 (Miki, T., Matsui, T., Heidaran, M. and Aaronson, S. A.,
unpublished observations) using poly-adenylated RNA extracted from
the human embryonic lung fibroblast cell line M426 (Aaronson, S. A.
and Todaro, G. J. 1968, Virology 36, 254-261), the initial source
of the growth factor. Screening of the library (9.times.10.sup.5
plaques) with the .sup.32P-labelled 26-mer oligonucleotides
identified 88 plaques which hybridized to both pools of
oligonucleotide probes.
[0136] Characterization and sequencing of selected cDNA clones. 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. Sequencing of a representative
smaller clone, designated clone 32, along with clone 49,
demonstrated that they were overlapping cDNAs (FIG. 6).
[0137] Description of the sequence encoding the KGF polypeptide.
Alignment of the two cDNAs (clones 3.2 and 49) established a
continuous sequence of 3.85 kb containing the complete KGF coding
sequence (FIG. 7). An ATG likely to be an initiation codon 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.
[0138] The sequence flanking the ATG codon did not conform to the
proposed GCC(G/A)CCATGG consensus for optimal initiation by
eukaryotic ribosomes (Kozak, M. (1987) Nucl. Acids Res. 13,
8125-8148), 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.
[0139] A 19-amino acid sequence that was homologous to the
experimentally determined N-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.
[0140] 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
(Lipman, D. J. and Pearson, R. W. (1985) Science 227, 1435-1443).
By this approach, a striking degree of relatedness between the
predicted primary structure of KGF and those of acidic and basic
FGF, as well as the related hst, FGF-5 and int-2-encoded proteins
was revealed.
[0141] Expression of mRNA transcripts of the KGF gene in human
cells. In preliminary attempts to examine expression of KGF mRNA in
human cells, a probe spanning the majority of the KGF coding
sequence (Probe A, FIG. 6) detected a single 2.4 kb transcript by
Northern blot analysis of total M426 RNA (FIG. 8). This was
considerably shorter than the length of the composite cDNA
sequence, 3.85 kb.
[0142] 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 B, FIG. 6), hybridized only to
the larger message (FIG. 8). Thus, it appears that the KGF gene is
transcribed as to alternate RNAs. Two other members of the FGF gene
family, bFGF (Abraham J. A., et al. (1986) Science 233, 545-548)
and int-2 (Mansour, S. L. and Martin, G. R. (1988) EMBO J. 1,
2035-2041), also express multiple RNAs, the significance of which
remains to be determined.
[0143] 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 FIG. 10, 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.
[0144] 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. 10). The EGF transcript was not expressed in
any of the same cell lines, and was only observed in kidney, among
the various tissues. Finally, the TGF.alpha. message was not
detected in any of the stromal fibroblast lines and was expressed
at varying levels in each of the epithelial cell lines. It was also
detected at low levels in kidney among the tissues examined (FIG.
10).
[0145] Inhibition of KGF mitogenic activity by heparin. 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 (Schreiber, et al., Proc. Natl. Acad. Sci. USA
82:6138-6142 (1985), Gospodarowizc et al., J. Cell Physiol.
128:475-485 (1986)). Despite binding tightly to bFGF, heparin had
minimal effects on its mitogenic activity (Gospodarowizc et al.,
supra). In view of the relatedness of KGF to the FGFs, the effect
of heparin on KGF mitogenic activity was examined. As shown in
Table 3, 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. TABLE-US-00003 TABLE 3 Effect of
Heparin on KGF Mitogen Activity. BALB/MK NIH/3T3 Growth Factor - +
- + KGF 150 9.5 <1 <1 aFGF 106 259 10.4 68 bFGF 30 124 45.7
70
[0146] 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 Rubin et
al., Proc. Natl. Acad. Sci. USA 86:802-806 (1989). Where indicated,
heparin was included in the culture media at a final concentration
of 20 .mu.g/ml. The concentration of all the growth factors was 50
ng/ml. The results represent fold stimulation of .sup.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.
[0147] Production of anti-KGF antibodies. 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
sequence; polyclonal antibodies raised in rabbits against both
naturally secreted KGF from human fibroblasts and recombinant KGF
produced in E. coli (see next section).
[0148] 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
BALB/MK bioassay.
[0149] 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. 7), 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 anti-bodies 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.
[0150] 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
BALB/MK bioassay.
[0151] Expression of KGF cDNA in E. coli. KGF cDNA was expressed to
produce polypeptide, in E. coli by placing its coding sequence
under control of the hybrid trk promoter (comprising elements of
trp and lac promoters), in the plasmid pKK233-2 (Amman, E. and
Brosius, J. (1985) Gene 40, 183). 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 (Sakai, R.
K., Scharf, S., Faloona, F., Mullis, K. B., Norn, G. T., Erlich, H.
A. and Arnheim, N. (1985) Science 230, 1350-1354). The fragment was
directionally inserted between two sites in the vector, namely the
NcoI site, made blunt ended by S1 nuclease digestion, and the
HindIII 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 trk promoter.
[0152] Several recombinants were tested for protein production by
the usual small scale methods. In brief, the clones were grown to
mid-exponential phase (OD.sub.595.about.0.5), treated with 1 mM
isopropyl .beta.-D-thiogalacto-pyranoside (IPTG) for 90 minutes,
and cell extracts were run on SDS-polyacrylamide gels for Western
blot analysis. All recombinants tested synthesized a protein that
was recognized by antibodies raised against an amino-terminal KGF
peptide. One recombinant was selected which showed the greatest
induction from IPTG, for further protein analyses.
[0153] One liter of bacteria was grown up in NZY broth containing
50 .mu.g/ml ampicillin and 12.5 .mu.g/ml tetracycline, to
OD.sub.5''-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.
[0154] 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
SDS-polyacrylamide gels and silver-staining.
[0155] 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 .mu.g of
Mono-S purified recombinant KGF.
[0156] Construction of a chimera containing KGF and aFGF sequences.
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.sub.2-terminal sequence of KGF grafted onto the C-terminal core
of aFGF was constructed, as follows. A Sau3AI restriction enzyme
site (GATC) in the 5' end of the KGF cDNA, within codons for
residues 76, 77, and 78 (Tyr, Leu, and Arg, respectively; see FIG.
7) was cut and joined to an homologous site in the aFGF cDNA within
codons for amino acids 39 (Arg) and 40. 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).
[0157] When recombinant E. coli 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 aFGF 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 aFGF. Thus the peptide
domains responsible for target cell specificity and heparin
sensitivity are clearly distinct and readily separable in KGF,
according to the practice of the present invention.
Discussion
[0158] 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.
[0159] 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.sub.4/PAGE at a higher molecular weight than predicted for
the purified protein. This may be accounted for by
glycosylation.
[0160] The FGFs are heparin-binding mitogens with broad target cell
specificities (Thomas, K. (1987) FASEB J. 1, 434-440). The hst gene
was identified as a transforming gene from a human stomach tumor
(Taira et al., Proc. Natl. Acad. Sci. USA 84: 2980-2984 (1987),
adjacent normal stomach tissue (Yoshida et al., Proc. Natl. Acad.
Sci. USA 84: 7305-7309 (1987), and from Kaposi's sarcoma
(Delli-Bovi et al., Proc. Natl. Acad. Sci. USA 84: 5660-5664
(1987), by standard NIH/3T3 transfection assays. The product of the
int-2 gene is expressed normally during mouse embryogenesis
(Jakobovits, A., Shackleford, G. M., Varmus, H. E. and Martin, G.
R. (1986) Proc. Natl. Acad. Sci. USA 83, 7806-7810) and aberrantly
after proviral integration of mouse mammary tumor virus (Peters,
G., Brookes, S. and Dickson, S. (1983) Cell 33, 364-377).
[0161] KGF is the sixth member of the fibroblast growth factor
family to be identified (Zhan, X., Bates, B., Hu, X. and Goldfarb,
M. (1988) Mol. Cell. Biol. 8, 3487-3495). While the name FGF-6 does
not seem suitable because KGF 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.
[0162] 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.
[0163] 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. 9). In the
case of int-2, the length of this sequence was 17 residues, while
in hst, the two homologous regions were contiguous. In KGF the
intervening sequence consisted of five amino acids.
[0164] In the aligned regions, the KGF amino acid 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 hst (human). In this same region, all
six proteins were identical at 19% of the residues, and allowing
for conservative substitutions, they showed 28% homology.
[0165] As shown in FIG. 9, the amino termini of these related
proteins are nonhomologous and of variable length. The primary KGF
and hot translation products contain hydrophobic N-terminal regions
which likely serve as signal sequences (von Heijne, G. (1986) Nucl.
Acids Res. 14, 4683-4690). The fact that this N-terminal domain is
not present in the mature KGF molecule (FIG. 7) further supports
this conclusion. In contrast, the FGFs are synthesized apparently
without signal peptides (Thomas, K. (1987) FASEB J. 1, 434-440).
The int-2 protein contains an atypically short region of N-terminal
hydrophobic residues (von Heijne, G. (1986) Nucl. Acids Res. 14,
4683-4690), but it is not known if the protein is secreted.
Moreover, the int-2 protein contains a long C-terminal extension
compared to the other family members.
[0166] Purified KGF contains five cysteine residues, two of which
are conserved throughout the family of FGF related proteins (FIG.
9). 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 (Schwarzbauer, J. E., Tamkum, J. M., Lemischka, I. R. and
Hynes, R. O. (1983) Cell 35, 421-431). Dibasic sites are also
common targets for proteolytic processing and such processing might
account for the microheterogeneity observed in some KGF
preparations (unpublished data).
[0167] 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 (Shaw, G. and Kamen, R. (1986) Cell 46, 659-667).
There was no classical AATAAA polyadenylation signal but two
variant sequences, AATTAAA and AATACA (Birnsteil, M. L.,
Busslinger, M. and Strub, K. (1985) Cell 41, 349-359), were
detected 24 and 19 nucleotides, respectively, upstream of the
poly(A) sequence at the 3' end of the cDNA.
[0168] It has been suggested that the heparin effect on acidic FGF
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 (Schrieber, A. B., Kenny, J., Kowalski, W.,
Friesel, J., Mehlman, T. and Maciag, T. (1985) Proc. Natl. Acad.
Sci. USA 82, 6138-6142, Gospodarowizc, O. and Cheng, J. (1986) J.
Cell Physiol. 128, 47.5-485). 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.
[0169] 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 (Thomas, K. A. and Giminez-Gallego, G. (1986) Trends
Biochem. Soc. 11, 81-84). FGF's are also mitogenic for glial
astrocytes and neuroblasts (Gensburger, C., Labourdette, G. and
Sensembrenner, M. (1987) FEBS Lett. 217, 1-5). The product of the
oncogene isolated from Kaposi's sarcoma, which is identical to hst,
also stimulates proliferation of NIH/3T3 and capillary endothelial
cells (Delli-Bovi, P., Curatola, A. M., Kern, F. G., Greco, A.,
Ittman, M. and Basilico, C. (1987) Cell 50, 729-737). 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 Rubin et al., Proc. Natl. Acad. Sci. USA 86:
802-806 (1989). It seems likely, therefore, that KGF acts through a
different cell surface receptor than the FGFs.
[0170] 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 FGF 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.
[0171] 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.
[0172] 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.
Sequence CWU 0
0
SEQUENCE LISTING <160> 3 <210> 1 <211> 3878
<212> DNA <213> Homo sapiens <220> <221>
CDS <222> (446)..(1030) <220> <221> polyA_signal
<222> (3824)..(3829) <220> <221> polyA_signal
<222> (3829)..(3834) <400> 1 acgcgctcac acacagagag
aaaatccttc tgcctgttga tttatggaaa caattatgat 60 tctgctggag
aacttttcag ctgagaaata gtttgtagct acagtagaaa ggctcaagtt 120
gcaccaggca gacaacagac atggaattct tatatatcca gctgttagca acaaaacaaa
180 agtcaaatag caaacagcgt cacagcaact gaacttacta cgaactgttt
ttatgaggat 240 ttatcaacag agttatttaa ggaggaatcc tgtgttgtta
tcaggaacta aaaggataag 300 gctaacaatt tggaaagagc aagtactctt
tcttaaatca atctacaatt cacagatagg 360 aagaggtcaa tgacctagga
gtaacaatca actcaagatt cattttcatt atgttattca 420 tgaacacccg
gagcactaca ctata atg cac aaa tgg ata ctg aca tgg atc 472 Met His
Lys Trp Ile Leu Thr Trp Ile 1 5 ctg cca act ttg ctc tac aga tca tgc
ttt cac att atc tgt cta gtg 520 Leu Pro Thr Leu Leu Tyr Arg Ser Cys
Phe His Ile Ile Cys Leu Val 10 15 20 25 ggt act ata tct tta gct tgc
aat gac atg act cca gag caa atg gct 568 Gly Thr Ile Ser Leu Ala Cys
Asn Asp Met Thr Pro Glu Gln Met Ala 30 35 40 aca aat gtg aac tgt
tcc agc cct gag cga cac aca aga agt tat gat 616 Thr Asn Val Asn Cys
Ser Ser Pro Glu Arg His Thr Arg Ser Tyr Asp 45 50 55 tac atg gaa
gga ggg gat ata aga gtg aga aga ctc ttc tgt cga aca 664 Tyr Met Glu
Gly Gly Asp Ile Arg Val Arg Arg Leu Phe Cys Arg Thr 60 65 70 cag
tgg tac ctg agg atc gat aaa aga ggc aaa gta aaa ggg acc caa 712 Gln
Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val Lys Gly Thr Gln 75 80
85 gag atg aag aat aat tac aat atc atg gaa atc agg aca gtg gca gtt
760 Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile Arg Thr Val Ala Val
90 95 100 105 gga att gtg gca atc aaa ggg gtg gaa agt gaa ttc tat
ctt gca atg 808 Gly Ile Val Ala Ile Lys Gly Val Glu Ser Glu Phe Tyr
Leu Ala Met 110 115 120 aac aag gaa gga aaa ctc tat gca aag aaa gaa
tgc aat gaa gat tgt 856 Asn Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu
Cys Asn Glu Asp Cys 125 130 135 aac ttc aaa gaa cta att ctg gaa aac
cat tac aac aca tat gca tca 904 Asn Phe Lys Glu Leu Ile Leu Glu Asn
His Tyr Asn Thr Tyr Ala Ser 140 145 150 gct aaa tgg aca cac aac gga
ggg gaa atg ttt gtt gcc tta aat caa 952 Ala Lys Trp Thr His Asn Gly
Gly Glu Met Phe Val Ala Leu Asn Gln 155 160 165 aag ggg att cct gta
aga gga aaa aaa acg aag aaa gaa caa aaa aca 1000 Lys Gly Ile Pro
Val Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr 170 175 180 185 gcc
cac ttt ctt cct atg gca ata act taa ttgcatatgg tatataaaga 1050 Ala
His Phe Leu Pro Met Ala Ile Thr 190 acccagttcc agcagggaga
tttctttaag tggactgttt tctttcttct caaaattttc 1110 tttcctttta
ttttttagta atcaagaaag gctggaaaaa ctactgaaaa actgatcaag 1170
ctggacttgt gcatttatgt ttgttttaag acactgcatt aaagaaagat ttgaaaagta
1230 tacacaaaaa tcagatttag taactaaagg ttgtaaaaaa ttgtaaaact
ggttgtacaa 1290 tcatgatgtt agtaacagta atttttttct taaattaatt
tacccttaag agtatgttag 1350 atttgattat ctgataatga ttatttaaat
attcctatct gcttataaaa tggctgctat 1410 aataataata atacagatgt
tgttatataa ggtatatcag acctacaggc ttctggcagg 1470 atttgtcaga
taatcaagcc acactaacta tggaaaatga gcagcatttt aaatgctttc 1530
tagtgaaaaa ttataatcta cttaaactct aatcagaaaa aaaattctca aaaaaactat
1590 tatgaaagtc aataaaatag ataatttaac aaaagtacag gattagaaca
tgcttatacc 1650 tataaataag aacaaaattt ctaatgctgc tcaagtggaa
agggtattgc taaaaggatg 1710 tttccaaaaa tcttgtatat aagatagcaa
cagtgattga tgataatact gtacttcatc 1770 ttacttgcca caaaataaca
ttttataaat cctcaaagta aaattgagaa atctttaagt 1830 ttttttcaag
taacataatc tatctttgta taattcatat ttgggaatat ggcttttaat 1890
aatgttcttc ccacaaataa tcatgctttt ttcctatggt tacagcatta aactctattt
1950 taagttgttt ttgaacttta ttgttttgtt atttaagttt atgttattta
taaaaaaaaa 2010 accttaataa gctgtatctg tttcatatgc ttttaatttt
aaaggaataa caaaactgtc 2070 tggctcaacg gcaagtttcc ctcccttttc
tgactgacac taagtctagc acacagcact 2130 tgggccagca aatcctggaa
gcagacaaaa ataagagcct gaagcaatgc ttacaataga 2190 tgtctcacac
agaacaatac aaatatgtaa aaactctttc accacatatt cttgccaatt 2250
aattggatca tataagtaaa atcattacaa atataagtat ttacaggatt ttaaagttag
2310 aatatatttg aatgcatggg tagaaaatat catattttaa aactatgtat
atttaaattt 2370 agtaattttc taatctctag aaatctctgc tgttcaaaag
gtggcagcac tgaaagttgt 2430 tttcctgtta gatggcaaga gcacaatgcc
caaaatagaa gatgcagtta agaataaggg 2490 gccctgaatg tcatgaaggc
ttgaggtcag cctacagata acaggattat tacaaggatg 2550 aatttccact
tcaaaagtct ttcattggca gatcttggta gcactttata tgttcaccaa 2610
tgggaggtca atatttatct aatttaaaag gtatgctaac cactgtggtt ttaatttcaa
2670 aatatttgtc attcaagtcc ctttacataa atagtatttg gtaatacatt
tatagatgag 2730 agttatatga aaaggctagg tcaacaaaaa caatagattc
atttaatttt cctgtggttg 2790 acctatacga ccaggatgta gaaaactaga
aagaactgcc cttcctcaga tatactcttg 2850 ggagagagca tgaatggtat
tctgaactat cacctgattc aaggactttg ctagctaggt 2910 tttgaggtca
ggcttcagta actgtagtct tgtgagcata ttgagggcag aggaggactt 2970
agtttttcat atgtgtttcc ttagtgccta gcagactatc tgttcataat cagttttcag
3030 tgtgaattca ctgaatgttt atagacaaaa gaaaatacac actaaaacta
atcttcattt 3090 taaaagggta aaacatgact atacagaaat ttaaatagaa
atagtgtata tacatataaa 3150 atacaagcta tgttaggacc aaatgctctt
tgtctatgga gttatacttc catcaaatta 3210 catagcaatg ctgaattagg
caaaaccaac atttagtggt aaatccattc ctggtagtat 3270 aagtcaccta
aaaaagactt ctagaaatat gtactttaat tatttgtttt tctcctattt 3330
ttaaatttat tatgcaaatt ttagaaaata aaatttgctc tagttacaca cctttagaat
3390 tctagaatat taaaactgta aggggcctcc atccctctta ctcatttgta
gtctaggaaa 3450 ttgagatttt gatacaccta aggtcacgca gctgggtaga
tatacagctg tcacaagagt 3510 ctagatcagt tagcacatgc tttctactct
tcgattatta gtattattag ctaatggtct 3570 ttggcatgtt tttgtttttt
atttctgttg agatatagcc tttacatttg tacacaaatg 3630 tgactatgtc
ttggcaatgc acttcataca caatgactaa tctatactgt gatgatttga 3690
ctcaaaagga gaaaagaaat tatgtagttt tcaattctga ttcctattca ccttttgttt
3750 atgaatggaa agctttgtgc aaaatataca tataagcaga gtaagccttt
taaaaatgtt 3810 ctttgaaaga taaaattaaa tacatgagtt tctaacaatt
agaaaaaaaa aaaaaaaaaa 3870 aaaaaaaa 3878 <210> 2 <211>
194 <212> PRT <213> Homo sapiens <400> 2 Met His
Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15
Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20
25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser
Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly
Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp
Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln
Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val
Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe
Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys
Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu
Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150
155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg
Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu
Pro Met Ala 180 185 190 Ile Thr <210> 3 <211> 14
<212> PRT <213> Artificial <220> <223>
Immunogenic Peptide corresponding to AA 33-45 of Fig 7 and SEQ ID 1
and where AA 45 N (ASN) is replaced with R (ARG) . <400> 3
Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Arg Arg 1 5 10
* * * * *