U.S. patent application number 12/036630 was filed with the patent office on 2008-10-16 for heregulin-like factor.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Mai Hijazi, C. Richter King, Steven M. Ruben, Paul E. Young.
Application Number | 20080254466 12/036630 |
Document ID | / |
Family ID | 46299544 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080254466 |
Kind Code |
A1 |
Young; Paul E. ; et
al. |
October 16, 2008 |
Heregulin-like Factor
Abstract
The present invention relates to a novel HLF protein which is a
member of the heregulin family. In particular, isolated nucleic
acid molecules are provided encoding the human HLF protein. HLF
polypeptides are also provided as are vectors, host cells and
recombinant methods for producing the same. The invention further
relates to screening methods for identifying agonists and
antagonists of HLF activity. Also provided are diagnostic methods
for detecting disorders of the regulation of cell growth and
therapeutic methods for treating disorders of the regulation of
cell growth.
Inventors: |
Young; Paul E.;
(Gaithersburg, MD) ; King; C. Richter;
(Washington, DC) ; Hijazi; Mai; (Washington,
DC) ; Ruben; Steven M.; (Brookeville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
Georgetown University Medical Center
Washington
DC
|
Family ID: |
46299544 |
Appl. No.: |
12/036630 |
Filed: |
February 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11354079 |
Feb 15, 2006 |
7335738 |
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12036630 |
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10609370 |
Jul 1, 2003 |
7049409 |
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11354079 |
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09097681 |
Jun 16, 1998 |
6727077 |
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10609370 |
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60049942 |
Jun 17, 1997 |
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Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/455; 435/6.1; 435/69.1; 530/350; 530/387.9;
536/23.1 |
Current CPC
Class: |
G01N 33/574 20130101;
C07K 14/4702 20130101; G01N 33/57488 20130101; C07H 21/04 20130101;
C07K 14/4756 20130101; G01N 2333/475 20130101 |
Class at
Publication: |
435/6 ; 536/23.1;
435/320.1; 435/455; 435/69.1; 530/350; 530/387.9 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 15/87 20060101 C12N015/87; C12P 21/04 20060101
C12P021/04; C07K 14/00 20060101 C07K014/00; C07K 16/18 20060101
C07K016/18 |
Claims
1. An isolated nucleic acid molecule nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence at least
95% identical to a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding the HLF polypeptide having the
amino acid sequence at positions 1 to 157 of SEQ ID NO:2) or the
complete amino acid sequence encoded by the cDNA clone contained in
ATCC Deposit No. 209123; (b) a nucleotide sequence encoding the
predicted extracellular domain of the HLF polypeptide having the
amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to 101 of SEQ
ID NO:2) or as encoded by the cDNA clone contained in ATCC Deposit
No. 209123; (c) a nucleotide sequence encoding the predicted
transmembrane domain of the HLF polypeptide having the amino acid
sequence in SEQ ID NO:2 (i.e., positions 102 to 121 of SEQ ID NO:2)
or as encoded by the cDNA clone contained in ATCC Deposit No.
209123; (d) a nucleotide sequence encoding the predicted
intracellular domain of the HLF polypeptide having the amino acid
sequence in SEQ ID NO:2 (i.e., positions 122 to 157 of SEQ ID NO:2)
or as encoded by the cDNA clone contained in ATCC Deposit No.
209123; (e) a nucleotide sequence encoding a soluble HLF
polypeptide having the extracellular and intracellular domains but
lacking the transmembrane domain; (f) the nucleotide sequence of
clone HAGFE38R (SEQ ID NO:4); (g) the nucleotide sequence of a
portion of the sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1)
wherein said portion comprises at least 50 contiguous nucleotides
from nucleotide about 1 to about 220 and from about 400 to 2199;
(h) the nucleotide sequence of a portion of the sequence shown in
FIGS. 1A and 1B (SEQ ID NO:1) wherein said portion consists of
residues 1 to 2199, 1 to 1500, 1 to 1000, 1 to 500, 1 to 250, 250
to 2199, 250 to 1500, 250 to 1000, 250 to 500, 500 to 2199, 500 to
1500, 500 to 1000, 1000 to 2199, and 1000 to 1500; and (i) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a) through (e) above.
2. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO:
1).
3. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO: 1)
encoding the HLF polypeptide having the amino acid sequence in
positions 1 to 157 of SEQ ID NO:2.
4. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO: 1)
encoding the extracellular domain of the HLF polypeptide having the
amino acid sequence from about 1 to about 101 in SEQ ID NO:2.
5. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence of the cDNA clone contained in
ATCC Deposit No. 209123.
6. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the HLF polypeptide having the
complete amino acid sequence encoded by the cDNA clone contained in
ATCC Deposit No. 209123.
7. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the extracellular domain of
the HLF polypeptide having the amino acid sequence encoded by the
cDNA clone contained in ATCC Deposit No. 209123.
8. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d), or (e) of claim 1
wherein said polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues.
9. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a HLF polypeptide having an amino acid sequence in (a), (b),
(c), (d), or (e) of claim 1.
10. The isolated nucleic acid molecule of claim 9, which encodes an
epitope-bearing portion of a HLF polypeptide wherein the amino acid
sequence of said portion is selected from the group of sequences in
SEQ ID NO:2 consisting of: about Ser-1 to about Thr-8, about Thr-9
to about Lys-18, about Thr-23 to about His-31, about Phe-32 to
about Leu-40, about Cys-43 to about Val-51, about Thr-56 to about
Tyr-68, about Gln-75 to about Leu-84, about Tyr-126 to about
Ala-135, about Ser-137 to about Leu-146, and about Ser-148 to about
Lys-157.
11. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
12. A recombinant vector produced by the method of claim 11.
13. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 12 into a host
cell.
14. A recombinant host cell produced by the method of claim 13.
15. A recombinant method for producing a HLF polypeptide,
comprising culturing the recombinant host cell of claim 14 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
16. An isolated HLF polypeptide comprising an amino acid sequence
at least 95% identical to a sequence selected from the group
consisting of: (a) the amino acid sequence of the HLF polypeptide
having the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,
positions 1 to 157 of SEQ ID NO:2) or the complete amino acid
sequence encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (b) the amino acid sequence of the predicted
extracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 1 to 101 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (c) the amino acid sequence of the predicted
transmembrane domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 102 to 121 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (d) the amino acid sequence of the predicted
intracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 122 to 157 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; and (e) the amino acid sequence of a soluble HLF
polypeptide having the extracellular and intracellular domains but
lacking the transmembrane domain.
17. The isolated HLF polypeptide of claim 16, which comprises an
epitope-bearing portion of the HLF protein, wherein said portion is
selected from the group consisting of: a polypeptide comprising
amino acid residues from about Ser-1 to about Thr-8, about Thr-9 to
about Lys-18, about Thr-23 to about His-31, about Phe-32 to about
Leu-40, about Cys-43 to about Val-51, about Thr-56 to about Tyr-68,
about Gln-75 to about Leu-84, about Tyr-126 to about Ala-135, about
Ser-137 to about Leu-146, and about Ser-148 to about Lys-157 of SEQ
ID NO:2.
18. An isolated antibody that binds specifically to a HLF
polypeptide of claim 16.
19. A method for diagnosing cancer in a human comprising, (a)
assaying HLF gene expression level in cells or body fluid of an
individual; and (b) comparing the HLF gene expression level with a
standard HLF gene expression level, whereby an increase or decrease
in the assayed HLF gene expression level compared to the standard
expression level is indicative of cancer in the tissue type
assayed.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of Ser. No. 11/354,079,
filed Feb. 15, 2006, which is a divisional of U.S. application Ser.
No. 10/609,370, filed Jul. 1, 2003, which is a divisional of U.S.
application Ser. No. 09/097,681, filed Jun. 16, 1998, now U.S. Pat.
No. 6,727,077, which claims benefit under 35 U.S.C. .sctn. 119(e)
of U.S. Provisional Application No. 60/049,942, filed on Jun. 17,
1997, which is herein incorporated by reference in its
entirety.
STATEMENT UNDER 37 C.F.R. .sctn. 1.77(b)(5)
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text document. The document is
entitled "PF383D3_SeqListing.txt", 141,312 bytes, created Feb. 5,
2008, and is hereby incorporated by reference in its entirety
herein.
FIELD OF THE INVENTION
[0003] The present invention relates to a novel human gene encoding
a polypeptide which is a novel member of the heregulin family. More
specifically, isolated nucleic acid molecules are provided encoding
a human polypeptide named heregulin-hke factor, hereinafter
referred to as "HLF". HLF polypeptides are also provided, as are
vectors, host cells and recombinant methods for producing the same.
Also provided are diagnostic methods for detecting disorders
related to primary cancers, and therapeutic methods for treating
such disorders. The invention further relates to screening methods
for identifying agonists and antagonists of HLF activity.
BACKGROUND OF THE INVENTION
[0004] The proto-oncogene termed erbB2 (or HER-2) encodes a 185 kDa
transmembrane tyrosine kinase molecule designated p185erbB2. The
overexpression of this receptor molecule correlates strongly with a
poor prognosis in a number of human cancers including, among
others, breast, ovarian, endometrium, fallopian tube, cervix, and
colon (Nowak, F., et al., Exp. Cell Res. 231:251-259; 1997;
Cirisano, F. D. and Karlan, B. Y., J. Soc. Gynecol. Investig.
3(3):99-105; 1996). Variously spliced transcripts of the heregulin
(HRG) gene have been found to indirectly stimulate p185erbB2
through transphosphorylation or receptor heterodimerization with
erbB3 and p180erbB4. A 45 kDa protein, designated HRG-alpha,
specifically induces tyrosine phosphorylation of p185erbB2 and has
been purified from the conditioned medium of a human breast tumor
cell line (Holmes, W. E., et al., Science 256:1205-1210; 1992). A
second, related HRG molecule of 52 kDa, which may be the product of
a novel gene, rather than a novel HRG gene splice product, has been
identified which exhibits similar characteristics including
induction of transient membrane ruffling, lamellipodia formation,
cell motility and proliferation of human breast cancer cells (Kung,
W., et al., Biochem. Biophys. Res. Commun. 202(3):1357-1365; 1994).
In addition, more recent studies have reported that heregulins can
induce tyrosine phosphorylation not only of p185erbB2, but of
several additional EGFR-related family members including erbB3 and
p180erbB4 (Tzahar, E., et al., J. Biol. Chem. 269:25226-25223;
1994; Plowman, G. D., et al., Nature 366:473-475; 1993).
[0005] Lewis and colleagues (Cancer Res. 56:1457-1465; 1996)
recently performed an extensive analysis of the effects of the
heregulin family of proteins on a panel of breast and ovarian tumor
cell lines. The biological responses to HRG were also compared to
EGF and to the growth-inhibitory anti-ErbB2 antibody 4D5. In nearly
all cases, HRG stimulation of DNA synthesis correlated with
positive effects on cell cycle progression and cell number and with
enhancement of colony formation in soft agar. In addition to the
effects of the heregulin family of proteins on breast and ovarian
cells, similar effects have also been recently observed on human
Schwann cells (Levi, A. D., et al., J. Neurosci. 15(2):1329-1340;
1995; Morrissey, T. K., et al., Proc. Natl. Acad. Sci. USA
92(5):1431-1435; 1995) suggesting that the heregulin family of
proteins play a key role in the genesis of a number of cancers.
[0006] The heregulin family of proteins consists at least of a
number of splice variants of heregulin, the Neu differentiating
factor, the glial growth factors-I, -II, and -III, and a protein
that stimulates muscle acetylcholine receptor synthesis (ARIA). In
addition to the obvious role such polypeptides may play in
oncogenic events, these proteins have also been exploited as
Pseudomonas exotoxin A fusion proteins to inhibit the growth of
several mammary carcinoma cell lines as well as to cause growth
retardation of transplanted human breast tumor cells in mice
(Jeschke, M., et al., Int. J. Cancer 60(5):730-739; 1995).
[0007] Thus, there is a need for polypeptides that function as
regulators of oncogenic events and existing tumors. Therefore,
there is a need for identification and characterization of such
human polypeptides which can play a role in detecting, preventing,
ameliorating or correcting such disorders.
SUMMARY OF THE INVENTION
[0008] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding at least a portion
of the HLF polypeptide having the complete amino acid sequence
shown in SEQ ID NO:2 or the complete amino acid sequence encoded by
the cDNA clone was deposited as DNA plasmid with the American Type
Culture Collection ("ATCC") on Jun. 19, 1997, and assigned ATCC
Deposit Number 209123. The ATCC is located at 10801 University
Boulevard, Manassas, Va. 20110-2209. The nucleotide sequence
determined by sequencing the deposited HLF clone, which is shown in
FIGS. 1A and 1B (SEQ ID NO: 1), contains an open reading frame
encoding a complete polypeptide of 157 amino acid residues,
beginning in frame with a serine residue at the amino-terminal end
of the polypeptide corresponding to nucleotide positions 2-4, and a
predicted molecular weight of about 17.7 kDa. Nucleic acid
molecules of the invention include those encoding the complete
amino acid sequence shown in SEQ ID NO:2, or the complete amino
acid sequence encoded by the cDNA clone in ATCC Deposit Number
209123, which molecules also can encode additional amino acids
fused to the N-terminus of the HLF amino acid sequence.
[0009] The HLF protein of the present invention shares sequence
homology with the translation product of the human mRNA for
heregulin (FIG. 2; SEQ ID NO:3), including the following conserved
domains: (a) the predicted extracellular domain of about 101 amino
acids; (b) the predicted transmembrane domain of about 19 amino
acids, and (c) the predicted intracellular domain of about 35 amino
acids. Heregulin is thought to be important in oncogenesis. The
homology between heregulin and HLF indicates that HLF may also be
involved in oncogenesis.
[0010] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide comprising a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the HLF polypeptide having the
complete amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to
157 of SEQ ID NO:2) or the complete amino acid sequence encoded by
the cDNA clone contained in ATCC Deposit No. 209123; (b) a
nucleotide sequence encoding the predicted extracellular domain of
the HLF polypeptide having the amino acid sequence in SEQ ID NO:2
(i.e., positions 1 to 101 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (c) a nucleotide
sequence encoding the predicted transmembrane domain of the HLF
polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e.,
positions 102 to 121 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (d) a nucleotide
sequence encoding the predicted intracellular domain of the HLF
polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e.,
positions 122 to 157 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (e) a nucleotide
sequence encoding a soluble HLF polypeptide having the
extracellular and intracellular domains but lacking the
transmembrane domain; and (f) a nucleotide sequence complementary
to any of the nucleotide sequences in (a) through (e) above.
[0011] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical to (or as stated in
another way, a nucleotide sequence at most 10% different, and more
preferably 5%, 4%, 3%, 2% or 1% different from), any of the
nucleotide sequences in (a) through (f) above, or a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide in (a) through (f) above. This polynucleotide which
hybridizes does not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence
consisting of only A residues or of only T residues. An additional
nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the
amino acid sequence of an epitope-bearing portion of a HLF
polypeptide having an amino acid sequence in (a) through (e)
above.
[0012] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of HLF polypeptides or peptides by
recombinant techniques.
[0013] The invention further provides an isolated HLF polypeptide
comprising an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of the HLF polypeptide
having the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,
positions 1 to 157 of SEQ ID NO:2) or the complete amino acid
sequence encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (b) the amino acid sequence of the predicted
extracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 1 to 101 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (c) the amino acid sequence of the predicted
transmembrane domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 102 to 121 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (d) the amino acid sequence of the predicted
intracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 122 to 157 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; and (e) the amino acid sequence of a soluble HLF
polypeptide having the extracellular and intracellular domains but
lacking the transmembrane domain. The polypeptides of the present
invention also include polypeptides having an amino acid sequence
at least 80% identical (or at most 20% different), more preferably
at least 90% identical (or at most 10% different), and still more
preferably 95%, 96%, 97%, 98% or 99% identical to (or 5%, 4%, 3%,
2% or 1% different from) those described in (a), (b), (c), (d), or
(e) above, as well as polypeptides having an amino acid sequence
with at least 90% similarity, and more preferably at least 95%
similarity, to those above.
[0014] An additional embodiment of this aspect of the invention
relates to a peptide or polypeptide which comprises the amino acid
sequence of an epitope-bearing portion of a HLF polypeptide having
an amino acid sequence described in (a), (b), (c), (d), or (e)
above. Peptides or polypeptides having the amino acid sequence of
an epitope-bearing portion of a HLF polypeptide of the invention
include portions of such polypeptides with at least six or seven,
preferably at least nine, and more preferably at least about 30
amino acids to about 50 amino acids, although epitope-bearing
polypeptides of any length up to and including the entire amino
acid sequence of a polypeptide of the invention described above
also are included in the invention.
[0015] In another embodiment, the invention provides an isolated
antibody that binds specifically to a HLF polypeptide having an
amino acid sequence described in (a), (b), (c), (d), or (e) above.
The invention further provides methods for isolating antibodies
that bind specifically to a HLF polypeptide having an amino acid
sequence as described herein. Such antibodies are useful
diagnostically or therapeutically as described below.
[0016] The invention also provides for pharmaceutical compositions
comprising HLF polypeptides, particularly human HLF polypeptides,
which may be employed, for instance, to treat many types of cancer.
Methods of treating individuals in need of HLF polypeptides are
also provided.
[0017] The invention further provides compositions comprising a HLF
polynucleotide or an HLF polypeptide for administration to cells in
vitro, to cells ex vivo and to cells in vivo, or to a multicellular
organism. In certain particularly preferred embodiments of this
aspect of the invention, the compositions comprise a HLF
polynucleotide for expression of a HLF polypeptide in a host
organism for treatment of disease. Particularly preferred in this
regard is expression in a human patient for treatment of a
dysfunction associated with aberrant endogenous activity of a
HLF
[0018] The present invention also provides a screening method for
identifying compounds capable of enhancing or inhibiting a
biological activity of the HLF polypeptide, which involves
contacting a receptor which is inhibited or enhanced by the HLF
polypeptide with the candidate compound in the presence of an HLF
polypeptide, assaying changes in tyrosine phosphorylation states of
the receptor and/or other molecules downstream in the corresponding
signal transduction cascade in the presence of the candidate
compound and of HLF polypeptide, and comparing the receptor
activation state to a standard level, the standard being assayed
when contact is made between the receptor and in the presence of
the HLF polypeptide and the absence of the candidate compound In
this assay, an increase in receptor activation state over the
standard downstream in the corresponding signal transduction
cascade in the presence of the candidate compound and of HLF
polypeptide, and comparing the receptor activation state to a
standard level, the standard being assayed when contact is made
between the receptor and in the presence of the HLF polypeptide and
the absence of the candidate compound In this assay, an increase in
receptor activation state over the standard indicates that the
candidate compound is an agonist of HLF activity and a decrease in
receptor activation state compared to the standard indicates that
the compound is an antagonist of HLF activity.
[0019] In another aspect, a screening assay for agonists and
antagonists is provided which involves determining the effect a
candidate compound has on HLF binding to a receptor. In particular,
the method involves contacting the receptor with an HLF polypeptide
and a candidate compound and determining whether HLF polypeptide
binding to the receptor is increased or decreased due to the
presence of the candidate compound. In this assay, an increase in
binding of HLF over the standard binding indicates that the
candidate compound is an agonist of HLF binding activity and a
decrease in HLF binding compared to the standard indicates that the
compound is an antagonist of HLF binding activity.
[0020] It has been discovered that HLF is expressed only in the
amygdala, whole brain, and primary breast culture tissue.
Therefore, nucleic acids of the invention are useful as
hybridization probes for differential identification of the
tissue(s) or cell type(s) present in a biological sample.
Similarly, polypeptides and antibodies directed to those
polypeptides are useful to provide immunological probes for
differential identification of the tissue(s) or cell type(s). In
addition, for a number of disorders of the above tissues or cells,
particularly of the neural system, significantly higher or lower
levels of HLF gene expression may be detected in certain tissues
(e.g., cancerous and wounded tissues) or bodily fluids (e.g.,
serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to a "standard" HLF
gene expression level, i.e., the HLF expression level in healthy
tissue from an individual not having the neural system disorder.
Thus, the invention provides a diagnostic method useful during
diagnosis of such a disorder, which involves: (a) assaying HLF gene
expression level in cells or body fluid of an individual; (b)
comparing the HLF gene expression level with a standard HLF gene
expression level, whereby an increase or decrease in the assayed
HLF gene expression level compared to the standard expression level
is indicative of disorder in the neural system.
[0021] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of HLF
activity in the body comprising administering to such an individual
a composition comprising a therapeutically effective amount of an
isolated HLF polypeptide of the invention or an agonist
thereof.
[0022] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
HLF activity in the body comprising, administering to such an
individual a composition comprising a therapeutically effective
amount of an HLF antagonist. Preferred antagonists for use in the
present invention are HLF-specific antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A and 1B shows the nucleotide sequence (SEQ ID NO:1)
and deduced amino acid sequence (SEQ ID NO:2) of HLF. An
extracellular epidermal growth factor (EGF) domain, conserved in
many other EGF-like polypeptides, is underlined in FIGS. 1A and
1B.
[0024] FIG. 2 shows the regions of identity between the amino acid
sequences of the HLF protein and translation product of the human
mRNA for heregulin (SEQ ID NO:3), determined by the computer
program Bestfit (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711) using the default
parameters.
[0025] FIG. 3 shows an analysis of the HLF amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic
Index--Jameson-Wolf" graph, the positive peaks indicate locations
of the highly antigenic regions of the HLF protein, i.e., regions
from which epitope-bearing peptides of the invention can be
obtained.
[0026] FIG. 4 shows a demonstration of the biochemical activity of
a recombinant EGF domain of the HLF protein (designated "H" in the
figure; as described in Example 5). The figure shows a Western blot
of MCF-7 cell lysates prepared from cultures which were treated or
mock-treated with recombinant EGF domain of the HLF protein or with
recombinant heregulin. The blots were immunoblotted with an
anti-phosphotyrosine monoclonal antibody.
[0027] FIG. 5 shows the amino acid sequences of the EGF/heregulin
family of growth factors and of the NRG-3 novel sequences.
Cysteines (C) defining the basic structure of the EGF domain and
highly conserved amino acids are in bold. Listed are sequences for
the EGF-like domains of transforming growth factor (TGF)-alpha (SEQ
ID NO:11); epidermal growth factor (EGF; SEQ ID NO:12);
heparin-binding EGF (HB-EGF; SEQ ID NO: 13); amphiregulin (Amph;
SEQ ID NO: 14); beta-cellulin (beta-cellulin; SEQ ID NO:15);
neuregulin (neuR; SEQ ID NO:16); human heregulins 1-alpha
(HRG-alpha-1; SEQ ID NO:17) and 1-beta (HRG-beta-1; SEQ ID NO:18);
heregulin-related gene (HRG)-2 (SEQ ID NO:19); and amino acids
29-80 of HLF of the present invention (amino acids 29-80 of SEQ ID
NO:2).
DETAILED DESCRIPTION
[0028] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a HLF polypeptide
having the amino acid sequence shown in SEQ ID NO:2, which was
determined by sequencing a cloned cDNA. The nucleotide sequence
shown in FIGS. 1A and 1B (SEQ ID NO: 1) was obtained by sequencing
the HAGFE38 clone, which was deposited on Jun. 19, 1997 at the
American Type Culture Collection, 12301 Park Lawn Drive, Rockville,
Md. 20852 (the ATCC is now located at 10801 University Blvd.,
Manassas, Va. 20110-2209), and given accession number ATCC 209123.
The deposited clone is contained in the pBluescript SK(-) plasmid
(Stratagene, La Jolla, Calif.).
[0029] The HLF protein of the present invention shares sequence
homology with the translation product of the human mRNA for
heregulin (FIG. 2; SEQ ID NO:3). Heregulin is thought to be an
important molecule in the activation pathways of the erbB family of
cell surface receptors. Altered expression of heregulin and related
ligand molecules, and/or the erbB family of receptor molecules can
often lead to the loss of regulation of cellular growth and
ultimately to oncogenesis. For example, the neu differentiation
factor (NDF) is a homologue of both heregulin and HLF. NDF is a
neuron/glia-specific signaling molecule which has been observed to
regulate survival, proliferation, and maturation of Schwann cell
precursors (Dong, Z., et al., Neuron 15:585-596; 1995; Marchionni,
M. A., et al., Nature 362:312-318; 1993). HLF is a member of the
same heregulin family of proteins and has, at least, activities
similar to those described above for heregulin and NDF.
[0030] Nucleic Acid Molecules
[0031] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90% identical
(or 10% different), more typically at least about 95% to at least
about 99.9% identical to (or at most about 5% to at most about 0.1%
different from) the actual nucleotide sequence of the sequenced DNA
molecule. The actual sequence can be more precisely determined by
other approaches including manual DNA sequencing methods well known
in the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0032] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0033] Using the information provided herein, such as the
nucleotide sequence in FIGS. 1A and 1B (SEQ ID NO: 1), a nucleic
acid molecule of the present invention encoding a HLF polypeptide
may be obtained using standard cloning and screening procedures,
such as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in FIGS. 1A and 1B (SEQ ID NO: 1) was discovered in a cDNA library
derived from human amygdala.
[0034] The determined nucleotide sequence of the HLF cDNA of FIGS.
1A and 1B (SEQ ID NO:1) contains an open reading frame encoding a
protein of 157 amino acid residues, with an amino-terminal serine
codon at nucleotide positions 2-4 of the nucleotide sequence in
FIG. 1A (SEQ ID NO: 1), and a deduced molecular weight of about
17.7 kDa. The amino acid sequence of the HLF protein shown in SEQ
ID NO:2 is about 32.7% identical to human mRNA for heregulin (FIG.
2). The nucleotide and amino acid sequence surface receptors, in
the regulation of cellular growth control, and ultimately in the
regulation of oncogenesis.
[0035] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, the actual
complete HLF polypeptide encoded by the deposited cDNA, which
comprises about 157 amino acids, may be somewhat longer or shorter.
More generally, the actual open reading frame may be anywhere in
the range of .+-.100 amino acids, .+-.20 amino acids, more likely
in the range of .+-.10 amino acids, of that predicted from the
serine codon at the N-terminus shown in FIG. 1A (SEQ ID NO:1). It
will further be appreciated that, depending on the analytical
criteria used for identifying various functional domains, the exact
"address" of the extracellular, EGF, transmembrane, and
intracellular domains of the HLF polypeptide may differ slightly
from the predicted positions above. For example, the exact location
of the HLF EGF domain in SEQ ID NO:2 may vary slightly (e.g., the
address may "shift" by about 1 to about 20 residues, more likely
about 1 to about 5 residues) depending on the criteria used to
define the domain. In this case, the ends of the transmembrane
domain and the beginning of the EGF domain were predicted on the
basis of the identification of the conserved cysteine residues at
positions 35, 43, 49, 62, 64, and 73 of SEQ ID NO:2, as shown in
FIG. 1A. In any event, as discussed further below, the invention
further provides polypeptides having various residues deleted from
the N-terminus of the complete polypeptide, including polypeptides
lacking one or more amino acids from the N-terminus of the
extracellular EGF domain described herein, which constitute soluble
forms of the extracellular EGF domain of the HLF protein.
[0036] Leader and Mature Sequences
[0037] In addition, methods for predicting whether a protein has a
secretory leader as well as the cleavage point for that leader
sequence are available. For instance, the method of McGeoch (Virus
Res. 3:271-286; 1985) uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje (Nucleic Acids Res.
14:4683-4690; 1986) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2 where
+1 indicates the amino terminus of the mature protein. The accuracy
of predicting the cleavage points of known mammalian secretory
proteins for each of these methods is in the range of 75-80% (von
Heinje, supra). However, the two methods do not always produce the
same predicted cleavage point(s) for a given protein.
[0038] In the present case, the deduced amino acid sequence of the
complete HLF polypeptide was analyzed by the computer program
PSORT, available from Dr. Kenta Nakai of the Institute for Chemical
Research, Kyoto University (Nakai, K. and Kanehisa, M., Genomics
14:897-911; 1992), which is an expert system for predicting the
cellular location of a protein based on the amino acid sequence. As
part of this computational prediction of localization, the methods
of McGeoch and von Heinje are incorporated. The analysis of the HLF
amino acid sequence by this program indicated that there appears to
be no N-terminal signal sequence associated with the HLF amino acid
sequence shown in SEQ ID NO:2, and that the HLF molecule, as shown
in SEQ ID NO:2, appears to be a type Ib membrane protein.
[0039] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0040] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0041] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF)
beginning in frame with a serine codon at positions 24 of the
nucleotide sequence shown in FIG. 1A (SEQ ID NO: 1).
[0042] In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode the HLF
protein. Of course, the genetic code and species-specific codon
preferences are well known in the art. Thus, it would be routine
for one skilled in the art to generate the degenerate variants
described above, for instance, to optimize codon expression for a
particular host (e.g., change codons in the human mRNA to those
preferred by a bacterial host such as E. coli).
[0043] In another aspect, the invention provides isolated nucleic
acid molecules encoding the HLF polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC Deposit No. 209123 on Jun. 19, 1997.
[0044] The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in FIGS. 1A and 1B
(SEQ ID NO: 1) or the nucleotide sequence of the HLF cDNA contained
in the above-described deposited clone, or a nucleic acid molecule
having a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as
probes for gene mapping, by in situ hybridization with chromosomes,
and for detecting expression of the HLF gene in human tissue, for
instance, by Northern blot analysis.
[0045] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. In particular, the invention provides a
polynucleotide having a nucleotide sequence representing the
portion of SEQ ID NO: 1 which consists of positions 1-2199 of SEQ
ID NO: 1.
[0046] In addition, the invention provides nucleic acid molecules
having nucleotide sequences related to a portion of SEQ ID NO: 1
which has been determined from the following related cDNA clone:
HAGFE38R.
[0047] Further, the invention includes a polynucleotide comprising
any portion of at least about 30 nucleotides, preferably at least
about 50 nucleotides, of SEQ ID NO: 1 from residue about 1 to about
220 and from about 400 to 2199. More preferably, the invention
includes a polynucleotide comprising nucleotide residues 1 to 2199,
1 to 1500, 1 to 1000, 1 to 500, 1 to 250, 250 to 2199, 250 to 1500,
250 to 1000, 250 to 500, 500 to 2199, 500 to 1500, 500 to 1000,
1000 to 2199, and 1000 to 1500.
[0048] More generally, by a fragment of an isolated nucleic acid
molecule having the nucleotide sequence of the deposited cDNA or
the nucleotide sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) is
intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which are
useful as diagnostic probes and primers as discussed herein. Of
course, larger fragments 50-300 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in FIGS. 1A and 1B (SEQ ID NO: 1). By a fragment
at least 20 nt in length, for example, is intended fragments which
include 20 or more contiguous bases from the nucleotide sequence of
the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A
and 1B (SEQ ID NO:1). Preferred nucleic acid fragments of the
present invention include nucleic acid molecules encoding
epitope-bearing portions of the HLF polypeptide as identified in
FIG. 3 and described in more detail below. Preferred nucleic acid
fragments of the present invention also include nucleic acid
molecules encoding Garnier-Robson and/or Chou-Fasman alpha, beta,
and/or turn regions, Garnier-Robson coil regions, Kyte-Doolittle
hydrophilic regions, Hopp-Woods hydrophobic regions, Eisenberg
alpha and/or beta amphipathic regions, Karplus-Schulz flexible
regions, Jameson-Wolf antigenic regions, and/or Emini surface
probability regions of the HLF polypeptide as identified in FIG. 3
or in a tabular representation of the data presented in FIG. 3.
[0049] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATCC
Deposit No. 209123, or, for example, any specific HLF
polynucleotide fragment described above (a non-limiting example is
a Chou-Fasman alpha turn region). By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0050] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below.
[0051] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A
and 1B (SEQ ID NO: 1)). Of course, a polynucleotide which
hybridizes only to a poly A sequence (such as the 3' terminal
poly(A) tract of the HLF cDNA shown in FIG. 1B (SEQ ID NO: 1)), or
to a complementary stretch of T (or U) residues, would not be
included in a polynucleotide of the invention used to hybridize to
a portion of a nucleic acid of the invention, since such a
polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0052] As indicated, nucleic acid molecules of the present
invention which encode an HLF polypeptide may include, but are not
limited to those encoding the amino acid sequence of the complete
polypeptide, by itself; and the coding sequence for the complete
polypeptide and additional sequences, such as those encoding an
added secretory leader sequence, such as a pre-, or pro- or
prepro-protein sequence.
[0053] Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0054] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz et al, Proc. Natl.
Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for convenient purification of the fusion protein. The
"HA" tag is another peptide useful for purification which
corresponds to an epitope derived from the influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984). As discussed below, other such fusion proteins include the
HLF fused to Fc at the N- or C-terminus.
[0055] Variant and Mutant Polynucleotides
[0056] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the HLF protein. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0057] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the HLF protein or portions thereof. Also especially
preferred in this regard are conservative substitutions.
[0058] Most highly preferred are nucleic acid molecules encoding
the EGF domain of the protein having the amino acid sequence shown
in SEQ ID NO:2 or the EGF domain of the HLF amino acid sequence
encoded by the deposited cDNA clone (nucleotides 77-280 of SEQ ID
NO: 1).
[0059] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the HLF polypeptide having the
complete amino acid sequence in SEQ ID NO:2 (i.e., positions 1 to
157 of SEQ ID NO:2) or the complete amino acid sequence encoded by
the cDNA clone contained in ATCC Deposit No. 209123; (b) a
nucleotide sequence encoding the predicted extracellular domain of
the HLF polypeptide having the amino acid sequence in SEQ ID NO:2
(i.e., positions 1 to 101 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (c) a nucleotide
sequence encoding the predicted transmembrane domain of the HLF
polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e.,
positions 102 to 121 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (d) a nucleotide
sequence encoding the predicted intracellular domain of the HLF
polypeptide having the amino acid sequence in SEQ ID NO:2 (i.e.,
positions 122 to 157 of SEQ ID NO:2) or as encoded by the cDNA
clone contained in ATCC Deposit No. 209123; (e) a nucleotide
sequence encoding a soluble HLF polypeptide having the
extracellular and intracellular domains but lacking the
transmembrane domain; and (f) a nucleotide sequence complementary
to any of the nucleotide sequences in (a) through (e) above.
[0060] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e) or (f), above, or a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e) or (f),
above. This polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues. An additional nucleic acid embodiment of the invention
relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of an
epitope-bearing portion of an HLF polypeptide having an amino acid
sequence in (a), (b), (c), (d) or (e), above.
[0061] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of HLF polypeptides or peptides by
recombinant techniques.
[0062] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding an HLF polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the HLF polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0063] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to
(or at most 10%, 5%, 4%, 3%, 2% or 1% different from), for
instance, the nucleotide sequence shown in FIGS. 1A and 1B or to
the nucleotides sequence of the deposited cDNA clone can be
determined conventionally using known computer programs such as the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0064] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to (or 5% different from) a reference
nucleotide sequence of the present invention, it is intended that
the nucleotide sequence of the polynucleotide is identical to the
reference sequence except that the polynucleotide sequence may
include up to five point mutations per each 100 nucleotides of the
reference nucleotide sequence encoding the HLF polypeptide. In
other words, to obtain a polynucleotide having a nucleotide
sequence at least 95% identical to a reference nucleotide sequence,
up to 5% of the nucleotides in the reference sequence may be
deleted or substituted with another nucleotide, or a number of
nucleotides up to 5% of the total nucleotides in the reference
sequence may be inserted into the reference sequence. The query
sequence may be an entire sequence shown in SEQ ID NO: 1, the ORF
(open reading frame), or any fragment specified as described
herein.
[0065] As a practical matter, whether any particular nucleic acid
molecule or polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99%
identical to (or at most 10%, 5%, 4%, 3%, 2% or 1% different from)
a nucleotide sequence of the presence invention can be determined
conventionally using known computer programs. A preferred method
for determining the best overall match between a query sequence (a
sequence of the present invention) and a subject sequence, also
referred to as a global sequence alignment, can be determined using
the FASTDB computer program based on the algorithm of Brutlag et
al. (Comp. App. Biosci. (1990) 6:237-245). In a sequence alignment
the query and subject sequences are both DNA sequences. An RNA
sequence can be compared by converting U's to T's. The result of
said global sequence alignment is in percent identity. Preferred
parameters used in a FASTDB alignment of DNA sequences to calculate
percent identity are: Matrix=Unitary, k-tuple=4, Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or
the length of the subject nucleotide sequence, whichever is
shorter.
[0066] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0067] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to be made for the purposes of the present invention.
[0068] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to (or
at most 10%, 5%, 4%, 3%, 2% or 1% different from) the nucleic acid
sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) or to the nucleic
acid sequence of the deposited cDNA, irrespective of whether they
encode a polypeptide having HLF activity. This is because even
where a particular nucleic acid molecule does not encode a
polypeptide having HLF activity, one of skill in the art would
still know how to use the nucleic acid molecule, for instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer.
Uses of the nucleic acid molecules of the present invention that do
not encode a polypeptide having HLF activity include, inter alia,
(1) isolating the HLF gene or allelic variants thereof in a cDNA
library; (2) in situ hybridization (e.g., "FISH") to metaphase
chromosomal spreads to provide precise chromosomal location of the
HLF gene, as described in Verma et al., Human Chromosomes: A Manual
of Basic Techniques, Pergamon Press, New York (1988); and Northern
Blot analysis for detecting HLF mRNA expression in specific
tissues.
[0069] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to (or
at most 10%, 5%, 4%, 3%, 2% or 1% different from) the nucleic acid
sequence shown in FIGS. 1A and 1B (SEQ ID NO: 1) or to the nucleic
acid sequence of the deposited cDNA which do, in fact, encode a
polypeptide having HLF protein activity. By "a polypeptide having
HLF activity" is intended polypeptides exhibiting activity similar,
but not necessarily identical, to an activity of the full-length or
soluble EGF domain of the HLF protein of the invention, as measured
in a particular biological assay. For example, the HLF protein of
the present invention can be assayed for activity by analyzing
changes in the phosphorylation state cell surface receptors. As
described by Marchionni and colleagues (Nature 362:312-318; 1993),
a tyrosine kinase activation assay may be used to determine such
activity. In this assay, a wide variety of cells and cell lines are
allowed to become quiescent in low serum medium. HLF protein, or
variants thereof, may then be added exogenously to the growth
medium. Cultured cells are then lysed in an SDS-based lysis buffer
and subject to SDS-PAGE. The proteins separated by SDS-PAGE are
then transferred to a membrane and immunoblotted with an
anti-phosphotyrosine antibody. Changes in tyrosine phosphorylation
state of cell surface receptor molecules may then be assessed by
comparing immunoblots of cell samples which were treated or not
treated with HLF, or a variant thereof. Such activity is useful for
determining the affect of HLF, or variants thereof, on the
stimulation of a wide variety of cell surface receptor molecules
and determining which signal transduction cascades may be initiated
by the binding of HLF, or a variant thereof.
[0070] HLF protein binding modulates the tyrosine phosphorylation
state and initiates a variety of signal transduction cascades in
erbB family or other cell surface receptor molecules in a
dose-dependent manner in the above-described assay. Thus, "a
polypeptide having HLF protein activity" includes polypeptides that
also exhibit any of the same binding and phosphorylation state
altering activities in the above-described assays in a
dose-dependent manner. Although the degree of dose-dependent
activity need not be identical to that of the HLF protein,
preferably, "a polypeptide having HLF protein activity" will
exhibit substantially similar dose-dependence in a given activity
as compared to the HLF protein (i.e., the candidate polypeptide
will exhibit greater activity or not more than about 25-fold less
and, preferably, not more than about tenfold less activity relative
to the reference HLF protein).
[0071] In addition, the binding of HLF, or variants thereof, to a
particular receptor molecule may be assayed by cross-linking HLF,
or an HLF variant, to whichever receptor it binds on the cell
surface and then immunoprecipitating the resulting ligand/receptor
complex with a specific antiserum. Such an assay will thereby
indicate a specific receptor binding profile for the HLF
protein(s). As described by Holmes and colleagues (Science
256:1205-1210; 1992), .sup.125I-labeled HLF, or HLF variant,
protein is cross-linked to any of a variety of cells or cell lines
by incubating a suspension of cells and 10.sup.6 CPM of
.sup.125I-labeled HLF, or HLF variant, proteins in Hank's balanced
salts (Life Technologies, Inc., Rockville, Md.) for 30 minutes at
22.degree. C. Bis(sulfosuccinimidyl) suberate is added to the cell
suspensions to a final concentration of 1 mM and the suspensions
are incubated for an additional 30 minutes. Cells are washed
Tris-buffered saline (TBS) and then lysed in TBS containing Triton
X-100 (0.5%). Immunoprecipitations are then performed using
portions of treated and mock-treated cultures combined with
antisera to specific cellular receptor molecules. Samples are then
prepared in SDS sample buffer, analyzed by SDS-PAGE (5.5%
polyacrylamide gels), and visualized by autoradiography.
[0072] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to (or at most 10%, 5%,
4%, 3%, 2% or 1% different from) the nucleic acid sequence of the
deposited cDNA or the nucleic acid sequence shown in FIGS. 1A and
1B (SEQ ID NO: 1) will encode a polypeptide "having HLF protein
activity." In fact, since degenerate variants of these nucleotide
sequences all encode the same polypeptide, this will be clear to
the skilled artisan even without performing the above described
comparison assay. It will be further recognized in the art that,
for such nucleic acid molecules that are not degenerate variants, a
reasonable number will also encode a polypeptide having HLF protein
activity. This is because the skilled artisan is fully aware of
amino acid substitutions that are either less likely or not likely
to significantly effect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid), as
further described below.
[0073] Vectors and Host Cells
[0074] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of HLF polypeptides or fragments thereof by
recombinant techniques. The vector may be, for example, a phage,
plasmid, viral or retroviral vector. Retroviral vectors may be
replication competent or replication defective. In the latter case,
viral propagation generally will occur only in complementing host
cells.
[0075] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0076] In one embodiment, the DNA of the invention is operatively
associated with an appropriate heterologous regulatory element
(e.g. a promoter and/or enhancer), such as the phage lambda PL
promoter, the E. coli lac, trp, phoA and tac promoters, the SV40
early and late promoters and promoters of retroviral LTRs, to name
a few. Other suitable promoters and enhancers will be known to the
skilled artisan.
[0077] In embodiments in which vectors contain expression
constructs, these constructs will further contain sites for
transcription initiation, termination and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the transcripts expressed by the constructs will preferably
include a translation initiating codon at the beginning and a
termination codon (UAA, UGA or UAG) appropriately positioned at the
end of the polypeptide to be translated.
[0078] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0079] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc., supra; pBS vectors,
Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,
pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic
vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from
Pharmacia. Other suitable vectors will be readily apparent to the
skilled artisan.
[0080] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0081] The polypeptide may be expressed in a modified form, such as
a fusion protein (comprising the polypeptide joined via a peptide
bond to a heterologous protein sequence (of a different protein)),
and may include not only secretion signals, but also additional
heterologous functional regions. Such a protein can be made by
ligating polynucleotides of the invention and the desired nucleic
acid sequence encoding the desired amino acid sequence to each
other, by methods known in the art, in the proper reading frame,
and expressing the fusion protein product by methods known in the
art. Alternatively, such a fusion protein can be made by protein
synthetic techniques, e.g. by use of a peptide synthesizer. For
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence in the host cell, during
purification, or during subsequent handling and storage. Also,
peptide moieties may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art. A preferred fusion
protein comprises a heterologous region from immunoglobulin that is
useful to stabilize and purify proteins. For example, EP-A-O 464
533 (Canadian counterpart 2045869) discloses fusion proteins
comprising various portions of constant region of immunoglobulin
molecules together with another human protein or part thereof. In
many cases, the Fc part in a fusion protein is thoroughly
advantageous for use in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP-A 0232 262). On
the other hand, for some uses it would be desirable to be able to
delete the Fc part after the fusion protein has been expressed,
detected and purified in the advantageous manner described. This is
the case when Fc portion proves to be a hindrance to use in therapy
and diagnosis, for example when the fusion protein is to be used as
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58
(1995) and K. Johanson et al., J. Biol. Chem. 270:9459-9471
(1995).
[0082] The HLF protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include: products purified from natural sources, including bodily
fluids, tissues and cells, whether directly isolated or cultured;
products of chemical synthetic procedures; and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. In addition,
polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated
processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is
removed with high efficiency from any protein after translation in
all eukaryotic cells. While the N-terminal methionine on
most-proteins also is efficiently removed in most prokaryotes, for
some proteins this prokaryotic removal process is inefficient,
depending on the nature of the amino acid to which the N-terminal
methionine is covalently linked.
[0083] Polypeptides and Fragments
[0084] The invention further provides an isolated HLF polypeptide
having the amino acid sequence encoded by the deposited cDNA, or
the amino acid sequence in SEQ ID NO:2, or a peptide or polypeptide
comprising a portion of the above polypeptides.
[0085] Variant and Mutant Polypeptides
[0086] To improve or alter the characteristics of HLF polypeptides,
protein engineering may be employed. Recombinant DNA technology
known to those skilled in the art can be used to create novel
mutant proteins or "muteins including single or multiple amino acid
substitutions, deletions, additions or fusion proteins. Such
modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
[0087] N-Terminal and C-Terminal Deletion Mutants
[0088] For instance, for many proteins, including the extracellular
domain of a membrane associated protein or the mature form(s) of a
secreted protein, it is known in the art that one or more amino
acids may be deleted from the N-terminus or C-terminus without
substantial loss of biological function. For instance, Ron and
colleagues (J. Biol. Chem., 268:2984-2988; 1993) reported modified
KGF proteins that had heparin binding activity even if 3, 8, or 27
amino-terminal amino acid residues were missing. In the present
case, since the protein of the invention is a member of the EGF
polypeptide family, deletions of N-terminal amino acids up to the
cysteine at position 35 of SEQ ID NO:2 may retain some biological
activity such as receptor binding and the initiation of the
corresponding signal transduction cascade. Polypeptides having
further N-terminal deletions including the cysteine residue at
position 35 of SEQ ID NO:2 would not be expected to retain such
biological activities because it is known that this residue in an
EGF-like, or heregulin, polypeptide is one of six conserved
cysteine residues required for both structure and biological
activity. That is to say, the first cysteine is required for
forming one of several disulfide bridges needed to provide
structural stability which is, in turn, necessary for receptor
binding and the instantiation of the signal transduction
cascade.
[0089] However, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification of loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or extracellular domain of the protein
generally will be retained when less than the majority of the
residues of the complete or extracellular domain of the protein are
removed from the N-terminus. Whether a particular polypeptide
lacking N-terminal residues of a complete protein retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0090] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of HLF shown in SEQ ID NO:2, up
to the cysteine residue at position number 35, and polynucleotides
encoding such polypeptides. In particular, the present invention
provides polypeptides comprising the amino acid sequence of
residues n-35 of SEQ ID NO:2, where n is an integer in the range of
1-35, and 35 is the position of the first residue from the
N-terminus of the complete HLF polypeptide (shown in SEQ ID NO:2)
believed to be required for the HLG protein to bind its receptor
and initiate the corresponding signal transduction cascade.
[0091] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues of 1-157, 2-157, 3-157, 4-157,
5-157, 6-157, 7-157, 8-157, 9-157, 10-157, 11-157, 12-157, 13-157,
14-157, 15-157, 16-157, 17-157, 18-157, 19-157, 20-157, 21-157,
22-157, 23-157, 24-157, 25-157, 26-157, 27-157, 28-157, 29-157,
30-157, 31-157, 32-157, 33-157, 34-157, and 35-157 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides also are provided.
[0092] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance,
Interferon-gamma shows up to ten times higher activities by
deleting 8-10 amino acid residues from the carboxy terminus of the
protein (Dobeli et al., J. Biotechnology 7:199-216; 1988). In the
present case, since the protein of the invention is a member of the
EGF or heregulin-like polypeptide family, deletions of C-terminal
amino acids up to the most carboxy-terminal cysteine of the
extracellular domain (position 73 of SEQ ID NO:2) may retain some
biological activity such as such as receptor binding and the
initiation of the corresponding signal transduction cascade.
Polypeptides having further C-terminal deletions including the
cysteine residue at position 73 of SEQ ID NO:2 would not be
expected to retain such biological activities because it is known
that this residue in an EGF-like, or heregulin-like, polypeptide is
one of six conserved cysteine residues required for both structure
and biological activity. That is to say, the first cysteine is
required for forming one of several disulfide bridges needed to
provide structural stability which is, in turn necessary for
receptor binding and the instantiation of the signal transduction
cascade.
[0093] However, even if deletion of one or more amino acids from
the C-terminus of a protein results in modification of loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or extracellular domain of the protein
generally will be retained when less than the majority of the
residues of the complete or extracellular domain protein are
removed from the C-terminus. Whether a particular polypeptide
lacking C-terminal residues of a complete protein retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0094] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the HLF shown in SEQ ID NO:2, up to
the cysteine residue at position 73 of SEQ ID NO:2, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides having the amino acid
sequence of residues 1-m of the amino acid sequence in SEQ ID NO:2,
where m is any integer in the range of 73 to 101, and residue 73 is
the position of the first residue from the C-terminus of the
complete HLF polypeptide (shown in SEQ ID NO:2) believed to be
required for receptor binding and initiation of the corresponding
signal transduction cascade.
[0095] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues 1-73, 1-74, 1-75, 1-76, 1-77,
1-78, 1-79, 1-80, 1-81, 1-82, 1-83, 1-84, 1-85, 1-86, 1-87, 1-88,
1-89, 1-90, 1-91, 1-92, 1-93, 1-94, 1-95, 1-96, 1-97, 1-98, 1-99,
1-100, and 1-101 of SEQ ID NO:2. Polynucleotides encoding these
polypeptides also are provided.
[0096] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini,
which may be described generally as having residues n-m of SEQ ID
NO:2, where n and m are integers as described above.
[0097] Also included are a nucleotide sequence encoding a
polypeptide consisting of a portion of the complete HLF amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No.
209123, where this portion excludes from 1 to about 34 amino acids
from the amino terminus of the complete amino acid sequence encoded
by the cDNA clone contained in ATCC Deposit No. 209123, or from 1
to about 83 amino acids from the carboxy terminus, or any
combination of the above amino terminal and carboxy terminal
deletions, of the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 209123. Polynucleotides
encoding all of the above deletion mutant polypeptide forms also
are provided.
[0098] As mentioned above, even if deletion of one or more amino
acids from the N-terminus of a protein results in modification of
loss of one or more biological functions of the protein, other
biological activities may still be retained. Thus, the ability of
the shortened HLF mutein to induce and/or bind to antibodies which
recognize the complete or mature of the protein generally will be
retained when less than the majority of the residues of the
complete or mature protein are removed from the N-terminus. Whether
a particular polypeptide lacking N-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art. It is not unlikely that an HLF mutein with a large
number of deleted N-terminal amino acid residues may retain some
biological or immunogenic activities. In fact, peptides composed of
as few as six HLF amino acid residues may often evoke an immune
response.
[0099] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the HLF shown in SEQ ID
NO:2, up to the asparagine residue at position number 152 and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n'-152 of SEQ ID NO:2, where n' is an integer
in the range of 2-152, and 153 is the position of the first residue
from the N-terminus of the complete HLF polypeptide believed to be
required for at least immunogenic activity of the HLF protein.
[0100] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues of S-2 to K-157; S-3 to K-157;
S-4 to K-157; S-5 to K-157; A-6 to K-157; T-7 to K-157; T-8 to
K-157; T-9 to K-157; T-10 to K-157; P-11 to K-157; E-12 to K-157;
T-13 to K-157; S-14 to K-157; T-15 to K-157; S-16 to K-157; P-17 to
K-157; K-18 to K-157; F-19 to K-157; H-20 to K-157; T-21 to K-157;
T-22 to K-157; T-23 to K-157; Y-24 to K-157; S-25 to K-157; T-26 to
K-157; E-27 to K-157; R-28 to K-157; S-29 to K-157; E-30 to K-157;
H-31 to K-157; F-32 to K-157; K-33 to K-157; P-34 to K-157; C-35 to
K-157; R-36 to K-157; D-37 to K-157; K-38 to K-157; D-39 to K-157;
L-40 to K-157; A-41 to K-157; Y-42 to K-157; C-43 to K-157; L-44 to
K-157; N-45 to K-157; D-46 to K-157; G-47 to K-157; E-48 to K-157;
C-49 to K-157; F-50 to K-157; V-51 to K-157; I-52 to K-157; E-53 to
K-157; T-54 to K-157; L-55 to K-157; T-56 to K-157; G-57 to K-157;
S-58 to K-157; H-59 to K-157; K-60 to K-157; H-61 to K-157; C-62 to
K-157; R-63 to K-157; C-64 to K-157; K-65 to K-157; E-66 to K-157;
G-67 to K-157; Y-68 to K-157; Q-69 to K-157; G-70 to K-157; V-71 to
K-157; R-72 to K-157; C-73 to K-157; D-74 to K-157; Q-75 to K-157;
F-76 to K-157; L-77 to K-157; P-78 to K-157; K-79 to K-157; T-80 to
K-157; D-81 to K-157; S-82 to K-157; 1-83 to K-157; L-84 to K-157;
S-85 to K-157; D-86 to K-157; P-87 to K-157; N-88 to K-157; H-89 to
K-157; L-90 to K-157; G-91 to K-157; I-92 to K-157; E-93 to K-157;
F-94 to K-157; M-95 to K-157; E-96 to K-157; S-97 to K-157; E-98 to
K-157; E-99 to K-157; V-100 to K-157; Y-101 to K-157; Q-102 to
K-157; R-103 to K-157; Q-104 to K-157; V-105 to K-157; L-106 to
K-157; S-107 to K-157; I-108 to K-157; S-109 to K-157; C-110 to
K-157; I-111 to K-157; I-112 to K-157; F-113 to K-157; G-114 to
K-157; I-115 to K-157; V-116 to K-157; 1-117 to K-157; V-118 to
K-157; G-119 to K-157; M-120 to K-157; F-121 to K-157; C-122 to
K-157; A-123 to K-157; A-124 to K-157; F-125 to K-157; Y-126 to
K-157; F-127 to K-157; K-128 to K-157; S-129 to K-157; K-130 to
K-157; R-131 to K-157; N-132 to K-157; 1-133 to K-157; T-134 to
K-157; A-135 to K-157; N-136 to K-157; S-137 to K-157; V-138 to
K-157; S-139 to K-157; E-140 to K-157; E-141 to K-157; R-142 to
K-157; W-143 to K-157; K-144 to K-157; G-145 to K-157; L-146 to
K-157; P-147 to K-157; S-148 to K-157; Q-149 to K-157; E-150 to
K-157; P-151 to K-157; and N-152 to K-157 of the HLF sequence shown
in SEQ ID NO:2. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0101] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened HLF mutein to induce and/or bind to
antibodies which recognize the complete or mature of the protein
generally will be retained when less than the majority of the
residues of the complete or mature protein are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that an HLF mutein
with a large number of deleted C-terminal amino acid residues may
retain some biological or immunogenic activities. In fact, peptides
composed of as few as six HLF amino acid residues may often evoke
an immune response.
[0102] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the HLF shown in SEQ ID
NO:2, up to the alanine residue at position number 6, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues 1-m' of SEQ ID NO:2, where m' is an integer in
the range of 7-156, and 6 is the position of the first residue from
the C-terminus of the complete HLF polypeptide believed to be
required for at least immunogenic activity of the HLF protein.
[0103] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues S-1 to D-156; S-1 to Q-155; S-1
to Q-154; S-1 to L-153; S-1 to N-152; S-1 to P-151; S-1 to E-150;
S-1 to Q-149; S-1 to S-148; S-1 to P-147; S-1 to L-146; S-1 to
G-145; S-1 to K-144; S-1 to W-143; S-1 to R-142; S-1 to E-141; S-1
to E-140; S-1 to S-139; S-1 to V-138; S-1 to S-137; S-1 to N-136;
S-1 to A-135; S-1 to T-134; S-1 to I-133; S-1 to N-132; S-1 to
R-131; S-1 to K-130; S-1 to S-129; S-1 to K-128; S-1 to F-127; S-1
to Y-126; S-1 to F-125; S-1 to A-124; S-1 to A-123; S-1 to C-122;
S-1 to F-121; S-1 to M-120; S-1 to G-119; S-1 to V-118; S-1 to
I-117; S-1 to V-116; S-1 to I-115; S-1 to G-114; S-1 to F-113; S-1
to I-112; S-1 to I-111; S-1 to C-110; S-1 to S-109; S-1 to I-108;
S-1 to S-107; S-1 to L-106; S-1 to V-105; S-1 to Q-104; S-1 to
R-103; S-1 to Q-102; S-1 to Y-101; S-1 to V-100; S-1 to E-99; S-1
to E-98; S-1 to S-97; S-1 to E-96; S-1 to M-95; S-1 to F-94; S-1 to
E-93; S-1 to I-92; S-1 to G-91; S-1 to L-90; S-1 to H-89; S-1 to
N-88; S-1 to P-87; S-1 to D-86; S-1 to S-85; S-1 to L-84; S-1 to
I-83; S-1 to S-82; S-1 to D-81; S-1 to T-80; S-1 to K-79; S-1 to
P-78; S-1 to L-77; S-1 to F-76; S-1 to Q-75; S-1 to D-74; S-1 to
C-73; S-1 to R-72; S-1 to V-71; S-1 to G-70; S-1 to Q-69; S-1 to
Y-68; S-1 to G-67; S-1 to E-66; S-1 to K-65; S-1 to C-64; S-1 to
R-63; S-1 to C-62; S-1 to H-61; S-1 to K-60; S-1 to H-59; S-1 to
S-58; S-1 to G-57; S-1 to T-56; S-1 to L-55; S-1 to T-54; S-1 to
E-53; S-1 to 1-52; S-1 to V-51; S-1 to F-50; S-1 to C49; S-1 to
E-48; S-1 to G-47; S-1 to D-46; S-1 to N-45; S-1 to L-44; S-1 to
C-43; S-1 to Y-42; S-1 to A-41; S-1 to L-40; S-1 to D-39; S-1 to
K-38; S-1 to D-37; S-1 to R-36; S-1 to C-35; S-1 to P-34; S-1 to
K-33; S-1 to F-32; S-1 to H-31; S-1 to E-30; S-1 to S-29; S-1 to
R-28; S-1 to E-27; S-1 to T-26; S-1 to S-25; S-1 to Y-24; S-1 to
T-23; S-1 to T-22; S-1 to T-21; S-1 to H-20; S-1 to F-19; S-1 to
K-18; S-1 to P-17; S-1 to S-16; S-1 to T-15; S-1 to S-14; S-1 to
T-13; S-1 to E-12; S-1 to P-11; S-1 to T-10; S-1 to T-9; S-1 to
T-8; S-1 to T-7; S-1 to A-6 of the HLF sequence shown in SEQ ID
NO:2. Polynucleotides encoding these polypeptides also are
provided.
[0104] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
an HLF polypeptide, which may be described generally as having
residues n'-m' of SEQ ID NO:2, where n' and m' are integers as
described above.
[0105] Also as mentioned above, even if deletion of one or more
amino acids from the N-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened HLF mutein to induce and/or bind to
antibodies which recognize the complete or mature of the protein
generally will be retained when less than the majority of the
residues of the complete or mature protein are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that an HLF mutein
with a large number of deleted N-terminal amino acid residues may
retain some biological or immunogenic activities. In fact, peptides
composed of as few as six HLF amino acid residues may often evoke
an immune response.
[0106] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the HLF shown in SEQ ID
NO:22, up to the aspartic acid residue at position number 715 and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n''-715 of SEQ ID NO:22, where n'' is an
integer in the range of 2-715, and 716 is the position of the first
residue from the N-terminus of the complete HLF polypeptide
believed to be required for at least immunogenic activity of the
HLF protein.
[0107] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues of S-2 to K-720; E-3 to K-720;
G-4 to K-720; A-5 to K-720; A-6 to K-720; A-7 to K-720; A-8 to
K-720; S-9 to K-720; P-10 to K-720; P-1 to K-720; G-12 to K-720;
A-13 to K-720; A-14 to K-720; S-15 to K-720; A-16 to K-720; A-17 to
K-720; A-18 to K-720; A-19 to K-720; S-20 to K-720; A-21 to K-720;
E-22 to K-720; E-23 to K-720; G-24 to K-720; T-25 to K-720; A-26 to
K-720; A-27 to K-720; A-28 to K-720; A-29 to K-720; A-30 to K-720;
A-31 to K-720; A-32 to K-720; A-33 to K-720; A-34 to K-720; G-35 to
K-720; G-36 to K-720; G-37 to K-720; P-38 to K-720; D-39 to K-720;
G-40 to K-720; G-41 to K-720; G-42 to K-720; E-43 to K-720; G-44 to
K-720; [0108] A-45 to K-720; A-46 to K-720; E-47 to K-720; P48 to
K-720; P-49 to K-720; R-50 to K-720; E-51 to K-720; L-52 to K-720;
R-53 to K-720; C-54 to K-720; S-55 to K-720; D-56 to K-720; C-57 to
K-720; I-58 to K-720; V-59 to K-720; W-60 to K-720; N-61 to K-720;
R-62 to K-720; Q-63 to K-720; Q-64 to K-720; T-65 to K-720; W-66 to
K-720; L-67 to K-720; C-68 to K-720; V-69 to K-720; V-70 to K-720;
P-71 to K-720; L-72 to K-720; F-73 to K-720; I-74 to K-720; G-75 to
K-720; F-76 to K-720; 1-77 to K-720; G-78 to K-720; L-79 to K-720;
G-80 to K-720; L-81 to K-720; S-82 to K-720; L-83 to K-720; M-84 to
K-720; L-85 to K-720; L-86 to K-720; K-87 to K-720; W-88 to K-720;
I-89 to K-720; V-90 to K-720; V-91 to K-720; G-92 to K-720; S-93 to
K-720; V-94 to K-720; K-95 to K-720; E-96 to K-720; Y-97 to K-720;
V-98 to K-720; P-99 to K-720; T-100 to K-720; D-101 to K-720; L-102
to K-720; V-103 to K-720; D-104 to K-720; S-105 to K-720; K-106 to
K-720; G-107 to K-720; M-108 to K-720; G-109 to K-720; Q-110, to
K-720; D-111 to K-720; P-112 to K-720; F-113 to K-720; F-114 to
K-720; L-115 to K-720; S-116 to K-720; K-117 to K-720; P-118 to
K-720; S-119 to K-720; S-120 to K-720; F-121 to K-720; P-122 to
K-720; K-123 to K-720; A-124 to K-720; M-125 to K-720; E-126 to
K-720; T-127 to K-720; T-128 to K-720; T-129 to K-720; T-130 to
K-720; T-131 to K-720; T-132 to K-720; S-133 to K-720; T-134 to
K-720; T-135 to K-720; S-136 to K-720; P-137 to K-720; A-138 to
K-720; T-139 to K-720; P-140 to K-720; S-141 to K-720; A-142 to
K-720; G-143 to K-720; G-144 to K-720; A-145 to K-720; A-146 to
K-720; S-147 to K-720; S-148 to K-720; R-149 to K-720; T-150 to
K-720; P-151 to K-720; N-152 to K-720; R-153 to K-720; I-154 to
K-720; S-155 to K-720; T-156 to K-720; R-157 to K-720; L-158 to
K-720; T-159 to K-720; T-160 to K-720; I-161 to K-720; T-162 to
K-720; R-163 to K-720; A-164 to K-720; P-165 to K-720; T-166 to
K-720; R-167 to K-720; F-168 to K-720; P-169 to K-720; G-170 to
K-720; H-171 to K-720; R-172 to K-720; V-173 to K-720; P-174 to
K-720; 1-175 to K-720; R-176 to K-720; A-177 to K-720; S-178 to
K-720; P-179 to K-720; R-180 to K-720; S-181 to K-720; T-182 to
K-720; T-183 to K-720; A-184 to K-720; R-185 to K-720; N-186 to
K-720; T-187 to K-720; A-188 to K-720; A-189 to K-720; P-190 to
K-720; A-191 to K-720; T-192 to K-720; V-193 to K-720; P-194 to
K-720; S-195 to K-720; T-196 to K-720; T-197 to K-720; A-198 to
K-720; P-199 to K-720; F-200 to K-720; F-201 to K-720; S-202 to
K-720; S-203 to K-720; S-204 to K-720; T-205 to K-720; L-206 to
K-720; G-207 to K-720; S-208 to K-720; R-209 to K-720; P-210 to
K-720; P-211 to K-720; V-212 to K-720; P-213 to K-720; G-214 to
K-720; T-215 to K-720; P-216 to K-720; S-217 to K-720; T-218 to
K-720; Q-219 to K-720; A-220 to K-720; M-221 to K-720; P-222 to
K-720; S-223 to K-720; W-224 to K-720; P-225 to K-720; T-226 to
K-720; A-227 to K-720; A-228 to K-720; Y-229 to K-720; A-230 to
K-720; T-231 to K-720; S-232 to K-720; S-233 to K-720; Y-234 to
K-720; L-235 to K-720; H-236 to K-720; D-237 to K-720; S-238 to
K-720; T-239 to K-720; P-240 to K-720; S-241 to K-720; W-242 to
K-720; T-243 to K-720; L-244 to K-720; S-245 to K-720; P-246 to
K-720; F-247 to K-720; Q-248 to K-720; D-249 to K-720; A-250 to
K-720; A-251 to K-720; S-252 to K-720; S-253 to K-720; S-254 to
K-720; S-255 to K-720; S-256 to K-720; S-257 to K-720; S-258 to
K-720; S-259 to K-720; S-260 to K-720; S-261 to K-720; T-262 to
K-720; T-263 to K-720; T-264 to K-720; T-265 to K-720; P-266 to
K-720; E-267 to K-720; T-268 to K-720; S-269 to K-720; T-270 to
K-720; S-271 to K-720; P-272 to K-720; K-273 to K-720; F-274 to
K-720; H-275 to K-720; T-276 to K-720; T-277 to K-720; T-278 to
K-720; Y-279 to K-720; S-280 to K-720; T-281 to K-720; E-282 to
K-720; R-283 to K-720; S-284 to K-720; E-285 to K-720; H-286 to
K-720; F-287 to K-720; K-288 to K-720; P-289 to K-720; C-290 to
K-720; R-291 to K-720; D-292 to K-720; K-293 to K-720; D-294 to
K-720; L-295 to K-720; A-296 to K-720; Y-297 to K-720; C-298 to
K-720; L-299 to K-720; N-300 to K-720; D-301 to K-720; G-302 to
K-720; E-303 to K-720; C-304 to K-720; F-305 to K-720; V-306 to
K-720; I-307 to K-720; E-308 to K-720; T-309 to K-720; L-310 to
K-720; T-311 to K-720; G-312 to K-720; S-313 to K-720; H-314 to
K-720; K-315 to K-720; H-316 to K-720; C-317 to K-720; R-318 to
K-720; C-319 to K-720; K-320 to K-720; E-321 to K-720; G-322 to
K-720; Y-323 to K-720; Q-324 to K-720; G-325 to K-720; V-326 to
K-720; R-327 to K-720; C-328 to K-720; D-329 to K-720; Q-330 to
K-720; F-331 to K-720; L-332 to K-720; P-333 to K-720; K-334 to
K-720; T-335 to K-720; D-336 to K-720; S-337 to K-720; I-338 to
K-720; L-339 to K-720; S-340 to K-720; D-341 to K-720; P-342 to
K-720; T-343 to K-720; D-344 to K-720; H-345 to K-720; L-346 to
K-720; G-347 to K-720; I-348 to K-720; E-349 to K-720; F-350 to
K-720; M-351 to K-720; E-352 to K-720; S-353 to K-720; E-354 to
K-720; E-355 to K-720; V-356 to K-720; Y-357 to K-720; Q-358 to
K-720; R-359 to K-720; Q-360 to K-720; V-361 to K-720; L-362 to
K-720; S-363 to K-720; I-364 to K-720; S-365 to K-720; C-366 to
K-720; 1-367 to K-720; I-368 to K-720; F-369 to K-720; G-370 to
K-720; I-371 to K-720; V-372 to K-720; 1-373 to K-720; V-374 to
K-720; G-375 to K-720; M-376 to K-720; F-377 to K-720; C-378 to
K-720; A-379 to K-720; A-380 to K-720; F-381 to K-720; Y-382 to
K-720; F-383 to K-720; K-384 to K-720; S-385 to K-720; K-386 to
K-720; K-387 to K-720; Q-388 to K-720; A-389 to K-720; K-390 to
K-720; Q-391 to K-720; 1-392 to K-720; Q-393 to K-720; E-394 to
K-720; Q-395 to K-720; L-396 to K-720; K-397 to K-720; V-398 to
K-720; P-399 to K-720; Q-400 to K-720; N-401 to K-720; G402 to
K-720; K-403 to K-720; S404 to K-720; Y405 to K-720; S-406 to
K-720; L-407 to K-720; K-408 to K-720; A-409 to K-720; S-410 to
K-720; S-411 to K-720; T-412 to K-720; M-413 to K-720; A-414 to
K-720; K-415 to K-720; S-416 to K-720; E-417 to K-720; N418 to
K-720; L-419 to K-720; V-420 to K-720; K-421 to K-720; S-422 to
K-720; H-423 to K-720; V-424 to K-720; Q-425 to K-720; L-426 to
K-720; Q-427 to K-720; N428 to K-720; Y-429 to K-720; S-430 to
K-720; K-431 to K-720; V-432 to K-720; E-433 to K-720; R-434 to
K-720; H-435 to K-720; P436 to K-720; V-437 to K-720; T-438 to
K-720; A-439 to K-720; L-440 to K-720; E-441 to K-720; K-442 to
K-720; M-443 to K-720; M-444 to K-720; E-445 to K-720; S-446 to
K-720; S447 to K-720; F-448 to K-720; V-449 to K-720; G450 to
K-720; P-451 to K-720; Q-452 to K-720; S453 to K-720; F-454 to
K-720; P455 to K-720; E-456 to K-720; V-457 to K-720; P-458 to
K-720; S459 to K-720; P460 to K-720; D-461 to K-720; R-462 to
K-720; G-463 to K-720; S-464 to K-720; Q-465 to K-720; S466 to
K-720; V-467 to K-720; K-468 to K-720; H-469 to K-720; H-470 to
K-720; R-471 to K-720; S-472 to K-720; L-473 to K-720; S474 to
K-720; S-475 to K-720; C-476 to K-720; C-477 to K-720; S-478 to
K-720; P-479 to K-720; G-480 to K-720; Q-481 to K-720; R-482 to
K-720; S-483 to K-720; G-484 to K-720; M-485 to K-720; L-486 to
K-720; H-487 to K-720; R-488 to K-720; N-489 to K-720; A-490 to
K-720; F-491 to K-720; R-492 to K-720; R-493 to K-720; T-494 to
K-720; P-495 to K-720; P-496 to K-720; S-497 to K-720; P-498 to
K-720; R-499 to K-720; S-500 to K-720; R-501 to K-720; L-502 to
K-720; G-503 to K-720; G-504 to K-720; 1-505 to K-720; V-506 to
K-720; G-507 to K-720; P-508 to K-720; A-509 to K-720; Y-510 to
K-720; Q-511 to K-720; Q-512 to K-720; L-513 to K-720; E-514 to
K-720; E-515 to K-720; S-516 to K-720; R-517 to K-720; I-518 to
K-720; P-519 to K-720; D-520 to K-720; Q-521 to K-720; D-522 to
K-720; T-523 to K-720; 1-524 to K-720; P-525 to K-720; C-526 to
K-720; Q-527 to K-720; G-528 to K-720; I-529 to K-720; E-530 to
K-720; V-531 to K-720; R-532 to K-720; K-533 to K-720; T-534 to
K-720; I-535 to K-720; S-536 to K-720; H-537 to K-720; L-538 to
K-720; P-539 to K-720; I-540 to K-720; Q-541 to K-720; L-542 to
K-720; W-543 to K-720; C-544 to K-720; V-545 to K-720; E-546 to
K-720; R-547 to K-720; P-548 to K-720; L-549 to K-720; D-550 to
K-720; L-551 to K-720; K-552 to K-720; Y-553 to K-720; S-554 to
K-720; S-555 to K-720; S-556 to K-720; G-557 to K-720; L-558 to
K-720; K-559 to K-720; T-560 to K-720; Q-561 to K-720; R-562 to
K-720; N-563 to K-720; T-564 to K-720; S-565 to K-720; I-566 to
K-720; N-567 to K-720; M-568 to K-720; Q-569 to K-720; L-570 to
K-720; P-571 to K-720; S-572 to K-720; R-573 to K-720; E-574 to
K-720; T-575 to K-720; N-576 to K-720; P-577 to K-720; Y-578 to
K-720; F-579 to K-720; N-580 to K-720; S-581 to K-720; L-582 to
K-720; E-583 to K-720; Q-584 to K-720; K-585 to K-720; D-586 to
K-720; L-587 to K-720; V-588 to K-720; G-589 to K-720; Y-590 to
K-720; S-591 to K-720; S-592 to K-720; T-593 to K-720; R-594 to
K-720; A-595 to K-720; S-596 to K-720; S-597 to K-720; V-598 to
K-720; P-599 to K-720; 1-600 to K-720; I-601 to K-720; P-602 to
K-720; S-603 to K-720; V-604 to K-720; G-605 to K-720; L-606 to
K-720; E-607 to K-720; E-608 to K-720; T-609 to K-720; C-610 to
K-720; L-611 to K-720; Q-612 to K-720; M-613 to K-720; P-614 to
K-720; G-615 to K-720; 1-616 to K-720; S-617 to K-720; E-618 to
K-720; V-619 to K-720; K-620 to K-720; S-621 to K-720; 1-622 to
K-720; K-623 to K-720; W-624 to K-720; C-625 to K-720; K-626 to
K-720; N-627 to K-720; S-628 to K-720; Y-629 to K-720; S-630 to
K-720; A-631 to K-720; D-632 to K-720; V-633 to K-720; V-634 to
K-720; N-635 to K-720; V-636 to K-720; S-637 to K-720; I-638 to
K-720; P-639 to K-720; V-640 to K-720; S-641 to K-720; D-642 to
K-720; C-643 to K-720; L-644 to K-720; I-645 to K-720; A-646 to
K-720; E-647 to K-720; Q-648 to K-720; Q-649 to K-720; E-650 to
K-720; V-651 to K-720; K-652 to K-720; I-653 to K-720; L-654 to
K-720; L-655 to K-720; E-656 to K-720; T-657 to K-720; V-658 to
K-720; Q-659 to K-720; E-660 to K-720; Q-661 to K-720; 1-662 to
K-720; R-663 to K-720; I-664 to K-720; L-665 to K-720; T-666 to
K-720; D-667 to K-720; A-668 to K-720; R-669 to K-720; R-670 to
K-720; S-671 to K-720; E-672 to K-720; D-673 to K-720; Y-674 to
K-720; E-675 to K-720; L-676 to K-720; A-677 to K-720; S-678 to
K-720; V-679 to K-720; E-680 to K-720; T-681 to K-720; E-682 to
K-720; D-683 to K-720; S-684 to K-720; A-685 to K-720; S-686 to
K-720; E-687 to K-720; N-688 to K-720; T-689 to K-720; A-690 to
K-720; F-691 to K-720; L-692 to K-720; P-693 to K-720; L-694 to
K-720; S-695 to K-720; P-696 to K-720; T-697 to K-720; A-698 to
K-720; K-699 to K-720; S-700 to K-720; E-701 to K-720; R-702 to
K-720; E-703 to K-720; A-704 to K-720; Q-705 to K-720; F-706 to
K-720; V-707 to K-720; L-708 to K-720; R-709 to K-720; N-710 to
K-720; E-711 to K-720; 1-712 to K-720; Q-713 to K-720; R-714 to
K-720; and D-715 to K-720 of the HLF sequence shown in SEQ ID
NO:22. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0109] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened HLF mutein to induce and/or bind to
antibodies which recognize the complete or mature of the protein
generally will be retained when less than the majority of the
residues of the complete or mature protein are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that an HLF mutein
with a large number of deleted C-terminal amino acid residues may
retain some biological or immunogenic activities. In fact, peptides
composed of as few as six HLF amino acid residues may often evoke
an immune response.
[0110] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the HLF shown in SEQ ID
NO:22, up to the alanine residue at position number 6, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues 1-m' of SEQ ID NO:22, where m'' is an integer
in the range of 7-718, and 6 is the position of the first residue
from the C-terminus of the complete HLF polypeptide believed to be
required for at least immunogenic activity of the HLF protein.
[0111] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues M-1 to T-719; M-1 to L-718; M-1
to A-717; M-1 to S-716; M-1 to D-715; M-1 to R-714; M-1 to Q-713;
M-1 to 1-712; M-1 to E-711; M-1 to N-710; M-1 to R-709; M-1 to
L-708; M-1 to V-707; M-1 to F-706; M-1 to Q-705; M-1 to A-704; M-1
to E-703; M-1 to R-702; M-1 to E-701; M-1 to S-700; M-1 to K-699;
M-1 to A-698; M-1 to T-697; M-1 to P-696; M-1 to S-695; M-1 to
L-694; M-1 to P-693; M-1 to L-692; M-1 to F-691; M-1 to A-690; M-1
to T-689; M-1 to N-688; M-1 to E-687; M-1 to S-686; M-1 to A-685;
M-1 to S-684; M-1 to D-683; M-1 to E-682; M-1 to T-681; M-1 to
E-680; M-1 to V-679; M-1 to S-678; M-1 to A-677; M-1 to L-676; M-1
to E-675; M-1 to Y-674; M-1 to D-673; M-1 to E-672; M-1 to S-671;
M-1 to R-670; M-1 to R-669; M-1 to A-668; M-1 to D-667; M-1 to
T-666; M-1 to L-665; M-1 to I-664; M-1 to R-663; M-1 to I-662; M-1
to Q-661; M-1 to E-660; M-1 to Q-659; M-1 to V-658; M-1 to T-657;
M-1 to E-656; M-1 to L-655; M-1 to L-654; M-1 to I-653; M-1 to
K-652; M-1 to V-651; M-1 to E-650; M-1 to Q-649; M-1 to Q-648; M-1
to E-647; M-1 to A-646; M-1 to I-645; M-1 to L-644; M-1 to C-643;
M-1 to D-642; M-1 to S-641; M-1 to V-640; M-1 to P-639; M-1 to
I-638; M-1 to S-637; M-1 to V-636; M-1 to N-635; M-1 to V-634; M-1
to V-633; M-1 to D-632; M-1 to A-631; M-1 to S-630; M-1 to Y-629;
M-1 to S-628; M-1 to N-627; M-1 to K-626; M-1 to C-625; M-1 to
W-624; M-1 to K-623; M-1 to 1-622; M-1 to S-621; M-1 to K-620; M-1
to V-619; M-1 to E-618; M-1 to S-617; M-1 to I-616; M-1 to G-615;
M-1 to P-614; M-1 to M-613; M-1 to Q-612; M-1 to L-611; M-1 to
C-610; M-1 to T-609; M-1 to E-608; M-1 to E-607; M-1 to L-606; M-1
to G-605; M-1 to V-604; M-1 to S-603; M-1 to P-602; M-1 to I-601;
M-1 to I-600; M-1 to P-599; M-1 to V-598; M-1 to S-597; M-1 to
S-596; M-1 to A-595; M-1 to R-594; M-1 to T-593; M-1 to S-592; M-1
to S-591; M-1 to Y-590; M-1 to G-589; M-1 to V-588; M-1 to L-587;
M-1 to D-586; M-1 to K-585; M-1 to Q-584; M-1 to E-583; M-1 to
L-582; M-1 to S-581; M-1 to N-580; M-1 to F-579; M-1 to Y-578; M-1
to P-577; M-1 to N-576; M-1 to T-575; M-1 to E-574; M-1 to R-573;
M-1 to S-572; M-1 to P-571; M-1 to L-570; M-1 to Q-569; M-1 to
M-568; M-1 to N-567; M-1 to 1-566; M-1 to S-565; M-1 to T-564; M-1
to N-563; M-1 to R-562; M-1 to Q-561; M-1 to T-560; M-1 to K-559;
M-1 to L-558; M-1 to G-557; M-1 to S-556; M-1 to S-555; M-1 to
S-554; M-1 to Y-553; M-1 to K-552; M-1 to L-551; M-1 to D-550; M-1
to L-549; M-1 to P-548; M-1 to R-547; M-1 to E-546; M-1 to V-545;
M-1 to C-544; M-1 to W-543; M-1 to L-542; M-1 to Q-541; M-1 to
I-540; M-1 to P-539; M-1 to L-538; M-1 to H-537; M-1 to S-536; M-1
to I-535; M-1 to T-534; M-1 to K-533; M-1 to R-532; M-1 to V-531;
M-1 to E-530; M-1 to I-529; M-1 to G-528; M-1 to Q-527; M-1 to
C-526; M-1 to P-525; M-1 to I-524; M-1 to T-523; M-1 to D-522; M-1
to Q-521; M-1 to D-520; M-1 to P-519; M-1 to I-518; M-1 to R-517;
M-1 to S-516; M-1 to E-515; M-1 to E-514; M-1 to L-513; M-1 to
Q-512; M-1 to Q-511; M-1 to Y-510; M-1 to A-509; M-1 to P-508; M-1
to G-507; M-1 to V-506; M-1 to I-505; M-1 to G-504; M-1 to G-503;
M-1 to L-502; M-1 to R-501; M-1 to S-500; M-1 to R-499; M-1 to
P498; M-1 to S-497; M-1 to P-496; M-1 to P495; M-1 to T-494; M-1 to
R-493; M-1 to R-492; M-1 to F-491; M-1 to A-490; M-1 to N489; M-1
to R-488; M-1 to H-487; M-1 to L-486; M-1 to M-485; M-1 to G484;
M-1 to S483; M-1 to R-482; M-1 to Q-481; M-1 to G480; M-1 to P479;
M-1 to S478; M-1 to C-477; M-1 to C-476; M-1 to S-475; M-1 to S474;
M-1 to L-473; M-1 to S472; M-1 to R-471; M-1 to H-470; M-1 to
H-469; M-1 to K-468; M-1 to V-467; M-1 to S-466; M-1 to Q-465; M-1
to S-464; M-1 to G463; M-1 to R-462; M-1 to D-461; M-1 to P460; M-1
to S-459; M-1 to P-458; M-1 to V-457; M-1 to E-456; M-1 to P455;
M-1 to F-454; M-1 to S453; M-1 to Q-452; M-1 to P451; M-1 to G450;
M-1 to V-449; M-1 to F-448; M-1 to S447; M-1 to S446; M-1 to E-445;
M-1 to M-444; M-1 to M-443; M-1 to K-442; M-1 to E-441; M-1 to
L-440; M-1 to A-439; M-1 to T-438; M-1 to V-437; M-1 to P-436; M-1
to H-435; M-1 to R-434; M-1 to E-433; M-1 to V432; M-1 to K-431;
M-1 to S430; M-1 to Y-429; M-1 to N428; M-1 to Q-427; M-1 to L-426;
M-1 to Q-425; M-1 to V-424; M-1 to H-423; M-1 to S422; M-1 to
K-421; M-1 to V-420; M-1 to L-419; M-1 to N-418; M-1 to E-417; M-1
to S-416; M-1 to K-415; M-1 to A-414; M-1 to M-413; M-1 to T-412;
M-1 to S-411; M-1 to S-410; M-1 to A-409; M-1 to K-408; M-1 to
L-407; M-1 to S-406; M-1 to Y-405; M-1 to S404; M-1 to K-403; M-1
to G402; M-1 to N-401; M-1 to Q-400; M-1 to P-399; M-1 to V-398;
M-1 to K-397; M-1 to L-396; M-1 to Q-395; M-1 to E-394; M-1 to
Q-393; M-1 to I-392; M-1 to Q-391; M-1 to K-390; M-1 to A-389; M-1
to Q-388; M-1 to K-387; M-1 to K-386; M-1 to S-385; M-1 to K-384;
M-1 to F-383; M-1 to Y-382; M-1 to F-381; M-1 to A-380; M-1 to
A-379; M-1 to C-378; M-1 to F-377; M-1 to M-376; M-1 to G-375; M-1
to V-374; M-1 to I-373; M-1 to V-372; M-1 to 1-371; M-1 to G-370;
M-1 to F-369; M-1 to I-368; M-1 to I-367; M-1 to C-366; M-1 to
S-365; M-1 to I-364; M-1 to S-363; M-1 to L-362; M-1 to V-361; M-1
to Q-360; M-1 to R-359; M-1 to Q-358; M-1 to Y-357; M-1 to V-356;
M-1 to E-355; M-1 to E-354; M-1 to S-353; M-1 to E-352; M-1 to
M-351; M-1 to F-350; M-1 to E-349; M-1 to I-348; M-1 to G-347; M-1
to L-346; M-1 to H-345; M-1 to D-344; M-1 to T-343; M-1 to P-342;
M-1 to D-341; M-1 to S-340; M-1 to L-339; M-1 to I-338; M-1 to
S-337; M-1 to D-336; M-1 to T-335; M-1 to K-334; M-1 to P-333; M-1
to L-332; M-1 to F-331; M-1 to Q-330; M-1 to D-329; M-1 to C-328;
M-1 to R-327; M-1 to V-326; M-1 to G-325; M-1 to Q-324; M-1 to
Y-323; M-1 to G-322; M-1 to E-321; M-1 to K-320; M-1 to C-319; M-1
to R-318; M-1 to C-317; M-1 to H-316; M-1 to K-315; M-1 to H-314;
M-1 to S-313; M-1 to K-312; M-1 to T-311; M-1 to L-310; M-1 to
T-309; M-1 to E-308; M-1 to 1-307; M-1 to V-306; M-1 to F-305; M-1
to C-304; M-1 to E-303; M-1 to G-302; M-1 to D-301; M-1 to N-300;
M-1 to L-299; M-1 to C-298; M-1 to Y-297; M-1 to A-296; M-1 to
L-295; M-1 to D-294; M-1 to K-293; M-1 to D-292; M-1 to R-291; M-1
to C-290; M-1 to P-289; M-1 to K-288; M-1 to F-287; M-1 to H-286;
M-1 to E-285; M-1 to S-284; M-1 to R-283; M-1 to E-282; M-1 to
T-281; M-1 to S-280; M-1 to Y-279; M-1 to T-278; M-1 to T-277; M-1
to T-276; M-1 to H-275; M-1 to F-274; M-1 to K-273; M-1 to P-272;
M-1 to S-271; M-1 to T-270; M-1 to S-269; M-1 to T-268; M-1 to
E-267; M-1 to P-266; M-1 to T-265; M-1 to T-264; M-1 to T-263; M-1
to T-262; M-1 to S-261; M-1 to S-260; M-1 to S-259; M-1 to S-258;
M-1 to S-257; M-1 to S-256; M-1 to S-255; M-1 to S-254; M-1 to
S-253; M-1 to S-252; M-1 to A-251; M-1 to A-250; M-1 to D-249; M-1
to Q-248; M-1 to F-247; M-1 to P-246; M-1 to S-245; M-1 to L-244;
M-1 to T-243; M-1 to W-242; M-1 to S-241; M-1 to P-240; M-1 to
T-239; M-1 to S-238; M-1 to D-237; M-1 to H-236; M-1 to L-235; M-1
to Y-234; M-1 to S-233; M-1 to S-232; M-1 to T-231; M-1 to A-230;
M-1 to Y-229; M-1 to A-228; M-1 to A-227; M-1 to T-226; M-1 to
P-225; M-1 to W-224; M-1 to S-223; M-1 to P-222; M-1 to M-221; M-1
to A-220; M-1 to Q-219; M-1 to T-218; M-1 to S-217; M-1 to P-216;
M-1 to T-215; M-1 to G-214; M-1 to P-213; M-1 to V-212; M-1 to
P-211; M-1 to P-210; M-1 to R-209; M-1 to S-208; M-1 to G-207; M-1
to L-206; M-1 to T-205; M-1 to S-204; M-1 to S-203; M-1 to S-202;
M-1 to F-201; M-1 to F-200; M-1 to P-199; M-1 to A-198; M-1 to
T-197; M-1 to T-196; M-1 to S-195; M-1 to P-194; M-1 to V-193; M-1
to T-192; M-1 to A-191; M-1 to P-190; M-1 to A-189; M-1 to A-188;
M-1 to T-187; M-1 to N-186; M-1 to R-185; M-1 to A-184; M-1 to
T-183; M-1 to T-182; M-1 to S-181; M-1 to R-180; M-1 to P-179; M-1
to S-178; M-1 to A-177; M-1 to R-176; M-1 to I-175; M-1 to P-174;
M-1 to V-173; M-1 to R-172; M-1 to H-171; M-1 to G-170; M-1 to
P-169; M-1 to F-168; M-1 to R-167; M-1 to T-166; M-1 to P-165; M-1
to A-164; M-1 to R-163; M-1 to T-162; M-1 to I-161; M-1 to T-160;
M-1 to T-159; M-1 to L-158; M-1 to R-157; M-1 to T-156; M-1 to
S-155; M-1 to I-154; M-1 to R-153; M-1 to N-152; M-1 to P-151; M-1
to T-150; M-1 to R-149; M-1 to S-148; M-1 to S-147; M-1 to A-146;
M-1 to A-145; M-1 to G-144; M-1 to G-143; M-1 to A-142; M-1 to
S-141; M-1 to P-140; M-1 to T-139; M-1 to A-138; M-1 to P-137; M-1
to S-136; M-1 to T-135; M-1 to T-134; M-1 to S-133; M-1 to T-132;
M-1 to T-131; M-1 to T-130; M-1 to T-129; M-1 to T-128; M-1 to
T-127; M-1 to E-126; M-1 to M-125; M-1 to A-124; M-1 to K-123; M-1
to P-122; M-1 to F-121; M-1 to S-120; M-1 to S-119; M-1 to P-118;
M-1 to K-117; M-1 to S-116; M-1 to L-115; M-1 to F-114; M-1 to
F-113; M-1 to P-112; M-1 to D-11; M-1 to Q-110; M-1 to G-109; M-1
to M-108; M-1 to G-107; M-1 to K-106; M-1 to S-105; M-1 to D-104;
M-1 to V-103; M-1 to L-102; M-1 to D-101; M-1 to T-100; M-1 to
P-99; M-1 to V-98; M-1 to Y-97; M-1 to E-96; M-1 to K-95; M-1 to
V-94; M-1 to S-93; M-1 to G-92; M-1 to V-91; M-1 to V-90; M-1 to
I-89; M-1 to W-88; M-1 to K-87; M-1 to L-86; M-1 to L-85; M-1 to
M-84; M-1 to L-83; M-1 to S-82; M-1 to L-81; M-1 to G-80; M-1 to
L-79; M-1 to G-78; M-1 to I-77; M-1 to F-76; M-1 to G-75; M-1 to
I-74; M-1 to F-73; M-1 to L-72; M-1 to P-71; M-1 to V-70; M-1 to
V-69; M-1 to C-68; M-1 to L-67; M-1 to W-66; M-1 to T-65; M-1 to
Q-64; M-1 to Q-63; M-1 to R-62; M-1 to N-61; M-1 to W-60; M-1 to
V-59; M-1 to 1-58; M-1 to C-57; M-1 to D-56; M-1 to S-55; M-1 to
C-54; M-1 to R-53; M-1 to L-52; M-1 to E-51; M-1 to R-50; M-1 to
P-49; M-1 to P-48; M-1 to E-47; M-1 to A-46; M-1 to A-45; M-1 to
G-44; M-1 to E-43; M-1 to G-42; M-1 to G-41; M-1 to G-40; M-1 to
D-39; M-1 to P-38; M-1 to G-37; M-1 to G-36; M-1 to G-35; M-1 to
A-34; M-1- to A-33; M-1 to A-32; M-1 to A-31; M-1 to A-30; M-1 to
A-29; M-1 to A-28; M-1 to A-27; M-1 to A-26; M-1 to T-25; M-1 to
G-24; M-1 to E-23; M-1 to E-22; M-1 to A-21; M-1 to S-20; M-1 to
A-19; M-1 to A-18; M-1 to A-17; M-1 to A-16; M-1 to S-15; M-1 to
A-14; M-1 to A-13; M-1 to G-12; M-1 to P-11; M-1 to P-10; M-1 to
S-9; M-1 to A-8; M-1 to A-7; M-1 to A-6 of the HLF sequence shown
in SEQ ID NO:22. Polynucleotides encoding these polypeptides also
are provided.
[0112] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
an HLF polypeptide, which may be described generally as having
residues n''-m'' of SEQ ID NO:22, where n'' and m'' are integers as
described above.
[0113] Other Mutants
[0114] In addition to terminal deletion forms of the protein
discussed above, it also will be recognized by one of ordinary
skill in the art that some amino acid sequences of the HLF
polypeptide can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0115] Thus, the invention further includes variations of the HLF
polypeptide which show substantial HLF polypeptide activity or
which include regions of HLF protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions selected according to
general rules known in the art so as have little effect on
activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided by Bowie
and colleagues (Science 247:1306-1310; 1990), wherein the authors
indicate that there are two main approaches for studying the
tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0116] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0117] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, (ii) one
in which one or more of the amino acid residues includes a
substituent group, (iii) one in which the extracellular domain of
the HLF polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), (iv) one in which the EGF domain of the HLF
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol), (v) one in which the additional amino acids
are fused to the extracellular form of the polypeptide, such as an
IgG Fc fusion region peptide or leader or secretory sequence or a
sequence which is employed for purification of the above form of
the polypeptide or a proprotein sequence, or (vi) one in which the
additional amino acids are fused to the EGF form of the
polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory sequence or a sequence which is employed for purification
of the above form of the polypeptide or a proprotein sequence. Such
fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein
[0118] Thus, the HLF of the present invention may include one or
more amino acid substitutions, deletions or additions, either from
natural mutations or human manipulation. As indicated, changes are
preferably of a minor nature, such as conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0119] In specific embodiments, the number of substitutions,
deletions or additions in the amino acid sequence of FIG. 1A and/or
any of the polypeptide fragments described herein is 30, 29, 28,
27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,
10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-10, 20-15, 15-10, 10-5
or 1-5.
[0120] Amino acids in the HLF protein of the present invention that
are essential for function can be identified by methods known in
the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in vitro
or in vitro proliferative activity.
[0121] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
[0122] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors. For example,
Ostade et al., Nature 361:266-268 (1993) describes certain
mutations resulting in selective binding of TNF-.alpha. to only one
of the two known types of TNF receptors. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith et al, J. Mol. Biol. 224:899-904
(1992) and de Vos et al. Science 255:306-312 (1992)).
[0123] Since HLF is a member of the EGF-related protein family, to
modulate rather than completely eliminate biological activities of
HLF, preferably mutations are made in sequences encoding amino
acids in the HLF conserved domain, i.e., in amino acid positions
about 26 to about 93 of SEQ ID NO:2, more preferably in residues
within this region which are not conserved in all members of the
EGF family. Also forming part of the present invention are isolated
polynucleotides comprising nucleic acid sequences which encode the
above HLF mutants.
[0124] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the HLF polypeptide
can be substantially purified by the one-step method described by
Smith and Johnson (Gene 67:3140; 1988). Polypeptides of the
invention also can be purified from natural or recombinant sources
using anti-HLF antibodies of the invention in methods which are
well known in the art of protein purification.
[0125] The invention further provides an isolated HLF polypeptide
comprising an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of the HLF polypeptide
having the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,
positions 1 to 157 of SEQ ID NO:2) or the complete amino acid
sequence encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (b) the amino acid sequence of the predicted
extracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 1 to 101 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (c) the amino acid sequence of the predicted
transmembrane domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 102 to 121 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; (d) the amino acid sequence of the predicted
intracellular domain of the HLF polypeptide having the amino acid
sequence shown in SEQ ID NO:2 (i.e., positions 122 to 157 of SEQ ID
NO:2) or as encoded by the cDNA clone contained in the ATCC Deposit
No. 209123; and (e) the amino acid sequence of a soluble HLF
polypeptide having the extracellular and intracellular domains but
lacking the transmembrane domain. The polypeptides of the present
invention also include polypeptides having an amino acid sequence
at least 80% identical, more preferably at least 90% identical, and
still more preferably 95%, 96%, 97%, 98% or 99% identical to (or at
most 20% different, more preferably at most 10% different, and
still more preferably 5%, 4%, 3%, 2% or 1% different from) those
described in (a), (b), (c), (d), or (e) above, as well as
polypeptides having an amino acid sequence with at least 90%
similarity, and more preferably at least 95% similarity, to those
above.
[0126] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. The polypeptides of
the invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to (or at most
20% different, more preferably at most 10% or 5% different, still
more preferably at most 4%, 3%, 2% or 1% different from) the
polypeptide encoded by the deposited cDNA or to the polypeptide of
SEQ ID NO:2, and also include portions of such polypeptides with at
least 30 amino acids and more preferably at least 50 amino
acids.
[0127] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Adv. Appl.
Math. 2:482-489; 1981) to find the best segment of similarity
between two sequences.
[0128] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of an
HLF polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the HLF
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0129] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to (or at most
10%, 5%, 4%, 3%, 2% or 1% different from), for instance, the amino
acid sequence shown in SEQ ID NO:2 or to the amino acid sequence
encoded by deposited cDNA clone can be determined conventionally
using known computer programs such the Bestfit program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, Wis.
53711). When using Bestfit or any other sequence alignment program
to determine whether a particular sequence is, for instance, 95%
identical to (or 5% different from) a reference sequence according
to the present invention, the parameters are set, of course, such
that the percentage of identity is calculated over the full length
of the reference amino acid sequence and that gaps in homology of
up to 5% of the total number of amino acid residues in the
reference sequence are allowed.
[0130] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to (or 5% different from) a query amino
acid sequence of the present invention, it is intended that the
amino acid sequence of the subject polypeptide is identical to the
query sequence except that the subject polypeptide sequence may
include up to five amino acid alterations per each 100 amino acids
of the query amino acid sequence. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a query amino acid sequence, up to 5% of the amino acid residues in
the subject sequence may be inserted, deleted, (indels) or
substituted with another amino acid. These alterations of the
reference sequence may occur at the amino or carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0131] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to (or at most
10%, 5%, 4%, 3%, 2% or 1% different from) for instance, the amino
acid sequences shown in SEQ ID NO:2 or to the amino acid sequence
encoded by deposited DNA clone can be determined conventionally
using known computer programs. A preferred method for determining
the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag et al. (Comp.
App. Biosci. (1990) 6:237-245). In a sequence alignment the query
and subject sequences are either both nucleotide sequences or both
amino acid sequences. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB amino
acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,
Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=500 or the length of the subject amino acid sequence,
whichever is shorter.
[0132] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. Whether a residue is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0133] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
matching/alignment of the first 10 residues at the N-terminus. The
10 unpaired residues represent 10% of the sequence (number of
residues at the N- and C-termini not matched/total number of
residues in the query sequence) so 10% is subtracted from the
percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are to made for the purposes of the
present invention.
[0134] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0135] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting HLF
protein expression as described below or as agonists and
antagonists capable of enhancing or inhibiting HLF protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" HLF protein binding proteins which
are also candidate agonists and antagonists according to the
present invention. The yeast two hybrid system is described in
Fields and Song, Nature 340:245-246 (1989).
[0136] Epitope-Bearing Portions
[0137] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes (Geysen et al., Proc. Natl.
Acad. Sci. USA 81:3998-4002; 1983).
[0138] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein (Sutcliffe, J. G.,
et al., Science 219:660-666; 1983). Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0139] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate HLF-specific
antibodies include: a polypeptide comprising amino acid residues
from about Ser-1 to about Thr-8, about Thr-9 to about Lys-18, about
Thr-23 to about His-31, about Phe-32 to about Leu-40, about Cys43
to about Val-51, about Thr-56 to about Tyr-68, about Gln-75 to
about Leu-84, about Tyr-126 to about Ala-135, about Ser-137 to
about Leu-146, and about Ser-148 to about Lys-157. These
polypeptide fragments have been determined to bear antigenic
epitopes of the HLF protein by the analysis of the Jameson-Wolf
antigenic index, as shown in FIG. 3, above.
The epitope-bearing peptides and polypeptides of the invention may
be produced by any conventional means. See, e.g., Houghten, R. A.
(1985) "General method for the rapid solid-phase synthesis of large
numbers of peptides: specificity of antigen-antibody interaction at
the level of individual amino acids." Proc. Natl. Acad. Sci. USA
82:5131-5135; this "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Pat. No. 4,631,211 to Houghten
et al. (1986).
[0140] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art. See, for instance, Geysen et al., supra. Further still,
U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method
of detecting or determining the sequence of monomers (amino acids
or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides,
as well as methods for using such oligopeptide sets and libraries
for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0141] Fusion Proteins
[0142] As one of skill in the art will appreciate, HLF polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. This has been shown, e.g., for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins (EP A 394,827; Traunecker et
al., Nature 331:84-86; 1988). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be
more efficient in binding and neutralizing other molecules than the
monomeric HLF protein or protein fragment alone (Fountoulakis, et
al., J. Biochem. 270:3958-3964; 1995).
[0143] Furthermore, HLF polypeptides of interest of the present
invention, for example the extracellular EGF-like domain shown in
FIG. 1A, can be combined with a recombinant toxin. Such a fusion
polypeptide can be used to target the toxin, for example
Pseudomonas exotoxin A, to a tumor through the efficient binding of
the extracellular or smaller soluble domains of the HLF molecule of
the present invention. In fact, Jeschke and colleagues (Int. J.
Cancer 60:730-739; 1995) and Fiddes and coworkers (Cell Growth
Differ. 6:1567-1577; 1995) have demonstrated that heregulin-toxin
fusion proteins can be utilized in such a fashion.
[0144] Antibodies
[0145] HLF-protein specific antibodies for use in the present
invention can be raised against the intact HLF protein or an
antigenic polypeptide fragment thereof, which may be presented
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse) or, if it is long enough (at least
about 25 amino acids), without a carrier.
[0146] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab')2
fragments) which are capable of specifically binding to HLF
protein. Fab and F(ab')2 fragments lack the Fc fragment of intact
antibody, clear more rapidly from the circulation, and may have
less non-specific tissue binding of an intact antibody (Wahl et
al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are
preferred.
[0147] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the HLF
protein or an antigenic fragment thereof can be administered to an
animal in order to induce the production of sera containing
polyclonal antibodies. In a preferred method, a preparation of HLF
protein is prepared and purified to render it substantially free of
natural contaminants. Such a preparation is then introduced into an
animal in order to produce polyclonal antisera of greater specific
activity.
[0148] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or HLF protein binding
fragments thereof). Such monoclonal antibodies can be prepared
using hybridoma technology (Kohler et al., Nature 256:495 (1975);
Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur.
J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp.
563-681). In general, such procedures involve immunizing an animal
(preferably a mouse) with a HLF protein antigen or, more
preferably, with a HLF protein-expressing cell. Suitable cells can
be recognized by their capacity to bind anti-HLF protein antibody.
Such cells may be cultured in any suitable tissue culture medium;
however, it is preferable to culture cells in Earle's modified
Eagle's medium supplemented with 10% fetal bovine serum
(inactivated at about 56.degree. C.), and supplemented with about
10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin,
and about 100 .mu.g/ml of streptomycin. The splenocytes of such
mice are extracted and fused with a suitable myeloma cell line. Any
suitable mryeloma cell line may be employed in accordance with the
present invention; however, it is preferable to employ the parent
myeloma cell line (SP20), available from the American Type Culture
Collection, Rockville, Md. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al. (Gastroenterology
80:225-232 (1981)). The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding the HLF protein antigen.
[0149] Alternatively, additional antibodies capable of binding to
the HLF protein antigen may be produced in a two-step procedure
through the use of anti-idiotypic antibodies. Such a method makes
use of the fact that antibodies are themselves antigens, and that,
therefore, it is possible to obtain an antibody which binds to a
second antibody. In accordance with this method, HLF-protein
specific antibodies are used to immunize an animal, preferably a
mouse. The splenocytes of such an animal are then used to produce
hybridoma cells, and the hybridoma cells are screened to identify
clones which produce an antibody whose ability to bind to the HLF
protein-specific antibody can be blocked by the HLF protein
antigen. Such antibodies comprise anti-idiotypic antibodies to the
HLF protein-specific antibody and can be used to immunize an animal
to induce formation of further HLF protein-specific antibodies.
[0150] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, HLF protein-binding fragments can be
produced through the application of recombinant DNA technology or
through synthetic chemistry.
[0151] For in vivo use of anti-HLF in humans, it may be preferable
to use "humanized" chimeric monoclonal antibodies. Such antibodies
can be produced using genetic constructs derived from hybridoma
cells producing the monoclonal antibodies described above. Methods
for producing chimeric antibodies are known in the art (Morrison,
Science 229:1202; 1985); Oi, et al., BioTechniques 4:214; 1986;
Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi, et al., EP
171496; Morrison, et al., EP 173494; Neuberger, et al., WO 8601533;
Robinson, et al., WO 8702671; Boulianne, et al., Nature 312:643;
1984; Neuberger et al., Nature 314:268; 1985).
[0152] Disorders Related to the Regulation of Cell Growth
[0153] Diagnosis
[0154] The present inventors have discovered that HLF is apparently
expressed detectably only in the amygdala, whole brain, and primary
breast culture tissue. For a number of disorders related to the
regulation of cell growth, substantially altered (increased or
decreased) levels of HLF gene expression can be detected in tissues
or other cells or bodily fluids (e.g., sera, plasma, urine,
synovial fluid or spinal fluid) taken from an individual having
such a disorder, relative to a "standard" HLF gene expression
level, that is, the HLF expression level in such tissues or bodily
fluids from an individual not having the disorder. Thus, the
invention provides a diagnostic method useful during diagnosis of a
disorder related to the regulation of cell growth, which involves
measuring the expression level of the gene encoding the HLF protein
in such tissues or other cells or bodily fluids from an individual
and comparing the measured gene expression level with a standard
HLF gene expression level, whereby an increase or decrease in the
gene expression level compared to the standard is indicative of a
such a disorder.
[0155] In particular, it is believed that certain tissues in
mammals with breast or brain cancers express significantly enhanced
levels of the HLF protein and mRNA encoding the HLF protein when
compared to a corresponding "standard" level. Further, it is
believed that enhanced levels of the HLF protein can be detected in
certain body fluids (e.g., sera, plasma, urine, and spinal fluid)
from mammals with such a cancer when compared to sera from mammals
of the same species not having the cancer.
[0156] Thus, the invention provides a diagnostic method useful
during diagnosis of a disorder of the regulation of cell growth,
including several types of cancers which involves measuring the
expression level of the gene encoding the HLF protein in tissues or
other cells or bodily fluids from an individual and comparing the
measured gene expression level with a standard HLF gene expression
level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of such a disorder.
[0157] Where a diagnosis of a disorder of the regulation of cell
growth, including diagnosis of a tumor, has already been made
according to conventional methods, the present invention is useful
as a prognostic indicator, whereby patients exhibiting enhanced HLF
gene expression will experience a worse clinical outcome relative
to patients expressing the gene at a level nearer the standard
level.
[0158] By "assaying the expression level of the gene encoding the
HLF protein" is intended qualitatively or quantitatively measuring
or estimating the level of the HLF protein or the level of the mRNA
encoding the HLF protein in a first biological sample either
directly (e.g., by determining or estimating absolute protein level
or mRNA level) or relatively (e.g., by comparing to the HLF protein
level or mRNA level in a second biological sample). Preferably, the
HLF protein level or mRNA level in the first biological sample is
measured or estimated and compared to a standard HLF protein level
or mRNA level, the standard being taken from a second biological
sample obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having a disorder of the regulation of cell growth. As will be
appreciated in the art, once a standard HLF protein level or mRNA
level is known, it can be used repeatedly as a standard for
comparison.
[0159] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains HLF protein or mRNA. As indicated,
biological samples include body fluids (such as sera, plasma,
urine, synovial fluid and spinal fluid) which contain free HLF
protein, or the extracellular or EGF domains of the HLF protein,
cancerous tissue, and other tissue sources found to express
complete HLF protein, or the extracellular or EGF domains of the
HLF protein, or an HLF receptor. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Where the biological sample is to include mRNA, a tissue biopsy is
the preferred source.
[0160] The present invention is useful for diagnosis or treatment
of various disorders of the regulation of cell growth in mammals,
preferably humans. Such disorders include breast cancer, brain
cancers, including neuroblastomas and glioblastomas, developmental
disorders, ovarian cancer, endometrial cancer, some types of colon
cancers, and the like.
[0161] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described by
Chomczynski and Sacchi (Anal. Biochem. 162:156-159; 1987). Levels
of mRNA encoding the HLF protein are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0162] Assaying HLF protein levels in a biological sample can occur
using antibody-based techniques. For example, HLF protein
expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting HLF protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0163] In addition to assaying HLF protein levels in a biological
sample obtained from an individual, HLF protein can also be
detected in vivo by imaging. Antibody labels or markers for in vivo
imaging of HLF protein include those detectable by X-radiography,
NMR or ESR. For X-radiography, suitable labels include
radioisotopes such as barium or cesium, which emit detectable
radiation but are not overtly harmful to the subject. Suitable
markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0164] A HLF protein-specific antibody or antibody fragment which
has been labeled with an appropriate detectable imaging moiety,
such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for immune system disorder. It will be understood in
the art that the size of the subject and the imaging system used
will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain HLF protein. In vivo tumor
imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
[0165] Treatment
[0166] As noted above, HLF polynucleotides and polypeptides are
useful for diagnosis of conditions involving abnormally high or low
expression of HLF activities. Given the cells and tissues where HLF
is expressed as well as the activities modulated by HLF, it is
readily apparent that a substantially altered (increased or
decreased) level of expression of HLF in an individual compared to
the standard or "normal" level produces pathological conditions
related to the bodily system(s) in which HLF is expressed and/or is
active.
[0167] It will also be appreciated by one of ordinary skill that,
since the HLF protein of the invention is a member of the EGF
family the extracellular domain of the protein may be released in
soluble form from the cells which express the HLF by proteolytic
cleavage. Therefore, when HLF-soluble extracellular domain is added
from an exogenous source to cells, tissues or the body of an
individual, the protein will exert its physiological activities on
its target cells of that individual.
[0168] Therefore, it will be appreciated that conditions caused by
a decrease in the standard or normal level of HLF activity in an
individual, particularly disorders of cellular growth regulation,
can be treated by administration of HLF polypeptide (in the form of
soluble extracellular domain or cells expressing the complete
protein. Thus, the invention also provides a method of treatment of
an individual in need of an increased level of HLF activity
comprising administering to such an individual a pharmaceutical
composition comprising an amount of an isolated HLF polypeptide of
the invention, particularly an extracellular form of the HLF
protein of the invention, effective to increase the HLF activity
level in such an individual.
[0169] An individual who is in need of increased HLF activity will
not express a sufficient amount of functional HLF protein,
administration of recombinant HLF protein, or more simply, of the
active extracellular or the active EGF domain, to such an
individual will result in the presence of a sufficient
concentration of HLF activity in the bloodstream. In addition, an
individual who has an abnormally increased level of HLF activity,
will require the use of an HLF antibody or antagonist, as described
in the present invention. The use of such HLF antagonists will
result in a therapeutic lowering of the effective level of HLF
activity in the bloodstream. As a result of such treatment, the
affected individual will have an effective concentration of HLF
activity which is much closer to that of what is deemed "normal".
Those of skill in the art will recognize other indications where
the ability to therapeutically adjust the level of effective HLF
activity is desirable.
[0170] It will be further appreciated by one of ordinary skill that
HLF may be used as an additive or supplement for the in vitro
culture of certain types of eukaryotic cells. Many cell types,
including primary cell cultures, are highly fastidious and require
a complex mixture of additives to the standard culture medium to
result in successful culture and survival of the cells. A number of
known growth factors and related molecules are currently used as
supplements to the medium of various cells. Such factors may
include molecules as epidermal growth factor (EGF), keratinocyte
growth factor (KGF), acidic fibroblast growth factor (aFGF),
insulin-like growth factor (IGF)-I, nerve growth factor (NGF), and
many others. Despite the availability and use of the collection of
growth factors listed above, a large number of cells and cell types
remain unculturable, either at all or for an extended period of
time. Since expression of HLF appears to be limited to the
amygdala, whole brain, and primary breast culture tissue or to
other neural cells and tissues, HLF is useful as an additive or
growth factor in the culture of neural and a number of other cells
and cell types.
[0171] It will be further appreciated by the skilled artisan, that
many cells and cell types require the absence of a specific growth
factor or related molecule from the culture medium. In the case of
culturing cells which require the absence of HLF from the culture
medium, antagonists or antibodies of HLF described herein may be
used to bind to and remove HLF from culture medium preparations
thus resulting in "HLF-free" culture media.
[0172] Formulations
[0173] The HLF polypeptide composition will be formulated and dosed
in a fashion consistent with good medical practice, taking into
account the clinical condition of the individual patient
(especially the side effects of treatment with HLF polypeptide
alone), the site of delivery of the HLF polypeptide composition,
the method of administration, the scheduling of administration, and
other factors known to practitioners. The "effective amount" of HLF
polypeptide for purposes herein is thus determined by such
considerations.
[0174] As a general proposition, the total pharmaceutically
effective amount of HLF polypeptide administered parenterally per
dose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day
of patient body weight, although, as noted above, this will be
subject to therapeutic discretion. More preferably, this dose is at
least 0.01 mg/kg/day, and most preferably for humans between about
0.01 and 1 mg/kg/day for the hormone. If given continuously, the
HLF polypeptide is typically administered at a dose rate of about 1
.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injections
per day or by continuous subcutaneous infusions, for example, using
a mini-pump. An intravenous bag solution may also be employed. The
length of treatment needed to observe changes and the interval
following treatment for responses to occur appears to vary
depending on the desired effect.
[0175] Pharmaceutical compositions containing the HLF of the
invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0176] The HLF polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or microcapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
HLF polypeptide compositions also include liposomally entrapped HLF
polypeptide. Liposomes containing HLF polypeptide are prepared by
methods known per se: DE 3,218,121; Epstein et al., Proc. Natl.
Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl.
Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP
88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.
Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the
liposomes are of the small (about 200-800 Angstroms) unilamellar
type in which the lipid content is greater than about 30 mol.
percent cholesterol, the selected proportion being adjusted for the
optimal HLF polypeptide therapy.
[0177] For parenteral administration, in one embodiment, the HLF
polypeptide is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0178] Generally, the formulations are prepared by contacting the
HLF polypeptide uniformly and intimately with liquid carriers or
finely divided solid carriers or both. Then, if necessary, the
product is shaped into the desired formulation. Preferably the
carrier is a parenteral carrier, more preferably a solution that is
isotonic with the blood of the recipient. Examples of such carrier
vehicles include water, saline, Riger's solution, and dextrose
solution. Non-aqueous vehicles such as fixed oils and ethyl oleate
are also useful herein, as well as liposomes.
[0179] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0180] The HLF polypeptide is typically formulated in such vehicles
at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of HLF polypeptide salts.
[0181] HLF polypeptide to be used for therapeutic administration
must be sterile. Sterility is readily accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic HLF polypeptide compositions generally are placed into
a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
[0182] HLF polypeptide ordinarily will be stored in unit or
multi-dose containers, for example, sealed ampoules or vials, as an
aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous HLF
polypeptide solution, and the resulting mixture is lyophilized. The
infusion solution is prepared by reconstituting the lyophilized HLF
polypeptide using bacteriostatic Water-for-Injection.
[0183] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
[0184] Agonists and Antagonists--Assays and Molecules
[0185] The invention also provides a method of screening compounds
to identify those which enhance or block the action of HLF on
cells, such as its interaction with HLF-binding molecules such as
receptor molecules. An agonist is a compound which increases the
natural biological functions of HLF or which functions in a manner
similar to HLF, while antagonists decrease or eliminate such
functions.
[0186] In another aspect of this embodiment the invention provides
a method for identifying a receptor protein or other ligand-binding
protein which binds specifically to a HLF polypeptide. For example,
a cellular compartment, such as a membrane or a preparation
thereof, may be prepared from a cell that expresses a molecule that
binds HLF. The preparation is incubated with labeled HLF. HLF and
complexes of HLF bound to the receptor or other binding protein are
isolated and characterized according to routine methods known in
the art. Alternatively, the HLF polypeptide may be bound to a solid
support so that binding molecules solubilized from cells are bound
to the column and then eluted and characterized according to
routine methods.
[0187] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
HLF, such as a molecule of a signaling or regulatory pathway
modulated by HLF. The preparation is incubated with labeled HLF in
the absence or the presence of a candidate molecule which may be a
HLF agonist or antagonist. The ability of the candidate molecule to
bind the binding molecule is reflected in decreased binding of the
labeled ligand. Molecules which bind gratuitously, i.e., without
inducing the effects of HLF on binding the HLF binding molecule,
are most likely to be good antagonists. Molecules that bind well
and elicit effects that are the same as or closely related to HLF
are agonists.
[0188] HLF-like effects of potential agonists and antagonists may
by measured, for instance, by determining activity of a second
messenger system following interaction of the candidate molecule
with a cell or appropriate cell preparation, and comparing the
effect with that of HLF or molecules that elicit the same effects
as HLF. Second messenger systems that may be useful in this regard
include but are not limited to AMP guanylate cyclase, ion channel
or phosphoinositide hydrolysis second messenger systems.
[0189] Another example of an assay for HLF antagonists is a
competitive assay that combines HLF and a potential antagonist with
membrane-bound HLF receptor molecules or recombinant HLF receptor
molecules under appropriate conditions for a competitive inhibition
assay. HLF can be labeled, such as by radioactivity, such that the
number of HLF molecules bound to a receptor molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0190] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing HLF-induced activities, thereby
preventing the action of HLF by excluding HLF from binding.
[0191] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression." CRC Press, Boca Raton, Fla. (1988). Triple helix
formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and Dervan et al., Science 251: 1360 (1991). The methods are based
on binding of a polynucleotide to a complementary DNA or RNA. For
example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design
an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to
a region of the gene involved in transcription thereby preventing
transcription and the production of HLF. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into HLF polypeptide. The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of HLF protein.
[0192] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described above. The antagonists may be employed for instance to
inhibit the binding to, and activation of, cell surface receptor
molecules belonging to the erbB family, as well as other known or
unknown cell surface receptor molecules. Consequently, inhibition
of such receptor binding will result in the indirect inhibition of
stimulation of the corresponding signal transduction pathways. Many
of the corresponding signal transduction pathways are involved in
the regulation of cell division and growth. The genesis or
acceleration of many cancers resulting from other related or
unrelated mechanisms is linked to abnormally increased levels of
cell surface receptor molecule stimulation. The activity of an HLF
antagonist will result in blocking an abnormally increased level of
HLF activity, and, in turn, diminish an abnormally increased level
of the stimulation of signal transduction pathways. This situation
will ultimately result in a return to the normal regulation of cell
division and growth and a corresponding diminution of the
corresponding oncogenic state. Thus, HLF antagonists of the present
invention may be employed to treat cancers. Any of the above
antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0193] Gene Mapping
[0194] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0195] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a HLF protein
gene. This can be accomplished using a variety of well known
techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose.
[0196] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Fluorescence
in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from
the cDNA as short as 50 or 60 bp. For a review of this technique,
see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,
Pergamon Press, New York (1988).
[0197] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0198] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0199] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1(a)
Expression and Purification of "GST-Tagged" EGF-Like Domain of HLF
in E. coli
[0200] The bacterial expression vector pGEX-3X was used for
bacterial expression in this example (Pharmacia, Inc., Uppsala,
Sweden). pGEX-3X encodes ampicillin antibiotic resistance ("Ampr")
and contains a bacterial origin of replication ("ori"), an IPTG
inducible promoter, and a sequence that encodes an N-terminal, in
frame, glutathione S-transferase (GST) tag that allows affinity
purification using one of the GST Purification Modules, and several
suitable single restriction enzyme cleavage sites. These elements
are arranged such that an inserted DNA fragment encoding a
polypeptide expresses that polypeptide with an N-terminal
GST-fusion protein.
[0201] The DNA sequence encoding the desired portion of the HLF
protein comprising the EGF-like domain of the HLF amino acid
sequence was amplified from the deposited cDNA clone using PCR
oligonucleotide primers which annealed to the amino and carboxy
terminal sequences of the desired portion of the HLF protein.
Additional nucleotides containing restriction sites to facilitate
cloning in the pGEX-3X vector were added to the 5' and 3' primer
sequences, respectively. For cloning the EGF-like domain of the HLF
protein, the 5' primer had the sequence 5'
GGCGGATCCCTCTTCTTCCTCCTCC 3' (SEQ ID NO:5) containing the
underlined Bam HI restriction site followed by 16 nucleotides of
the amino terminal coding sequence of the EGF-like domain of the
HLF sequence in SEQ ID NO:2. The 3' primer had the sequence 5'
GGCGAATTCTAAACTTCTTCACTCTCCATGAATTCAATCCCC 3' (SEQ ID NO:6)
containing the underlined Eco RI restriction site followed by 33
nucleotides complementary to the 3' end of the EGF-like domain of
the HLF DNA sequence in FIG. 1A.
[0202] The amplified HLF DNA fragment and the vector pGEX-3X were
digested with BamHI and Eco RI and the digested DNAs were then
ligated together. Insertion of the HLF DNA into the restricted
pGEX-3X vector placed the HLF protein coding region downstream from
the IPTG-inducible promoter and in frame with an initiating AUG and
the N-terminal GST fusion tag.
[0203] The ligation mixture was transformed into competent E. coli
cells using standard procedures such as those described in Sambrook
et al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
Plasmid DNA was isolated from resistant colonies and the identity
of the cloned DNA confirmed by restriction analysis, PCR and DNA
sequencing.
[0204] Clones containing the desired constructs were grown
overnight ("O/N") in liquid culture in LB media supplemented with
ampicillin (100 .mu.g/ml). The O/N culture was used to inoculate a
large culture, at a dilution of approximately 1:25 to 1:250. The
cells were grown to an optical density at 600 nm ("OD600") of
approximately 0.4. Isopropyl-.beta.-D-thiogalactopyranoside
("IPTG") was then added to a final concentration of 0.1 mM to
induce transcription from the lac repressor sensitive promoter, by
inactivating the lacI repressor. Cells subsequently were incubated
further for 3 to 4 hours. Cells then were harvested by
centrifugation, resuspended in 1.times.PBS, and lysed by
sonication.
[0205] The expressed GST-HLF(EGF domain) fusion protein was
purified using glutathione sepharose 4B essentially as described by
the manufacturer (Pharmacia, Uppsala, Sweden). Briefly, cell
lysates were combined with the glutathione sepharose 4B. The
mixture was pelleted by centrifugation and washed. The GST fusion
portion of the polypeptide was cleaved by the addition of thrombin
site-specific protease for 18 hours. Following cleavage, thrombin
was bound to p-Aminobenzmnidine agarose beads. The
thrombin-p-Aminobenzmidine agarose bead complexes and the
GST-glutathione sepharose complexes were pelleted by
centrifugation. The supernatant then contained the purified EGF
domain of the HLF protein. Purity of the protein preparation was
analyzed by SDS-PAGE. The purified protein was then stored frozen
at -20.degree. C.
Example 2
Cloning and Expression of HLF protein in a Baculovirus Expression
System
[0206] In this illustrative example, the plasmid shuffle vector
pA2GP is used to insert the cloned DNA encoding the mature protein,
lacking its naturally associated secretory signal (leader)
sequence, into a baculovirus to express the mature HLF protein,
using a baculovirus leader and standard methods as described in
Summers et al., A Manual of Methods for Baculovirus Vectors and
Insect Cell Culture Procedures, Texas Agricultural Experimental
Station Bulletin No. 1555 (1987). This expression vector contains
the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by the secretory
signal peptide (leader) of the baculovirus gp67 protein and
convenient restriction sites such as Bam HI, Xba I and Asp 718. The
polyadenylation site of the simian virus 40 (SV40) is used for
efficient polyadenylation. For easy selection of recombinant virus,
the plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate viable virus that expresses the cloned polynucleotide.
[0207] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0208] The cDNA sequence encoding the mature HLF protein in the
deposited clone, lacking the AUG initiation codon and the naturally
associated leader sequence shown in SEQ ID NO:2, is amplified using
PCR oligonucleotide primers corresponding to the 5' and 3'
sequences of the gene. The 5' primer has the sequence 5'
GGCGGATCCCCTCTTCTTCCTCCTCC-3' (SEQ ID NO:7) containing the
underlined Bam HI restriction enzyme site followed by 16
nucleotides of the sequence of the mature HLF protein shown in SEQ
ID NO:2, beginning with the indicated N-terminus of the
extracellular domain of the HLF protein. The 3' primer has the
sequence 5 GGCGGTACCTAAACTTCTTCACTCTCCATGAATTCAATCCCC 3' (SEQ ID
NO:8) containing the underlined Asp 718 restriction site followed
by 33 nucleotides complementary to the 3' coding sequence in FIG.
1A.
[0209] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with Bam HI and Asp
718 and again is purified on a 1% agarose gel. This fragment is
designated herein F1.
[0210] The plasmid is digested with the restriction enzymes Bam HI
and Asp 718 and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This
vector DNA is designated herein "V1".
[0211] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Statagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria are identified that contain the plasmid
with the human HLF gene by digesting DNA from individual colonies
using Bam HI and Asp 718 and then analyzing the digestion product
by gel electrophoresis. The sequence of the cloned fragment is
confirmed by DNA sequencing. This plasmid is designated herein
pA2GPHLF.
[0212] Five .mu.g of the plasmid pA2GPHLF is co-transfected with
1.0 .mu.g of a commercially available linearized baculovirus DNA
("BaculoGold.TM. baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofection method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84: 7413-7417 (1987). One .mu.g of
BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid pA2GPHLF are
mixed in a sterile well of a microtiter plate containing 50 .mu.l
of serum-free Grace's medium (Life Technologies Inc., Gaithersburg,
Md.). Afterwards, 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium
are added, mixed and incubated for 15 minutes at room temperature.
Then the transfection mixture is added drop-wise to Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate is then incubated for 5
hours at 27.degree. C. The transfection solution is then removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. Cultivation is then continued at
27.degree. C. for four days.
[0213] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10). After appropriate incubation, blue stained plaques are
picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 .mu.l of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at
4.degree. C. The recombinant virus is called V-HLF.
[0214] To verify the expression of the HLF gene Sf9 cells are grown
in Grace's medium supplemented with 10% heat-inactivated FBS. The
cells are infected with the recombinant baculovirus V-HLF at a
multiplicity of infection ("MOI") of about 2. If radiolabeled
proteins are desired, 6 hours later the medium is removed and is
replaced with SF-900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Rockville, Md.). After 42
hours, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[0215] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the extracellular domain of the HLF
protein.
Example 3
Cloning and Expression of HLF in Mammalian Cells
[0216] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and
pBC12MI (ATCC 67109). Mammalian host cells that could be used
include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[0217] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0218] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins. The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[0219] The expression plasmid, pHLFHA, is made by cloning a portion
of the cDNA encoding the extracellular domain of the HLF protein
into the expression vector pcDNAI/Amp or pcDNAIII (which can be
obtained from Invitrogen, Inc.). To produce a soluble, secreted
form of the polypeptide, the extracellular domain is fused to the
secretory leader sequence of the human IL-6 gene.
[0220] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37:767 (1984). The fusion of the HA tag to the
target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[0221] A DNA fragment encoding the extracellular domain of the HLF
polypeptide is cloned into the polylinker region of the vector so
that recombinant protein expression is directed by the CMV
promoter. The plasmid construction strategy is as follows. The HLF
cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above for
construction of vectors for expression of HLF in E. coli. Suitable
primers include the following, which are used in this example. The
5' primer, containing the underlined Bam HI site, a Kozak sequence,
an AUG start codon, a sequence encoding the secretory leader
peptide from the human IL-6 gene, and 16 nucleotides of the 5'
coding region of the extracellular domain of the HLF polypeptide,
has the following sequence:
TABLE-US-00002 5' GCCGGATCCGCCACCATGAAC
[0222] TCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTG
CCTGCTGCCTTCCCTGCCCCAGTCTCTTCTTCCTCCTCC 3' (SEQ ID NO:9). The 3'
primer, containing the underlined Xba I and 33 of nucleotides
complementary to the 3' coding sequence immediately before the stop
codon, has the following sequence: 5' GGCTCTAGATAAACTTCTTCAC
[0223] TCTCCATGAATTCAATCCCC 3' (SEQ ID NO:10).
[0224] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with Bam HI and Xba I and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037), and the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis or other
means for the presence of the fragment encoding the extracellular
domain of the HLF polypeptide
[0225] For expression of recombinant HLF, COS cells are transfected
with an expression vector, as described above, using DEAE-DEXTRAN,
as described, for instance, in Sambrook et al., Molecular Cloning:
a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring
Harbor, N.Y. (1989). Cells are incubated under conditions for
expression of HLF by the vector.
[0226] Expression of the HLF-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow et al., Antibodies: A Laboratory Manual, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing .sup.35S-cysteine for 8
hours. The cells and the media are collected, and the cells are
washed and the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson et al. cited above. Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0227] The vector pC4 is used for the expression of HLF
polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr
(ATCC Accession No. 37146). To produce a soluble, secreted form of
the polypeptide, the extracellular domain is fused to the secretory
leader sequence of the human IL-6 gene. The plasmid contains the
mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that
are transfected with these plasmids can be selected by growing the
cells in a selective medium (alpha minus MEM, Life Technologies)
supplemented with the chemotherapeutic agent methotrexate. The
amplification of the DHFR genes in cells resistant to methotrexate
(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R.
M., Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem.
253:1357-1370, Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys.
Acta, 1097:107-143, Page, M. J. and Sydenham, M. A. 1991,
Biotechnology 9:64-68). Cells grown in increasing concentrations of
MTX develop resistance to the drug by overproducing the target
enzyme, DHFR, as a result of amplification of the DHFR gene. If a
second gene is linked to the DHFR gene, it is usually co-amplified
and over-expressed. It is known in the art that this approach may
be used to develop cell lines carrying more than 1,000 copies of
the amplified gene(s). Subsequently, when the methotrexate is
withdrawn, cell lines are obtained which contain the amplified gene
integrated into one or more chromosome(s) of the host cell.
[0228] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rouse
Sarcoma Virus (Cullen, et al., Molecular and Cellular Biology,
March 1985:438-447) plus a fragment isolated from the enhancer of
the immediate early gene of human cytomegalovirus (CMV) (Boshart et
al., Cell 41:521-530 (1985)). Downstream of the promoter are the
following single restriction enzyme cleavage sites that allow the
integration of the genes: BamHI, Xba I, and Asp718. Behind these
cloning sites the plasmid contains the 3' intron and
polyadenylation site of the rat preproinsulin gene. Other high
efficiency promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. Clontech's Tet-Off and Tet-On gene expression systems and
similar systems can be used to express the HLF polypeptide in a
regulated way in mammalian cells (Gossen, M., & Bujard, H.
1992, Proc. Natl. Acad. Sci. USA 89:5547-5551). For the
polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell
lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more
than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[0229] The plasmid pC4 is digested with the restriction enzymes Bam
HI and Asp 718 and then dephosphorylated using calf intestinal
phosphates by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0230] The DNA sequence encoding the extracellular domain of the
HLF polypeptide is amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the desired portion of
the gene. The 5' primer containing the underlined Bam HI site, a
Kozak sequence, an AUG start codon, a sequence encoding the
secretory leader peptide from the human IL-6 gene, and 16
nucleotides of the 5' coding region of the extracellular domain of
the HLF polypeptide, has the following sequence (where Kozak is in
italics): 5' GCCGGATCCGCCA
CCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTG
GGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTCTCTTCTTCCTCCTCC 3'(SEQ ID
NO:9). The 3' primer, containing the underlined Asp 718 restriction
site and 33 nucleotides complementary to the 3' coding sequence
immediately before the stop codon as shown in FIG. 1A (SEQ ID NO:
1), has the following sequence: 5'
GGCGGTACCTAAACTTCTTCACTCTCCATGAATTCAATCCCC 3' (SEQ ID NO:8).
[0231] The amplified fragment is digested with the endonucleases
Bam HI and Asp 718 and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0232] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using lipofectin
(Felgner et al., supra). The plasmid pSV2-neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that
confers resistance to a group of antibiotics including G418. The
cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418.
After 2 days, the cells are typsinized and seeded in hybridoma
cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml G418. After
about 10-14 days single clones are trypsinized and then seeded in
6-well petri dishes or 10 ml flasks using different concentrations
of methotrexate (50 nM, 100 nM, 200 nM, 400 DM, 800 DM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 .mu.M, 2 .mu.M, 5 .mu.M, 10 mM,
20 mM). The same procedure is repeated until clones are obtained
which grow at a concentration of 100-200 .mu.M. Expression of the
desired gene product is analyzed, for instance, by SDS-PAGE and
Western blot or by reversed phase HPLC analysis.
Example 4
Tissue Distribution of HLF mRNA Expression
[0233] Northern blot analysis is carried out to examine HLF gene
expression in human tissues, using methods described by, among
others, Sambrook et al., cited above. A cDNA probe containing the
entire nucleotide sequence of the HLF protein (SEQ ID NO: 1) is
labeled with .sup.32P using the rediprime.TM. DNA labeling system
(Amersham Life Science), according to manufacturer's instructions.
After labeling, the probe is purified using a CHROMA SPIN-100.TM.
column (Clontech Laboratories, Inc.), according to manufacturer's
protocol number PT1200-1. The purified labeled probe is then used
to examine various human tissues for HLF mRNA.
[0234] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from Clontech and are examined with the labeled probe using
ExpressHyb.TM. hybridization solution (Clontech) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
Example 5
Analysis of erbB Receptor Family Activation
[0235] To test for the ability of recombinant EGF domain of the HLF
protein (as produced in Example 1) to activate erbB family members,
a tyrosine kinase activation assay was used as follows. In this
analysis, a human breast cancer cell line (MCF-7) was allowed to
become quiescent by extended culture in low serum medium. Exogenous
recombinant EGF domain of the HLF protein (10 mg.mL) or recombinant
heregulin (0.1 mg.mL) were added to the growth medium, and cell
culture was continued in the presence or absence of exogenous
protein for 30 minutes. Cells were harvested and lysed by the
addition of SDS-containing sample buffer (1% SDS, 0.15M Tris, pH
8.6, 5% BME, and 1 mM sodium ortho-vanadate).
[0236] Cell lysates were then subject to SDS-PAGE on 16-20%
Tris-glycine gradient gels (Novex). Subsequently,
electrophoretically separated proteins were transferred to a Hybond
ECL nitrocellulose membrane (Amersham). Tyrosine phosphate
containing proteins were identified by immunoblotting using
anti-phosphotyrosine antibodies.
[0237] As shown in FIG. 4, there is a clear increase in the
tyrosine phosphorylation of proteins in the size range of
approximately 185 kDa in samples prepared from cultures which were
grown in medium which contained the recombinant EGF domain of the
HLF protein. The erbB family of cell surface receptor molecules
consists of at least four members, all of which are roughly the
molecular mass of the proteins observed to increase in tyrosine
phosphorylation in this analysis. Furthermore, treatment of MCF-7
cells with recombinant heregulin in this analysis produced a
similar result with regard to a change in the tyrosine
phosphorylation state of cellular proteins.
[0238] These results strongly suggest that recombinant EGF domain
of the HLF protein was able to activate phosphorylation of at least
one of the members of the erbB family of cell surface receptors
expressed in these cells.
Example 6
HLF in Breast Cancer Cells, Activation of Multiple erbB
Proteins
[0239] Introduction
[0240] Increased activity of members of the erbB family has been
implicated in the development of cancer. Different molecular
mechanisms of activation have been identified. The ligands of the
EGF/Heregulin family are inappropriately expressed in breast
cancers. EGF, alpha-TGF, amphiregulin and heregulin are expressed
in breast cancers containing appropriate receptors thus leading to
autocrine growth stimulation. The importance of autocrine growth
stimulation is required for the transformation of NIH/3T3 cells
with high levels of EGF receptor, since full morphological
transformation requires the co-expression of alpha-TGF. The
causative role of autocrine growth stimulation by alpha-TGF in
breast cancer is demonstrated in experiments using transgenic
animals where the expression of alpha-TGF acts synergistically to
produce frequent breast cancers. These findings indicate that the
co-incident expression of erbB receptor proteins with their ligands
can result in aberrant cell growth. Moreover, in 20% of breast
cancers amplification of the erbB2 gene results in overexpression
of p185erb.sup.B-2. In these cancers activation of signalling has
been thought to be independent of ligand activation. Overexpression
of p185erb.sup.B-2 is an oncogenic event in experimental systems.
To date no ligand has been isolated that binds only to erbB2.
However, recent studies show that erbB2 can form part of a receptor
for heregulin. Overexpression of the erbB1 (EGFR) protein is
common, although gene amplification is infrequent in breast
cancer.
[0241] The erbB receptors bind their ligands as dimers--formed from
two identical erbB proteins (homodimers) or from two different
proteins (heterodimers). EGF can bind homodimers of erbB1 (EGFR) or
heterodimers of erbB1 and erbB2. Similarly, Heregulin1b can bind
homodimers of erbB4 or heterodimers of erbB2 and erbB3. Other
ligands of the EGF/Heregulin family have receptors formed by homo
and heterodimers of erbB proteins. Ligand binding and dimer
formation leads to increased autophosphorylation of the receptor
proteins and substrates activating intracellular signalling
pathways.
[0242] The cellular consequences of receptor stimulation by members
of the EGF/Heregulin family vary with the ligand and cellular
context. EGF and alpha-TGF can stimulate the growth of many cells
in culture, but in cases of breast cancers that overexpress the EGF
Receptor, EGF can be growth inhibitory at concentrations above
approximately 10 nM. In a similar way, heregulin can both stimulate
growth of some human cancer cells as well as inhibit those that
overexpress erbB2. The display of erbB proteins on breast cancer
cells is not uniform. Many cells lack one or more of the family and
others greatly overexpress erbB1 or erbB2. Heregulin clearly has
effects on cell morphology as evidenced by changes in the actin
cytoskeleton. Heregulin seems to play a number of specialized roles
in appropriate regulation the neuro-muscular junction, between
neuronal and glial cells and in Schwann cell development. In
prenatal development heregulin and erbB2 and erbB4 control in
morphogenesis of brain and heart. These findings indicate that
members of the EGF/Heregulin family can have differing effects on
cell phenotype.
[0243] In this study we characterize a new ligand for the
EGF/Heregulin family of growth factors. Our results indicate that
HLF binds and activates multiple members of the erbB family of
receptors. We demonstrate that HLF is expressed in a human breast
cancer cell line and can alter the growth of human breast cancer
cell lines. These results indicate that HLF may have in vivo
effects on the growth of the normal and malignant breast epithelial
cells.
[0244] Results
[0245] HLF Contains an EGF Like Domain.
[0246] The ligands of the EGF/Heregulin family have a well-defined
sequence similarity which we used to identify HLF. FIG. 5 shows a
compilation of known ligands of the EGF/Heregulin family; all
contain 6 cysteines. Between the fourth and sixth cysteine is the
common EGF-like folding motif containing a conserved hydrophobic
amino acid (Y37 (Tyr-68 of SEQ ID NO:2)) and a conserved glycine
(G39 (Gly-70 of SEQ ID NO:2)). This region apparently forms a very
stable core structure that is used in many extracellular proteins.
Sequence similarity among the ligands is not limited to this
folding motif. There is an exactly conserved arginine (R41 (Arg-72
of SEQ ID NO:2)) and hydrophobic amino acids at positions 14 and 16
(Asn-45 and Asp46 of SEQ ID NO:2). A hydrophobic amino acid that is
required for binding activity is found at position 46 or 47 (Leu-77
or Pro-78 of SEQ ID NO:2). The number of amino acids between
cysteines is similar among the ligands with the notable exception
of loop B and loop C. Heregulins have a loop C which is three amino
acids longer than the EGF-like ligands. The overall sequence
similarity among the EGF/Heregulin family members is 19-42% (except
between Hrg1a and Hrg1b which are derived from the same gene).
Recently, NRG2 has been identified in rat brain. NRG2 is most
closely related to Hrg1b with sequence identity of 42% in the EGF
like domain.
[0247] Using a consensus sequence derived from EGF and Heregulin
sequences we screened the HGS database of over 800,000 sequences.
As shown in FIG. 5, one cDNA encoded the HLF sequence which has
34-38% similarity to EGF and Heregulin Family within the EGF-like
domain. Importantly, in the HLF sequence, all of the conserved
cysteine residues, the R41, and the G39 (Gly-70 and Arg-72 of SEQ
ID NO:2, respectively) are exactly conserved. There is additional
sequence conservation in NRG-3, notably hydrophobic amino acids at
positions 13, 15, 37 and 46-47 (Leu-44, Asp-46, Tyr-68, and
Leu-77-Pro-78 of SEQ ID NO:2, respectively). The length of the B
and C loops are more similar to heregulin than EGF. Within the
coding frame defined by our current cDNA clone there is a sequence
of hydrophobic amino acids that is consistent with a transmembrane
domain C-terminal to the EGF-like domain. This structure is similar
to the transmembrane domains found in alpha-TGF, EGF, heregulin and
other ligand precursor proteins (not shown in FIG. 5). The sequence
attributes of the HLF cDNA make it a strong candidate as encoding a
novel growth factor binding one or several of the erbB family of
receptors. NGR-2 is 36% identical to HLF in the highly conserved
EGF-like domain therefore they are products of distinct genes.
Don-1 has also been recently and independently identified by
sequence similarity to EGF/Heregulin and is apparently the product
of the same gene as NRG-2.
[0248] Demonstration that HLF Activates ErbB Family Proteins.
[0249] In order to obtain an initial estimation for the action of
HLF as a ligand for erbB family of receptors we generated
recombinant protein in E. coli using a GST fusion system (see also
Example 1). The EGF-like domain of HLF was released and purified
from the GST by thrombin cleavage. The resulting protein contained
a single polypeptide when analyzed by SDS-PAGE.
[0250] To test for the ability of recombinant HLF to activate
receptors of the erbB family we used a tyrosine kinase activation
assay. Tyrosine phosphate containing proteins were then identified
by immunoblotting using anti-phosphotyrosine antibodies. We found a
clear increase in the tyrosine phosphorylation at .about.p185 when
recombinant HLF is applied to MCF-7. In MCF-7 cells recombinant
heregulin-1b results in a large increase in tyrosine phosphorylated
proteins at about this size. These results indicate that
recombinant HLF is able to activate phosphorylation of at least one
of the members of the erbB family expressed in MCF-7 cells.
[0251] HLF Activates Multiple erbB Proteins.
[0252] In order to begin the analysis of the HLF receptor we used
an experimental system where the display of erbB proteins can be
controlled. The 32D cell is a murine myeloid cell line which is
devoid of expression of genes of the erbB family. Growth of 32D
cells is dependent on the IL-3 present in WEHI conditioned media.
When expression constructs encoding an erbB protein are introduced
into 32D cells the resulting cell can survive in the absence of
IL-3 if an appropriate EGF/Heregulin family member is present. For
example, the introduction of EGF Receptor expression leads to
growth of 32D cells in the presence of EGF or alpha-TGF and
introduction of erbB4 allows growth in heregulin. Similar
experimental systems have been used to examine the receptor
specificity of the newly discovered NRG-2. We also show that growth
of 32D cells in the presence of HLF occurs only when EGF Receptor
or erbB4 are present singly or when erbB2 and erbB3 are present in
combination. The expression of erbB2 or erbB3 alone does not lead
to HLF induced growth. To confirm that this growth stimulation was
the result of receptor activation we determined whether HLF induces
the tyrosine phosphorylation of EGF Receptor, erbB4 or erbB2 and
erbB3 when expressed together. We show the appearance of an
appropriate sized band when cell lysates of these 32D cells are
probed by antiphosphotyrosine antibodies. These results are strong
evidence that HLF can activate erbB1 homodimers (the EGF receptor),
erbB4 homodimers, and erbB2+erbB3 heterodimers.
[0253] The results of 32D experiments indicate that the receptor
binding pattern of HLF is complex. In order to confirm that HLF
activates proteins other than erbB4 in MCF-7 cells we
immunoprecipitated erbB3 and determined the level of tyrosine
phosphorylation by immunoblot. We also observed that erbB3 is
phosphorylated on tyrosine as a consequence of HLF stimulation.
[0254] Biological Activity of HLF.
[0255] The effects of the EGF/Heregulin family vary significantly.
Differing cellular phenotypes can be induced by different ligands
in the same cell system and the same ligand can cause differing
effects among different cells. Mitogenic activity of HLF has been
detected in 32D cell experiments. The MCF-7 cell is dependent on
estrogens in the media either in the form of phenol red or present
in the fetal bovine serum Little proliferation is seen in phenol
red free media containing serum treated with charcoal to remove
steroids. Heregulin is able to promote growth in the absence of
estrogen. When HLF is added there is also a clear growth
stimulation. Growth inhibitory effects have also been observed. HLF
inhibits the growth of the breast cancer cell line MDA-MB-468.
These cells overexpress the EGF receptor and can be stimulated by
EGF at low concentrations (<10 nM) and growth inhibited at
higher concentrations (>10 nM). HLF was found to inhibit growth
of MDA-MB-468 under conditions similar to those producing growth
stimulation of 32D cells containing EGF Receptor. No growth
suppression or stimulation are seen when HLF is applied to MCF-7
cells when they are grown in media containing agonists for the
estrogen receptor.
[0256] HLF mRNA Expression in Breast Cancer.
[0257] Preliminary experiments using northern blotting methods
showed a weak signal for HLF mRNA in adult brain with a size of
approximately 2 kD (data not shown). Similar northern blot results
are reported in the recent HLF study. Because of the weakness of
this signal we have used RT-PCR to detect HLF mRNA. We have
confirmed expression in the brain and detect equivalent signals in
samples of normal and breast cancer tissue. RT-PCR employed two
primer sets. The two primer sets generated concordant results. This
indicates that the bands observed by RT-PCR were due to actual HLF
mRNA. In addition all assays included control reactions lacking
reverse transcriptase in order to detect the presence of
contaminating DNA. The observed band at 340 bp corresponds to the
predicted size based on the HLF cDNA. It was cloned sequenced and
shown to contain HLF coding information. Bands at 500 bp and 120 bp
were also sequenced. These do not contain HLF coding information
and thus likely represent mispriming by the RT-PCR oligonucleotides
on unrelated mRNAs. These results are strong evidence that HLF can
be expressed in human breast cancer cell lines.
[0258] Discussion
[0259] Our results suggest that HLF can bind and activate erbB1 and
heterodimers of erbB2+erbB3. Taken together the available data
suggests that the precise receptor binding and activation profile
of HLF is complex. Our results demonstrate that erbB3 can be
phosphorylated as a consequence of HLF binding. The HLF induced
increases in tyrosine phosphorylation on erbB3 suggests that the
erbB3 protein can be part of an HLF receptor. Our studies of 32D
cells supports this conclusion where erbB2 is the other member of
the heterodimeric receptor with erbB3. Still to be determined is
whether HLF can bind to erbB1+erbB3 heterodimers or erbB3+erbB4
heterodimers or erbB2+erbB4 heterodimers. Our preliminary data does
conclusively demonstrate that HLF is a new ligand for the erbB
family of receptors. These results suggest that HLF may have a
receptor specificity somewhat analogous to b-cellulin.
[0260] In adult tissue expression levels of HLF are low but
detectable using sensitive methods such as RT-PCR. HLF is expressed
at the highest levels in brain where it is likely to play a
critical role in morphogenesis. Interestingly, we identify HLF
expression a breast cancer cell line, MCF-7, that clearly has
receptors that can be activated by HLF. Our results also show that
HLF can cause alteration of growth of MCF-7 cancer cells in vitro.
The ability to cause growth of MCF-7 cells in the absence of
estrogen is similar to that previously reported for heregulin. Our
results suggest that effects on cell phenotype by HLF may depend on
the cell line. MDA-MB-468 which has high levels of EGFR are growth
inhibited by HLF in vitro.
[0261] The results in this paper together with those recently
reported earlier identify HLF as a new ligand for the erbB family
of growth factor receptors and suggest a role for HLF in the growth
regulation of normal and malignant breast epithelial cells.
[0262] Materials and Methods
[0263] Preparation of Recombinant HLF. Preparation of recombinant
HLF is also described in Example 1. In the case of the protein
produced in this Example, the coding segment containing the
EGF-like domain of HLF (nucleotide 79 to 279 of HGS38) were
amplified by PCR and inserted into the pGEX3 plasmid for expression
as a fusion protein with bacterial glutathione S transferase.
Protein was prepared using standard methods. Bacteria were cultured
to an OD.sub.600 of approximately 0.4 and induced to express
recombinant protein by addition of 0.1 mM IPTG. Bacteria were
collected by centrifugation resuspended in 1.times.PBS and lysed by
sonication. Recombinant protein was collected by incubation with
glutathione beads. After washing the recombinant HLF protein was
cleaved from the GST bound to the beads by thrombin cleavage for 18
hours. Thrombin was removed by incubation with p-Aminobenzamidine
agarose beads. Refolding followed the methods used for the
preparation of recombinant antibody fragments. Briefly, recombinant
HLF was denatured in 6 M guanidine HCL containing 65 mM DTE. This
was rapidly diluted 100 fold to a final protein concentration of
100 .mu.g/ml into 0.4 M Arginine, 0.1 M Tris pH 8.0, 0.9 mM
oxidized glutathione 2.0 mM EDTA. Refolding was allowed to proceed
for 24 hours at 4.degree. C. Refolded protein was extensively
dialyzed against PBS using 3000 kDa cutoff membranes. Protein
preparations were stored at -20.degree. C.
[0264] Detection of Receptor Activation Byphosphotyrosine
Immunoblot.
[0265] Cells were starved (24 hours for MCF-7, 4 hours for 32D
derived cell lines) before addition of the indicated amounts of
growth factors. Total cell lysates were prepared by addition of SDS
PAGE sample buffer (1% SDS, 0.15M Tris pH 8.6, 5% BME and 1 mM
Sodium OrthoVanadate) directly to cells. Cell lysates and were run
on 8-16% Tris-Glycine gradient gels (Novex). Proteins were
transferred onto Hybond ECL nitrocellulose membranes (Amersham) and
were immunoblotted with anti-phosphotyrosine MAb.
[0266] 32D Cell Experiments.
[0267] 32D cells containing expression constructs for erbB1, erbB2,
erbB3, erbB4 and erbB2 and erbB3 together were grown in IL-3
containing (WEHI conditioned media) or HRG-1-beta prior to the
experiment. Expression of the erbB proteins was verified by FACS
analysis using erbB-specific antisera. Cells (10.sup.4 per well)
were plated in 24 well dishes in the absence of IL-3 containing
media (DMEM, 10% FCS) or in the presence of the indicated growth
factors, heregulin-1beta (100 ng/ml), EGF (100 ng/ml), and HLF (10
g/ml). Cells were allowed to grow for 3 days and viable cells
counted using a hemocytometer.
[0268] Immunoprecipitation and Immunoblot of erbB3.
[0269] Cells were plated in 80 cm.sup.2 dishes (DMEM+10% FCS) for
until 80% confluent. Cells were then allowed to become quiescent in
serum free media (DMEM) for 24 hours. Cells were then stimulated
with the indicated growth factors, heregulin-1beta (1 .mu.g/ml),
and HLF (10 .mu.g/ml) for 15 minutes. Cells were lysed in 1% Triton
X100 in PBS containing 1 mM Sodium orthovanadate. Nuclei were
removed by centrifugation. erbB3 proteins were immunoprecipitated
(2 hours at 40.degree. C.) using monoclonal anti-erbB3 antibodies
(Neomarkers) and collected on protein A sepharose. Proteins were
released by incubation in 1% SDS containing PAGE sample buffer at
100.degree. C. and electrophoresed on 8-16% gels (Novagen).
Proteins were transferred to nitrocellulose. Proteins containing
pTyr were detected using monoclonal anti-phosphotyrosine antibodies
(Oncogene Science) and the ECL detection system (Amersham).
[0270] Growth Assays.
[0271] Cells were plated in IMEM+10% FBS at 3000 cells per well in
96 well dishes. Cells were allowed to become quiescent in serum
free IMEM for 24 hours and growth factors EGF (2 ng/ml) and HLF (10
.mu.g/ml) were added to the media. Growth of cells at 1, 3, and 5
days was monitored using the XTT assay method. XTT was added at 10
.mu.g/ml in IMEM and PMS (1.5 mg/ml in PBS) to 25% of volume of
well for 4 hours at 37.degree. C. OD monitored at 540 nm.
[0272] Detection of HLF mRNA.
[0273] Total RNA was extracted from cultured cells using the
RNazolB method (Tel-Test, CS-104). The final RNA pellet was
resuspended in 135 .mu.l DEPC treated H.sub.20. DNase treatment was
performed using the SNAP RNA isolation kit (Invitrogen, K1950-01).
Briefly, 10.times. DNase buffer and RNase free DNase I was added to
each sample and incubated for 20 min at 37.degree. C. RNA
purification was performed as indicated in the kit. Concentration
of each sample was determined, samples were dried and resuspended
to give a final concentration of 2 .mu.g/ul.
[0274] RT-PCR was performed using 2 .mu.g of total RNA in the Gene
Amp RNA PCR Core kit (Perkin Elmer, N808-0143). cDNA was
synthesized using the downstream primer 5'-CCA CGA TGA CAA TTC CAA
AG-3' (SEQ ID NO:20). Samples were reverse transcribed 1 h at
37.degree. C. RT was heat inactivated 5 min at 99.degree. C.,
samples were cooled on ice. PCR was performed with the entire RT
reaction using the upstream primer 5'-TAC CAC CAC CAC ACC AGA AA-3'
(SEQ ID NO:21). The reaction was performed for 40 cycles, 1 min at
94.degree. C., 1 min 30 sec at 58.degree. C., 2 min at 72.degree.
C. followed by an extension for 8 min at 72.degree. C. Samples were
electrophoresed on an agarose gel and visualized with ethidium
bromide staining.
[0275] Confirmation of the sequence of the bands was performed by
purifying the bands from agarose gel slices (Wizard PCR preps DNA
purification system, Promeoa, A7170) and cloning into a TA vector
(Invitrogen, K2000-JIO) for automated sequencing. Bands of unknown
identity present in the reaction products were cloned and sequenced
in a similar fashion.
[0276] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0277] The entire disclosure of all publications (including
patents, patent applications, journal articles, manuscripts,
laboratory manuals, books, or other documents) cited herein are
hereby incorporated by reference.
Sequence CWU 1
1
2212199DNAHomo sapiens 1ctcttcttcc tcctccgcta ccaccaccac accagaaact
agcaccagcc ccaaatttca 60tacgacgaca tattccacag agcgatccga gcacttcaaa
ccctgccgag acaaggacct 120tgcatactgt ctcaatgatg gcgagtgctt
tgtgatcgaa accctgaccg gatcccataa 180acactgtcgg tgcaaagaag
gctaccaagg agtccgttgt gatcaatttc tgccgaaaac 240tgattccatc
ttatcggatc caaaccactt ggggattgaa ttcatggaga gtgaagaagt
300ttatcaaagg caggtgctgt caatttcatg tatcatcttt ggaattgtca
tcgtgggcat 360gttctgtgca gcattctact tcaaaagcaa aaggaatatt
acagcaaatt ctgtgtctga 420ggaaagatgg aagggtctgc cttcccagga
gcccaatctg caacaagaca aataatgcct 480aacaatggat taatgatgtc
tactattctg caacttacat ctcatttctt tctaatgcat 540tggaccagag
aaatttaaaa ctcaaatgaa ctgtaaagtt tccacactga cactgttggg
600ctaatagtat tcccatgtgc aaggcatgca tcttttcttc cccagagcaa
tgcctctcat 660gagagagcta atggtattgc aatcagctgc tgattgtttt
ctctgttccc attttctggg 720tgaaggaaga aagagcaaaa aagtgtgtgc
ttgtgagaga ggagggatgg tagataggca 780gaggcaggct cagaatggaa
ggaccacgta tcttggaata ttactaagtc aggacttgag 840tgaaaaaaga
ctaaaggtaa gcaaattata aaaggattta ggaaacgcag tccggtattg
900gatattgctt aaagaaaatt cccttataag tttatacttc caagactctg
aattggatta 960ctgcaaacat cattaagtgt ttctaattta atcccatgag
agtaatggaa tccttgctct 1020gagacatgca ctcttacttt ttcaggatga
tttaccagac tagaacctcc tgatttcccc 1080ttttttgtgt gtgtgaatga
acccctgata aaatcttgtg gctgtaacat gctccttaaa 1140atgctgatat
gatagattta tttttaacaa taggctatag attagctgtt aggaagcaaa
1200tagattatta caacaggatt aaagcaacta agagtgctag agataaaagt
ctcccaaata 1260attggaaaga taaaagaaat atcttaaaaa acagagctac
atcacactga tattgtaaat 1320tcaaaatggg taatgaagct caaagcctcc
aaagcttgca gcaagtgctg gtgaattgct 1380tgggaagatg caactagtgt
aatcttttac ctttgggtca atgttctgat tcttttgcag 1440cttctgctca
caagactgag cttgcttgat ggtatcggga aagatatgaa cattttgcgt
1500gtgcctccac atgcagccac cacagtgtcc gtggaagata gcttttatga
acttcattta 1560cagaggagga aatggaggct caacaagttt aggaaattat
tagggtagca aaactagtgg 1620gtagcagagt gggattcaaa tcccagtccc
tgtgatacaa taagccacgc tctgtagggt 1680gctactgact ggagaagctc
attgctaaga ccggccatgt gctccactga cggcactatc 1740tttgtcagag
acgttggaag acaggcaaaa ttcaagggca tgattctact gggaaagttg
1800tcagaatcaa aatggagtca tttgtgttaa aaaccctgac aaatagagcc
ggagaaggac 1860atgaagggag cagtcacgta ggcaaatgcc tgattacaag
aactatcaca aaagtctgtg 1920aaaaccgcag ctttgcatga agactattgc
agccttacac gcacgaaaat agttctgcaa 1980ggacatatgc ccagcaactt
cctgtccacc cttggactgg ctcctccttt cttgggatcc 2040ttgcagccaa
ggatagtgac ctcaaatcag ttgtgtacct aacgtttcct gtcttcctag
2100tgataaaaca tagtttccta tatcgtgtgt attcccattg caacacttat
ttccaaataa 2160atattttctt ttagagtctc aaaaaaaaaa aaaaaaaaa
21992157PRTHomo sapiens 2Ser Ser Ser Ser Ser Ala Thr Thr Thr Thr
Pro Glu Thr Ser Thr Ser1 5 10 15Pro Lys Phe His Thr Thr Thr Tyr Ser
Thr Glu Arg Ser Glu His Phe20 25 30Lys Pro Cys Arg Asp Lys Asp Leu
Ala Tyr Cys Leu Asn Asp Gly Glu35 40 45Cys Phe Val Ile Glu Thr Leu
Thr Gly Ser His Lys His Cys Arg Cys50 55 60Lys Glu Gly Tyr Gln Gly
Val Arg Cys Asp Gln Phe Leu Pro Lys Thr65 70 75 80Asp Ser Ile Leu
Ser Asp Pro Asn His Leu Gly Ile Glu Phe Met Glu85 90 95Ser Glu Glu
Val Tyr Gln Arg Gln Val Leu Ser Ile Ser Cys Ile Ile100 105 110Phe
Gly Ile Val Ile Val Gly Met Phe Cys Ala Ala Phe Tyr Phe Lys115 120
125Ser Lys Arg Asn Ile Thr Ala Asn Ser Val Ser Glu Glu Arg Trp
Lys130 135 140Gly Leu Pro Ser Gln Glu Pro Asn Leu Gln Gln Asp
Lys145 150 1553645PRTHomo sapiens 3Met Ser Glu Arg Lys Glu Gly Arg
Gly Lys Gly Lys Gly Lys Lys Lys1 5 10 15Glu Arg Gly Ser Gly Lys Lys
Pro Glu Ser Ala Ala Gly Ser Gln Ser20 25 30Pro Ala Leu Pro Pro Gln
Leu Lys Glu Met Lys Ser Gln Glu Ser Ala35 40 45Ala Gly Ser Lys Leu
Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser50 55 60Ser Leu Arg Phe
Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys65 70 75 80Asn Lys
Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu85 90 95Leu
Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys100 105
110Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile
Thr115 120 125Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala
Ser Thr Glu130 135 140Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg
Ile Ser Val Ser Thr145 150 155 160Glu Gly Ala Asn Thr Ser Ser Ser
Thr Ser Thr Ser Thr Thr Gly Thr165 170 175Ser His Leu Val Lys Cys
Ala Glu Lys Glu Lys Thr Phe Cys Val Asn180 185 190Gly Gly Glu Cys
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr195 200 205Leu Cys
Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr210 215
220Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu
Ala225 230 235 240Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr
Gly Ile Cys Ile245 250 255Ala Leu Leu Val Val Gly Ile Met Cys Val
Val Ala Tyr Cys Lys Thr260 265 270Lys Lys Gln Arg Lys Lys Leu His
Asp Arg Leu Arg Gln Ser Leu Arg275 280 285Ser Glu Arg Asn Asn Met
Met Asn Ile Ala Asn Gly Pro His His Pro290 295 300Asn Pro Pro Pro
Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys305 310 315 320Asn
Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser325 330
335Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr Thr
Val340 345 350Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr
Glu Ser Ile355 360 365Leu Ser Glu Ser His Ser Val Ile Val Met Ser
Ser Val Glu Asn Ser370 375 380Arg His Ser Ser Pro Thr Gly Gly Pro
Arg Gly Arg Leu Asn Gly Thr385 390 395 400Gly Gly Pro Arg Glu Cys
Asn Ser Phe Leu Arg His Ala Arg Glu Thr405 410 415Pro Asp Ser Tyr
Arg Asp Ser Pro His Ser Glu Arg Tyr Val Ser Ala420 425 430Met Thr
Thr Pro Ala Arg Met Ser Pro Val Asp Phe His Thr Pro Ser435 440
445Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro Pro Val Ser Ser
Met450 455 460Thr Val Ser Met Pro Ser Met Ala Val Ser Pro Phe Met
Glu Glu Glu465 470 475 480Arg Pro Leu Leu Leu Val Thr Pro Pro Arg
Leu Arg Glu Lys Lys Phe485 490 495Asp His His Pro Gln Gln Phe Ser
Ser Phe His His Asn Pro Ala His500 505 510Asp Ser Asn Ser Leu Pro
Ala Ser Pro Leu Arg Ile Val Glu Asp Glu515 520 525Glu Tyr Glu Thr
Thr Gln Glu Tyr Glu Pro Ala Gln Glu Pro Val Lys530 535 540Lys Leu
Ala Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro Asn Gly His545 550 555
560Ile Ala Asn Arg Leu Glu Val Asp Ser Asn Thr Ser Ser Gln Ser
Ser565 570 575Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu
Asp Thr Pro580 585 590Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser
Leu Glu Ala Thr Pro595 600 605Ala Phe Arg Leu Ala Asp Ser Arg Thr
Asn Pro Ala Gly Arg Phe Ser610 615 620Thr Gln Glu Glu Ile Gln Ala
Arg Leu Ser Ser Val Ile Ala Asn Gln625 630 635 640Asp Pro Ile Ala
Val6454536DNAHomo sapiensmisc_feature(315)n equals a,t,g, or c
4ggcacagctc ttcttcctcc tccgctacca ccaccacacc agaaactagc accagcccca
60aatttcatac gacgacatat tccacagagc gatccgagca cttcaaaccc tgccgagaca
120aggaccttgg catactgtct caatgatggc gagtgctttg tgatcgaaac
cctgaccgga 180tcccattaaa cactgtcggt gcaaagaagg ctaccaagga
gtccgttgtg atcaatttct 240gccgaaaact gattccatct tatcggatcc
aaaccacttg gggattggaa ttcatgggag 300agtgaagaag ttttnnccaa
agggcaggtg ntgtncaatt tccaagtgnn caactttggg 360gattggtncn
tcgtgggggc ntgtttnngg tggcagcatt tcntaactnc caaaaagcca
420aaaagggatt tttnaccggc aaatttccgt gntctgaagg gaaaattggg
aagggtcttg 480ccctttcccc aggaggccca attnggncaa caaggccaat
natggcntaa caaggg 536525DNAArtificial sequence5' primer for cloning
the EGF like domain of the HLF protein 5ggcggatccc tcttcttcct cctcc
25642DNAArtificial sequence3' primer for cloning the EGF like
domain of the HLF protein 6ggcgaattct aaacttcttc actctccatg
aattcaatcc cc 42726DNAArtificial sequence5' primer for amplifying
the mature HLF sequence 7ggcggatccc ctcttcttcc tcctcc
26842DNAArtificial sequence3' primer for amplifying the mature HLF
sequence 8ggcggtacct aaacttcttc actctccatg aattcaatcc cc
429120DNAArtificial sequence5' primer, containing a Bam HI site, a
Kozak sequence,an AUG start codon, a sequence encoding the
secretory leader peptide from the human IL-6 gene, and 16
nucleotides of the 5' coding region of the extracellular domain of
the HLF polypeptide 9gccggatccg ccaccatgaa ctccttctcc acaagcgcct
tcggtccagt tgccttctcc 60ctggggctgc tcctggtgtt gcctgctgcc ttccctgccc
cagtctcttc ttcctcctcc 1201042DNAArtificial sequence3' primer,
containing the Xba I and 33 nucleotides complementaryto the 3'
coding sequence OF HLF immediately before the stop codon
10ggctctagat aaacttcttc actctccatg aattcaatcc cc 421148PRTHomo
sapiens 11Ser His Phe Asn Asp Cys Pro Asp Ser His Thr Gln Phe Cys
Phe His1 5 10 15Gly Thr Cys Arg Phe Leu Val Gln Glu Asp Lys Pro Ala
Cys Val Cys20 25 30His Ser Gly Tyr Val Gly Ala Arg Cys Glu His Ala
Asp Leu Leu Ala35 40 451250PRTHomo sapiens 12Arg Asn Ser Asp Ser
Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu1 5 10 15His Asp Gly Val
Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys20 25 30Asn Cys Val
Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu35 40 45Lys
Trp501348PRTHomo sapiens 13Gly Lys Lys Arg Asp Pro Cys Leu Arg Lys
Tyr Lys Asp Phe Cys Ile1 5 10 15His Gly Glu Cys Lys Tyr Val Lys Glu
Leu Arg Ala Pro Ser Cys Ile20 25 30Cys His Pro Gly Tyr Gly Gly Glu
Arg Cys His Gly Leu Ser Leu Pro35 40 451449PRTHomo sapiens 14Arg
Lys Lys Lys Asn Pro Cys Asn Ala Glu Phe Gln Asn Phe Cys Ile1 5 10
15His Gly Glu Cys Lys Tyr Ile Glu His Leu Glu Ala Val Thr Cys Lys20
25 30Cys Gln Gln Glu Tyr Phe Gly Glu Arg Cys Gly Glu Lys Ser Met
Lys35 40 45Thr1549PRTHomo sapiens 15Lys Gly His Phe Ser Arg Cys Pro
Lys Gln Tyr Lys His Tyr Cys Ile1 5 10 15Lys Gly Arg Cys Arg Phe Val
Val Ala Glu Gln Thr Pro Ser Cys Val20 25 30Cys Asp Glu Gly Tyr Ile
Gly Ala Arg Cys Glu Arg Val Asp Leu Phe35 40 45Tyr1652PRTHomo
sapiens 16Thr Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe
Cys Val1 5 10 15Asn Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn
Pro Ser Arg20 25 30Tyr Leu Cys Lys Cys Pro Gly Phe Thr Gly Ala Arg
Cys Thr Glu Asn35 40 45Val Pro Met Lys501753PRTHomo sapiens 17Thr
Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val1 5 10
15Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg20
25 30Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr
Glu35 40 45Asn Val Pro Met Lys501853PRTHomo sapiens 18Thr Ser His
Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val1 5 10 15Asn Gly
Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg20 25 30Tyr
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn35 40
45Tyr Val Met Ala Ser501950PRTHomo sapiens 19Ser Gly His Ala Arg
Lys Cys Asn Glu Thr Ala Lys Ser Tyr Cys Val1 5 10 15Asn Gly Gly Val
Cys Tyr Tyr Ile Glu Gly Ile Asn Gln Leu Ser Cys20 25 30Lys Cys Pro
Val Gly Tyr Thr Gly Asp Arg Cys Gln Gln Phe Ala Met35 40 45Val
Asn502020DNAArtificial sequencedownstream primer for synthesizing
HLF cDNA 20ccacgatgac aattccaaag 202120DNAArtificial
sequenceupstream primer for HLF PCR 21taccaccacc acaccagaaa
2022720PRTHomo sapiens 22Met Ser Glu Gly Ala Ala Ala Ala Ser Pro
Pro Gly Ala Ala Ser Ala1 5 10 15Ala Ala Ala Ser Ala Glu Glu Gly Thr
Ala Ala Ala Ala Ala Ala Ala20 25 30Ala Ala Gly Gly Gly Pro Asp Gly
Gly Gly Glu Gly Ala Ala Glu Pro35 40 45Pro Arg Glu Leu Arg Cys Ser
Asp Cys Ile Val Trp Asn Arg Gln Gln50 55 60Thr Trp Leu Cys Val Val
Pro Leu Phe Ile Gly Phe Ile Gly Leu Gly65 70 75 80Leu Ser Leu Met
Leu Leu Lys Trp Ile Val Val Gly Ser Val Lys Glu85 90 95Tyr Val Pro
Thr Asp Leu Val Asp Ser Lys Gly Met Gly Gln Asp Pro100 105 110Phe
Phe Leu Ser Lys Pro Ser Ser Phe Pro Lys Ala Met Glu Thr Thr115 120
125Thr Thr Thr Thr Ser Thr Thr Ser Pro Ala Thr Pro Ser Ala Gly
Gly130 135 140Ala Ala Ser Ser Arg Thr Pro Asn Arg Ile Ser Thr Arg
Leu Thr Thr145 150 155 160Ile Thr Arg Ala Pro Thr Arg Phe Pro Gly
His Arg Val Pro Ile Arg165 170 175Ala Ser Pro Arg Ser Thr Thr Ala
Arg Asn Thr Ala Ala Pro Ala Thr180 185 190Val Pro Ser Thr Thr Ala
Pro Phe Phe Ser Ser Ser Thr Leu Gly Ser195 200 205Arg Pro Pro Val
Pro Gly Thr Pro Ser Thr Gln Ala Met Pro Ser Trp210 215 220Pro Thr
Ala Ala Tyr Ala Thr Ser Ser Tyr Leu His Asp Ser Thr Pro225 230 235
240Ser Trp Thr Leu Ser Pro Phe Gln Asp Ala Ala Ser Ser Ser Ser
Ser245 250 255Ser Ser Ser Ser Ser Thr Thr Thr Thr Pro Glu Thr Ser
Thr Ser Pro260 265 270Lys Phe His Thr Thr Thr Tyr Ser Thr Glu Arg
Ser Glu His Phe Lys275 280 285Pro Cys Arg Asp Lys Asp Leu Ala Tyr
Cys Leu Asn Asp Gly Glu Cys290 295 300Phe Val Ile Glu Thr Leu Thr
Gly Ser His Lys His Cys Arg Cys Lys305 310 315 320Glu Gly Tyr Gln
Gly Val Arg Cys Asp Gln Phe Leu Pro Lys Thr Asp325 330 335Ser Ile
Leu Ser Asp Pro Thr Asp His Leu Gly Ile Glu Phe Met Glu340 345
350Ser Glu Glu Val Tyr Gln Arg Gln Val Leu Ser Ile Ser Cys Ile
Ile355 360 365Phe Gly Ile Val Ile Val Gly Met Phe Cys Ala Ala Phe
Tyr Phe Lys370 375 380Ser Lys Lys Gln Ala Lys Gln Ile Gln Glu Gln
Leu Lys Val Pro Gln385 390 395 400Asn Gly Lys Ser Tyr Ser Leu Lys
Ala Ser Ser Thr Met Ala Lys Ser405 410 415Glu Asn Leu Val Lys Ser
His Val Gln Leu Gln Asn Tyr Ser Lys Val420 425 430Glu Arg His Pro
Val Thr Ala Leu Glu Lys Met Met Glu Ser Ser Phe435 440 445Val Gly
Pro Gln Ser Phe Pro Glu Val Pro Ser Pro Asp Arg Gly Ser450 455
460Gln Ser Val Lys His His Arg Ser Leu Ser Ser Cys Cys Ser Pro
Gly465 470 475 480Gln Arg Ser Gly Met Leu His Arg Asn Ala Phe Arg
Arg Thr Pro Pro485 490 495Ser Pro Arg Ser Arg Leu Gly Gly Ile Val
Gly Pro Ala Tyr Gln Gln500 505 510Leu Glu Glu Ser Arg Ile Pro Asp
Gln Asp Thr Ile Pro Cys Gln Gly515 520 525Ile Glu Val Arg Lys Thr
Ile Ser His Leu Pro Ile Gln Leu Trp Cys530 535 540Val Glu Arg Pro
Leu Asp Leu Lys Tyr Ser Ser Ser Gly Leu Lys Thr545 550 555 560Gln
Arg Asn Thr Ser Ile Asn Met Gln Leu Pro Ser Arg Glu Thr Asn565 570
575Pro Tyr Phe Asn Ser Leu Glu Gln Lys Asp Leu Val Gly Tyr Ser
Ser580 585 590Thr Arg Ala Ser Ser Val Pro Ile Ile Pro Ser Val Gly
Leu Glu Glu595 600 605Thr Cys Leu Gln Met Pro Gly Ile Ser Glu Val
Lys Ser Ile Lys Trp610 615 620Cys Lys Asn Ser Tyr Ser Ala Asp Val
Val Asn Val Ser Ile Pro Val625 630
635 640Ser Asp Cys Leu Ile Ala Glu Gln Gln Glu Val Lys Ile Leu Leu
Glu645 650 655Thr Val Gln Glu Gln Ile Arg Ile Leu Thr Asp Ala Arg
Arg Ser Glu660 665 670Asp Tyr Glu Leu Ala Ser Val Glu Thr Glu Asp
Ser Ala Ser Glu Asn675 680 685Thr Ala Phe Leu Pro Leu Ser Pro Thr
Ala Lys Ser Glu Arg Glu Ala690 695 700Gln Phe Val Leu Arg Asn Glu
Ile Gln Arg Asp Ser Ala Leu Thr Lys705 710 715 720
* * * * *