U.S. patent application number 09/955363 was filed with the patent office on 2002-11-21 for method for producing dimerized polypeptide fusions.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Bell, Lillian Anne, Kindsvogel, Wayne R., Sledziewski, Andrzej Z..
Application Number | 20020173621 09/955363 |
Document ID | / |
Family ID | 27538286 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020173621 |
Kind Code |
A1 |
Sledziewski, Andrzej Z. ; et
al. |
November 21, 2002 |
Method for producing dimerized polypeptide fusions
Abstract
Methods for producing secreted receptor analogs and biologically
active peptide dimers are disclosed. The methods for producing
secreted receptor analogs and biologically active peptide dimers
utilize a DNA sequence encoding a receptor analog or a peptide
requiring dimerization for biological activity joined to a
dimerizing protein. The receptor analog includes a ligand-binding
domain. Polypeptides comprising essentially the extracellular
domain of a human PDGF receptor fused to dimerizing proteins, the
portion being capable of binding human PDGF or an isoform thereof,
are also disclosed. The polypeptides may be used within methods for
determining the presence of and for purifying human PDGF or
isoforms thereof.
Inventors: |
Sledziewski, Andrzej Z.;
(Seattle, WA) ; Bell, Lillian Anne; (Seattle,
WA) ; Kindsvogel, Wayne R.; (Seattle, WA) |
Correspondence
Address: |
Gary E. Parker
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
27538286 |
Appl. No.: |
09/955363 |
Filed: |
September 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09955363 |
Sep 18, 2001 |
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09435059 |
Oct 25, 1999 |
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09435059 |
Oct 25, 1999 |
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08980400 |
Nov 26, 1997 |
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08980400 |
Nov 26, 1997 |
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08477329 |
Jun 7, 1995 |
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08477329 |
Jun 7, 1995 |
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08180195 |
Jan 11, 1994 |
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08180195 |
Jan 11, 1994 |
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07634510 |
Dec 27, 1990 |
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07634510 |
Dec 27, 1990 |
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07347291 |
May 2, 1989 |
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07347291 |
May 2, 1989 |
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07146877 |
Jan 22, 1988 |
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Current U.S.
Class: |
530/350 ;
435/455; 435/69.7 |
Current CPC
Class: |
C07K 14/72 20130101;
C07K 14/71 20130101; C07K 2317/52 20130101; C07K 2319/30 20130101;
C07K 2317/56 20130101 |
Class at
Publication: |
530/350 ;
435/69.7; 435/455 |
International
Class: |
C07K 014/705; C12P
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 18, 1989 |
EP |
89100787.4 |
Claims
1. A method for producing a secreted, biologically active dimerized
polypeptide fusion, comprising: introducing into a eukaryotic host
cell a DNA construct comprising a transcriptional promoter
operatively linked to a secretory signal sequence followed
downstream by and in proper reading frame with a DNA sequence
encoding a non-immunoglobulin polypeptide requiring dimerization
for biological activity joined to a dimerizing protein; growing
said host cell in an appropriate growth medium under physiological
conditions to allow the secretion of a dimerized polypeptide fusion
encoded by said DNA sequence; and isolating said dimerized
polypeptide fusion from said host cell.
2. A method for producing a secreted, biologically active dimerized
polypeptide fusion, comprising: introducing into a eukaryotic host
cell a first DNA construct comprising a transcriptional promoter
operatively linked to a first secretory signal sequence followed
downstream by and in proper reading frame with a first DNA sequence
encoding a non-immunoglobulin polypeptide requiring dimerization
for biological activity joined to an immunoglobulin light chain
constant region; introducing into said host cell a second DNA
construct comprising a transcriptional promoter operatively linked
to a second secretory signal sequence followed downstream by and in
proper reading frame with a second DNA sequence encoding an
immunoglobulin heavy chain constant region domain selected from the
group consisting of C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4;
growing said host cell in an appropriate growth medium under
physiological conditions to allow the secretion of a biologically
active dimerized polypeptide fusion encoded by said first and
second DNA sequences; and isolating said biologically active
dimerized polypeptide fusion from said host cell.
3. The method of claim 2 wherein said second DNA sequence further
encodes an immunoglobulin hinge region and wherein said hinge
region is joined to said immunoglobulin heavy chain constant
region.
4. The method of claim 2 wherein said second DNA sequence further
encodes an immunoglobulin variable region and wherein said variable
region is joined upstream of and in proper reading frame with said
immunoglobulin heavy chain constant region domain.
5. The method of claim 2 wherein said host cell is a fungal cell or
a cultured mammalian cell.
6. The method of claim 2 wherein said host cell is a cultured
rodent myeloma cell line.
7. The method of claim 2 wherein said non-immunoglobulin
polypeptide requiring dimerization for biological activity is
selected from the group consisting of a polypeptide comprising the
amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine, number 29, to lysine, number 531, a polypeptide
comprising the amino acid sequence of FIG. 1 (sequence ID Numbers 1
and 2) from isoleucine, number 29, to methionine, number 441, and a
polypeptide comprising the amino acid sequence of FIG. 11 (Sequence
ID Numbers 35 and 36) from glutamine, number 24 to glutamic acid,
number 524.
8. A method for producing a secreted, biologically active dimerized
polypeptide fusion, comprising: introducing into a eukaryotic host
cell a first DNA construct comprising a transcriptional promoter
operatively linked to a first secretory signal sequence followed
downstream by and in proper reading frame with a first DNA sequence
encoding a non-immunoglobulin polypeptide requiring dimerization
for biological activity joined to an immunoglobulin heavy chain
constant region domain selected from the group consisting of
C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4; introducing into said
host cell a second DNA construct comprising a transcriptional
promoter operatively linked to a second secretory signal sequence
followed downstream by and in proper reading frame with a second
DNA sequence encoding an immunoglobulin light chain constant
region; growing said host cell in an appropriate growth medium
under physiological conditions to allow the secretion of a
biologically active dimerized polypeptide fusion encoded by said
first and second DNA sequences; and isolating said biologically
active dimerized polypeptide fusion from said host cell.
9. The method of claim 8 wherein said first DNA sequence further
encodes an immunoglobulin hinge region and wherein said hinge
region is joined to said immunoglobulin constant region.
10. The method of claim 8 wherein said second DNA sequence further
encodes an immunoglobulin variable region and wherein said variable
region is joined upstream of and in proper reading frame with said
immunoglobulin light chain constant region domain.
11. The method of claim 8 wherein said host cell is a fungal cell
or a cultured mammalian cell.
12. The method of claim 8 wherein said host cell is a cultured
rodent myeloma cell line.
13. The method of claim 8 wherein said non-immunoglobulin
polypeptide requiring dimerization for biological activity is
selected from the group consisting of a polypeptide comprising the
amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine, number 29, to lysine, number 531 a polypeptide
comprising the amino acid sequence of FIG. 1 (Sequence ID Numbers 1
and 2) from isoleucine, number 29, to methionine, number 441, and a
polypeptide comprising the amino acid sequence of FIG. 11 (Sequence
ID Numbers 35 and 36) from glutamine, number 24 to glutamic acid,
number 524.
14. A method for producing a secreted receptor analog, comprising:
introducing into a eukaryotic host cell a DNA construct comprising
a transcriptional promoter operatively linked to at least one
secretory signal sequence followed downstream by and in proper
reading frame with a DNA sequence encoding a ligand-binding domain
of a receptor requiring dimerization for biological activity joined
to a dimerizing protein; growing said host cell in an appropriate
growth medium under physiological conditions to allow the secretion
of a receptor analog encoded by said DNA sequence; and isolating
said receptor analog from said host cell.
15. A method for determining the presence of PDGF or isoforms
thereof in a biological sample, comprising: incubating a
polypeptide comprising a PDGF receptor analog fused to a dimerizing
protein with a biological sample suspected of comprising PDGF or an
isoform thereof under physiological conditions to allow the
formation of receptor/ligand complexes; and detecting the presence
of the receptor/ligand complexes as an indication of the presence
of human PDGF or an isoform thereof.
16. The method of claim 15 wherein the polypeptide is tagged with a
label selected from the group consisting of radionuclides,
fluorophores, enzymes, and luminescers.
17. The method of claim 15 wherein the biological sample is
selected from the group consisting of blood, urine, plasma, serum,
platelet and other cell lysates, platelet releasates, cell
suspensions, cell-conditioned culture media and chemically or
physically separated portions thereof.
18. The method of claim 15 wherein said human PDGF receptor analog
is selected from the group consisting of the amino acid sequence of
FIG. 1 (sequence ID Numbers 1 and 2) from isoleucine, number 29, to
methionine, number 441, joined to a dimerizing protein, the amino
acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine, number 29, to lysine, number 531, joined to a
dimerizing protein and the amino acid sequence of FIG. 11 (Sequence
ID Numbers 35 and 36) from glutamine, number 24 to glutamic acid,
number 524, joined to a dimerizing protein.
19. The method of claim 15 wherein said dimerizing protein
comprises at least a portion of a protein selected from the group
consisting of an immunoglobulin light chain, an immunoglobulin
heavy chain and yeast invertase, wherein said portion associates as
a dimer in a covalent or a noncovalent manner.
20. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a eukaryotic host cell a DNA construct
comprising a transcriptional promoter operatively linked to a
secretory signal sequence followed downstream in proper reading
frame by a DNA sequence encoding a ligand-binding domain of a PDGF
receptor; growing said host cell in an appropriate growth medium
under physiological conditions to allow the secretion of a PDGF
receptor analog encoded by said DNA sequence; and isolating said
PDGF receptor analog from said host cell.
21. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a cultured rodent myeloma cell a DNA
construct comprising a transcriptional promoter operatively linked
to a PDGF receptor signal sequence followed downstream by and in
proper reading frame with a DNA sequence encoding the amino acid
sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,
number 24, to glutamic acid, number 524, joined to a dimerizing
protein, wherein said dimerizing protein is an immunoglobulin
constant region selected from the group consisting of C.sub.H1,
C.sub.H2, C.sub.H3, C.sub.H4 and C.sub..kappa. joined to an
immunoglobulin hinge region; growing said cultured rodent myeloma
cell in an appropriate growth medium under physiological conditions
to allow the secretion of a PDGF receptor analog encoded by said
DNA sequence; and isolating the PDGF receptor analog from said
cultured rodent myeloma cell.
22. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a cultured rodent myeloma cell a first
DNA construct comprising a transcriptional promoter operatively
linked to a PDGF receptor signal sequence followed downstream by
and in proper reading frame with a first DNA sequence encoding the
amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine, number 29, to lysine, number 531 joined to an
immunoglobulin light chain constant region; introducing into said
cultured rodent myeloma cell a second DNA construct comprising a
transcriptional promoter operatively linked to a PDGF receptor
signal sequence followed downstream by and in proper reading frame
with a second DNA sequence encoding the amino acid sequence of FIG.
1 (sequence ID Numbers 1 and 2) from isoleucine number 29, to
lysine, number 531 joined to an immunoglobulin heavy chain constant
region domain selected from the group consisting of C.sub.H1,
C.sub.H2, C.sub.H3, and C.sub.H4 joined to an immunoglobulin hinge
region; growing said cultured rodent myeloma cell in an appropriate
growth medium under physiological conditions to allow the secretion
of a PDGF receptor analog encoded by said first and second DNA
sequences; and isolating said PDGF receptor analog from said
cultured rodent myeloma cell.
23. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a cultured rodent myeloma cell a first
DNA construct comprising a mouse V.sub.H promoter operatively
linked to a PDGF receptor signal sequence followed downstream of
and in proper reading frame with a DNA sequence encoding the amino
acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from
glutamine, number 24 to glutamic acid, number 524, joined to an
immunoglobulin heavy chain constant region domain selected from the
group consisting of C.sub.H1, CH.sup.2, C.sub.H3 and C.sub.H4
joined to an immunoglobulin hinge region; introducing into said
cultured rodent myeloma cell a second DNA construct comprising a
mouse V.sub..kappa. promoter operatively linked to a PDGF receptor
signal sequence followed downstream of and in proper reading frame
with a DNA sequence encoding the amino acid sequence of FIG. 11
(Sequence ID Numbers 35 and 36) from glutamine, number 24 to
glutamic acid, number 524, joined to an immunoglobulin light chain
constant region; growing said cultured rodent myeloma cell in an
appropriate growth medium under physiological conditions to allow
the secretion of a PDGF receptor analog encoded by said first and
second DNA sequences; and isolating said PDGF receptor analog from
said cultured myeloma cell.
24. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a cultured rodent myeloma cell a first
DNA construct comprising a mouse V.sub.H promoter operatively
linked to a PDGF receptor signal sequence followed downstream of
and in proper reading frame with a DNA sequence encoding the amino
acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine number 29, to lysine, number 531, joined to an
immunoglobulin heavy chain constant region domain selected from the
group consisting of C.sub.H1, C.sub.H2, C.sub.H3 and C.sub.H4
joined to an immunoglobulin hinge region; introducing into said
cultured rodent myeloma cell a second DNA construct comprising a
mouse V.sub..kappa. promoter operatively linked to a PDGF receptor
signal sequence followed downstream of and in proper reading frame
with a DNA sequence encoding the amino acid sequence of FIG. 11
(Sequence ID Numbers 35 and 36) from glutamine, number 24 to
glutamic acid, number 524, joined to an immunoglobulin light chain
constant region; growing said cultured rodent myeloma cell in an
appropriate growth medium under physiological conditions to allow
the secretion of a PDGF receptor analog encoded by said first and
second DNA sequences; and isolating said PDGF receptor analog from
said cultured myeloma cell.
25. A method for producing a secreted PDGF receptor analog,
comprising: introducing into a cultured rodent myeloma cell a first
DNA construct comprising a mouse V.sub.H promoter operatively
linked to a PDGF receptor signal sequence followed downstream of
and in proper reading frame with a DNA sequence encoding the amino
acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from
glutamine, number 24 to glutamic acid, number 524, joined to an
immunoglobulin heavy chain constant region domain selected from the
group consisting of C.sub.H1, C.sub.H2, C.sub.H3 and C.sub.H4
joined to an immunoglobulin hinge region; introducing into said
cultured rodent myeloma cell a second DNA construct comprising a
mouse V.sub..kappa. promoter operatively linked to a PDGF receptor
signal sequence followed downstream of and in proper reading frame
with a DNA sequence encoding the amino acid sequence of FIG. 1
(Sequence ID Numbers 1 and 2) from isoleucine number 29, to lysine,
number 531, joined to an immunoglobulin light chain constant
region; growing said cultured rodent myeloma cell in an appropriate
growth medium under physiological conditions to allow the secretion
of a PDGF receptor analog encoded by said first and second DNA
sequences; and isolating said PDGF receptor analog from said
cultured myeloma cell.
26. A method for determining the presence of PDGF or an isoform
thereof in a biological sample comprising the steps of: incubating
a polypeptide comprising a PDGF receptor analog joined to a
dimerizing protein with a biological sample suspected of containing
PDGF or an isoform thereof under conditions that allow the
formation of receptor/ligand complexes; and detecting the presence
of receptor/ligand complexes, and therefrom determining the
presence of human PDGF or an isoform thereof.
27. The method according to claim 26 wherein said biological sample
is selected from the group consisting of blood, urine, plasma,
serum, platelet and other cell lysates, platelet releasates, cell
suspensions, cell-conditioned culture media, and chemically or
physically separated portions thereof.
28. A method for purifying PDGF or an isoform thereof from a
sample, comprising: immobilizing a polypeptide comprising a PDGF
receptor analog fused to a dimerizing protein on a substrate;
contacting a sample comprising PDGF or an isoform thereof with the
immobilized polypeptide under conditions such that the PDGF or
isoform thereof binds to the polypeptide; and eluting the PDGF or
isoform thereof from the polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 07/347,291, filed May 2, 1989, which is a
continuation-in-part application of U.S. application Ser. No.
146,877, filed Jan. 22, 1988, now abandoned.
TECHNICAL FIELD
[0002] The present invention is generally directed toward the
expression of proteins, and more specifically, toward the
expression of growth factor receptor analogs and biologically
active dimerized polypeptide fusions.
BACKGROUND OF THE INVENTION
[0003] In higher eucaryotic cells, the interaction between
receptors and ligands (e.g., hormones) is of central importance in
the transmission of and response to a variety of extracellular
signals. It is generally accepted that hormones and growth factors
elicit their biological functions by binding to specific
recognition sites (receptors) in the plasma membranes of their
target cells. Upon ligand binding, a receptor undergoes a
conformational change, triggering secondary cellular responses that
result in the activation or inhibition of intracellular processes.
The stimulation or blockade of such an interaction by
pharmacological means has important therapeutic implications for a
wide variety of illnesses.
[0004] Ligands fall into two classes: those that have stimulatory
activity, termed agonists; and those that block the effects
elicited by the original ligands, termed antagonists. The discovery
of agonists that differ in structure and composition from the
original ligand may be medically useful. In particular, agonists
that are smaller than the original ligand may be especially useful.
The bioavailability of these smaller agonists may be greater than
that of the original ligand. This may be of particular importance
for topical applications and for instances when diffusion of the
agonist to its target sites is inhibited by poor circulation.
Agonists may also have slightly different spectra of biological
activity and/or different potencies, allowing them to be used in
very specific situations. Agonists that are smaller and chemically
simpler than the native ligand may be produced in greater quantity
and at lower cost. The identification of antagonists which
specifically block, for example, growth factor receptors has
important pharmaceutical applications. Antagonists that block
receptors against the action of endogenous, native ligand may be
used as therapeutic agents for conditions including
atherosclerosis, autocrine tumors, fibroplasia and keloid
formation.
[0005] The discovery of new ligands that may be used in
pharmaceutical applications has centered around designing compounds
by chemical modification, complete synthesis, and screening
potential ligands by complex and costly screening procedures. The
process of designing a new ligand usually begins with the
alteration of the structure of the original effector molecule. If
the original effector molecule is known to be chemically simple,
for example, a catecholamine or prostaglandin, the task is
relatively straightforward. However, if the ligand is structurally
complex, for example, a peptide hormone or a growth factor, finding
a molecule which is functionally equivalent to the original ligand
becomes extremely difficult.
[0006] Currently, potential ligands are screened using radioligand
binding methods (Lefkowitz et al., Biochem. Biophys. Res. Comm. 60:
703-709, 1974; Aurbach et al., Science 186: 1223-1225, 1974; Atlas
et al., Proc. Natl. Acad. Sci. USA 71: 4246-4248, 1974). Potential
ligands can be directly assayed by binding the radiolabeled
compounds to responsive cells, to the membrane fractions of
disrupted cells, or to solubilized receptors. Alternatively,
potential ligands may be screened by their ability to compete with
a known labeled ligand for cell surface receptors.
[0007] The success of these procedures depends on the availability
of reproducibly high quality preparations of membrane fractions or
receptor molecules, as well as the isolation of responsive cell
lines. The preparation of membrane fractions and soluble receptor
molecules involves extensive manipulations and complex purification
steps. The isolation of membrane fractions requires gentle
manipulation of the preparation, a procedure which does not lend
itself to commercial production. It is very difficult to maintain
high biological activity and biochemical purity of receptors when
they are purified by classical protein chemistry methods.
Receptors, being integral membrane proteins, require cumbersome
purification procedures, which include the use of detergents and
other solvents that interfere with their biological activity. The
use of these membrane preparations in ligand binding assays
typically results in low reproducibility due to the variability of
the membrane preparations.
[0008] As noted above, ligand binding assays require the isolation
of responsive cell lines. Often, only a limited subset of cells is
responsive to a particular agent, and such cells may be responsive
only under certain conditions. In addition, these cells may be
difficult to grow in culture or may possess a low number of
receptors. Currently available cell types responsive to
platelet-derived growth factor (PDGF), for example, contain only a
low number (up to 4.times.10.sup.5; see Bowen-Pope and Ross, J
Biol. Chem. 257: 5161-5171, 1982) of receptors per cell, thus
requiring large numbers of cells to assay PDGF analogs or
antagonists.
[0009] Presently, only a few naturally-occurring secreted
receptors, for example, the interleukin-2 receptor (IL-2-R) have
been identified. Rubin et al. (J. Immun. 135: 3172-3177, 1985) have
reported the release of large quantities of IL-2-R into the culture
medium of activated T-cell lines. Bailon et al. (Bio/Technology 5:
1195-1198, 1987) have reported the use of a matrix-bound
interleukin-2 receptor to purify recombinant interleukin-2.
[0010] Three other receptors have been secreted from mammalian
cells. The insulin receptor (Ellis et al., J Cell Biol. 150: 14a,
1987), the HIV-1 envelope glyco-protein cellular receptor CD4
(Smith et al., Science 238: 1704-1707, 1987), the murine IL-7
receptor (Cell 60: 941-951, 1990) and the epidermal growth factor
(EGF) receptor (Livneh et al., J. Biol. Chem. 261: 12490-12497,
1986) have been secreted from mammalian cells using truncated cDNAs
that encode portions of the extracellular domains.
[0011] Naturally-occurring, secreted receptors have not been widely
identified, and there have been only a limited number of reports of
secreted recombinant receptors. Secreted receptors may be used in a
variety of assays, which include assays to determine the presence
of ligand in biological fluids and assays to screen for potential
agonists and antagonists. Current methods for ligand screening and
ligand/receptor binding assays have been limited to those using
preparations of whole cells or cell membranes for as a source for
receptor molecules. The low reproducibility and high cost of
producing such preparations does not lend itself to commercial
production. There is therefore a need in the art for a method of
producing secreted receptors. There is a further need in the art
for an assay system that permits high volume screening of compounds
that may act on higher eucaryotic cells via specific surface
receptors. This assay system should be rapid, inexpensive and
adaptable to high volume screening. The present invention discloses
such a method and assay system, and further provides other related
advantages.
DISCLOSURE OF INVENTION
[0012] Briefly stated, the present invention discloses methods for
producing secreted receptor analogs, including receptor analogs and
secreted platelet-derived growth factor receptor (PDGF-R) analogs.
In addition, the present invention discloses methods for producing
secreted biologically active dimerized polypeptide fusions.
[0013] Within one aspect of the invention a method for producing a
secreted PDGF-R analog is disclosed, comprising (a) introducing
into a eukaryotic host cell a DNA construct comprising a
transcriptional promoter operatively linked to a secretory signal
sequence followed downstream of and in proper reading frame with a
DNA sequence encoding at least a portion of the ligand-binding
domain of a PDGF-R, the portion including a ligand-binding domain;
(b) growing the host cell in an appropriate growth medium under
physiological conditions to allow the secretion of a PDGF-R analog
encoded by said DNA sequence; and (c) isolating the PDGF-R analog
from the host cell.
[0014] Within one embodiment of the present invention, a PDGF-R
analog comprising the amino acid sequence of FIG. 1 (Sequence ID
Numbers 1 and 2) from isoleucine, number 29, to methionine, number
441, is secreted. Within another embodiment, a PDGF-R analog
comprising the amino acid sequence of FIG. 1 (Sequence ID Numbers 1
and 2) from isoleucine, number 29 to lysine, number 531 is
secreted. Within yet another embodiment of the invention, a PDGF-R
analog comprising the amino acid sequence of FIG. 11 (Sequence ID
Numbers 35 and 36) from glutamine, number 24 to glutamic acid,
number 524 is secreted.
[0015] Yet another aspect of the present invention discloses a
method for producing a secreted, biologically active dimerized
polypeptide fusion. The method generally comprises a) introducing
into a eukaryotic host cell a DNA construct comprising a
transcriptional promoter operatively linked to a secretory signal
sequence followed downstream by and in proper reading frame with a
DNA sequence encoding a non-immunoglobulin polypeptide requiring
dimerization for biological activity joined to a dimerizing
protein; (b) growing the host cell in an appropriate growth medium
under physiological conditions to allow the secretion of a
dimerized polypeptide fusion encodes by said DNA sequence; and (c)
isolating the biologically active dimerized polypeptide fusion from
the host cell.
[0016] Within one embodiment, the dimerizing protein is yeast
invertase. Within another embodiment, the dimerizing protein is at
least a portion of an immunoglobulin light chain. Within another
embodiment, the dimerizing protein is at least a portion of an
immunoglobulin heavy chain.
[0017] In another aspect of the invention, a method is disclosed
for producing a secreted, biologically active dimerized polypeptide
fusion, comprising (a) introducing into a eukaryotic host cell a
first DNA construct comprising a transcriptional promoter
operatively linked to a first secretory signal sequence followed
downstream by and in proper reading frame with a first DNA sequence
encoding a non-immunoglobulin polypeptide requiring dimerization
for biological activity joined to an immunoglobulin light chain
constant region; (b) introducing into the host cell a second DNA
construct comprising a transcriptional promoter operatively linked
to a second secretory signal sequence followed downstream by and in
proper reading frame with a second DNA sequence encoding an
immunoglobulin heavy chain constant region domain selected from the
group consisting of C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4; (c)
growing the host cell in an appropriate growth medium under
physiological conditions to allow the secretion of a dimerized
polypeptide fusion encoded by said first and second DNA sequences;
and (d) isolating the dimerized polypeptide fusion from the host
cell. In one embodiment, the second DNA sequence further encodes an
immunoglobulin heavy chain hinge region wherein the hinge region is
joined to the heavy chain constant region domain. In a preferred
embodiment, the second DNA sequence further encodes an
immunoglobulin variable region joined upstream of and in proper
reading frame with the immunoglobulin heavy chain constant
region.
[0018] In another aspect of the invention, a method is disclosed
for producing a secreted, biologically active dimerized polypeptide
fusion, comprising (a) introducing into a eukaryotic host cell a
first DNA construct comprising a transcriptional promoter
operatively linked to a first secretory signal sequence followed
downstream by and in proper reading frame with a first DNA sequence
encoding a non-immunoglobulin polypeptide requiring dimerization
for biological activity joined to an immunoglobulin heavy chain
constant region domain selected from the group consisting of
C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4; (b) introducing into
the host cell a second DNA construct comprising a transcriptional
promoter operatively linked to a second secretory signal sequence
followed downstream by and in proper reading frame with a second
DNA sequence encoding an immunoglobulin light chain constant
region; (c) growing the host cell in an appropriate growth medium
under physiological conditions to allow the secretion of a
dimerized polypeptide fusion encoded by said first and second DNA
sequences; and (d) isolating the dimerized polypeptide fusion from
the host cell. In one embodiment, the first DNA sequence further
encodes an immunoglobulin heavy chain hinge region wherein the
hinge region is joined to the heavy chain constant region domain.
In a preferred embodiment, the second DNA sequence further encodes
an immunoglobulin variable region joined upstream of and in proper
reading frame with the immunoglobulin light chain constant
region.
[0019] In another aspect of the invention, a method is disclosed
for producing a secreted, biologically active dimerized polypeptide
fusion, comprising (a) introducing into a eukaryotic host cell a
DNA construct comprising a transcriptional promoter operatively
linked to a secretory signal sequence followed downstream by and in
proper reading frame with a DNA sequence encoding a
nonimmunoglobulin polypeptide requiring dimerization for biological
activity joined to an immunoglobulin heavy chain constant region
domain selected from the group consisting of C.sub.H1, C.sub.H2,
C.sub.H3, and CH.sub.4; (b) growing the host cell in an appropriate
growth medium under physiological conditions to allow the secretion
of a dimerized polypeptide fusion encoded by said first and second
DNA sequences; and (c) isolating the biologically active dimerized
polypeptide fusion from the host cell. In one embodiment, the DNA
sequence further encodes an immunoglobulin heavy chain hinge region
wherein the hinge region is joined to the heavy chain constant
region domain.
[0020] In another aspect of the invention, a method is disclosed
for producing a secreted, biologically active dimerized polypeptide
fusion, comprising (a) introducing into a eukaryotic host cell a
first DNA construct comprising a transcriptional promoter
operatively linked to a first secretory signal sequence followed
downstream by and in proper reading frame with a first DNA sequence
encoding a first polypeptide chain of a non-immunoglobulin
polypeptide dimer requiring dimerization for biological activity
joined to an immunoglobulin heavy chain constant region domain,
selected from the group consisting of C.sub.H1, C.sub.H2, C.sub.H3,
and C.sub.H4; (b) introducing into the host cell a second DNA
construct comprising a transcriptional promoter operatively linked
to a second secretory signal sequence followed downstream by and in
proper reading frame with a second DNA sequence encoding a second
polypeptide chain of the non-immunoglobulin polypeptide dimer
joined to an immunoglobulin light chain constant region domain; (c)
growing the host cell in an appropriate growth medium under
physiological conditions to allow the secretion of a dimerized
polypeptide fusion encoded by said first and second DNA sequences
wherein said dimerized polypeptide fusion exhibits biological
activity characteristic of said non-immunoglobulin polypeptide
dimer; and (d) isolating the dimerized polypeptide fusion from the
host cell. In one embodiment the first DNA sequence further encodes
an immunoglobulin heavy chain hinge region domain wherein the hinge
region is joined to the immunoglobulin heavy chain constant region
domain.
[0021] Within one embodiment of the present invention, a
biologically active dimerized polypeptide fusion comprising the
amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from
isoleucine, number 29, to methionine, number 441, is secreted.
Within another embodiment, a biologically active dimerized
polypeptide fusion comprising the amino acid sequence of FIG. 1
(Sequence ID Numbers 1 and 2) from isoleucine, number 29 to lysine,
number 531 is secreted. Within another embodiment of the invention,
a biologically active dimerized polypeptide fusion comprising the
amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from
glutamine, number 24 to glutamic acid, number 524 is secreted.
Within yet another embodiment of the invention, a biologically
active dimerized polypeptide fusion comprising the amino acid
sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine,
number 29 to lysine, number 531 dimerized to the amino acid
sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,
number 24 to glutamic acid, number 524 is secreted.
[0022] In yet another aspect of the invention, a method is
disclosed for producing a secreted receptor analog, comprising (a)
introducing into a eukaryotic host cell a DNA construct comprising
a transcriptional promoter operatively linked to at least one
secretory signal sequence followed downstream by and in proper
reading frame with a DNA sequence encoding a ligand-binding domain
of a receptor requiring dimerization for biological activity joined
to a dimerizing protein; (b) growing the host cell in an
appropriate growth medium under physiological conditions to allow
the secretion of a receptor analog encoded by said DNA sequence;
and (c) isolating the receptor analog from the host cell.
[0023] In yet another aspect of the invention, a method is
disclosed for producing a secreted receptor analog, comprising (a)
introducing into a eukaryotic host cell a first DNA construct
comprising a transcriptional promoter operatively linked to a first
secretory signal sequence followed downstream by and in proper
reading frame with a first DNA sequence encoding a ligand-binding
domain of a receptor requiring dimerization for biological activity
joined to an immunoglobulin light chain constant region; (b)
introducing into the host cell a second DNA construct comprising a
transcriptional promoter operatively linked to a second secretory
signal sequence followed downstream by and in proper reading frame
with a second DNA sequence encoding an immunoglobulin heavy chain
constant region domain, selected from the group consisting of
C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4; (c) growing the host
cell in an appropriate growth medium under physiological conditions
to allow the secretion of a receptor analog encoded by said first
and second DNA sequences; and (d) isolating the receptor analog
from the host cell. In one embodiment, the second DNA sequence
further encodes an immunoglobulin heavy chain hinge region wherein
the hinge region is joined to the heavy chain constant region
domain. In a preferred embodiment, the second DNA sequence further
encodes an immunoglobulin variable region joined upstream of and in
proper reading frame with the immunoglobulin heavy chain constant
region.
[0024] In another aspect of the invention, a method is disclosed
for producing a secreted receptor analog, comprising (a)
introducing into a eukaryotic host cell a DNA construct comprising
a transcriptional promoter operatively linked to a secretory signal
sequence followed downstream by and in proper reading frame with a
DNA sequence encoding a ligand-binding domain of a receptor
requiring dimerization for biological activity joined to an
immunoglobulin heavy chain constant region domain, selected from
the group C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4; (b) growing
the host cell in an appropriate growth medium under physiological
conditions to allow the secretion of the receptor analog; and (c)
isolating the receptor analog from the host cell. In one
embodiment, the DNA sequence further encodes an immunoglobulin
heavy chain hinge region wherein the hinge region is joined to the
heavy chain constant region domain.
[0025] In another aspect of the invention, a method is disclosed
for producing a secreted receptor analog, comprising (a)
introducing into a eukaryotic host cell a first DNA construct
comprising a transcriptional promoter operatively linked to a first
secretory signal sequence followed downstream of and in proper
reading frame with a first DNA sequence encoding a ligand-binding
domain of a receptor requiring dimerization for biological activity
joined to an immunoglobulin heavy chain constant region domain,
selected from the group C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4;
(b) introducing into the host cell a second DNA construct
comprising a transcriptional promoter operatively linked to a
second secretory signal sequence followed downstream by and in
proper reading frame with a second DNA sequence encoding an
immunoglobulin light chain constant region; (c) growing the host
cell in an appropriate growth medium under physiological conditions
to allow the secretion of a receptor analog encoded by said first
and second DNA sequences; and (d) isolating the receptor analog
from the host cell. In one embodiment, the first DNA sequence
further encodes an immunoglobulin heavy chain hinge region wherein
the hinge region is joined to the heavy chain constant region
domain. In a preferred embodiment, the second DNA sequence further
encodes an immunoglobulin variable region joined upstream of and in
proper reading frame with the immunoglobulin light chain constant
region.
[0026] In another aspect of the invention, a method is disclosed
for producing a secreted receptor analog, comprising (a)
introducing into a eukaryotic host cell a first DNA construct
comprising a transcriptional promoter operatively linked to a first
secretory signal sequence followed downstream in proper reading
frame by a first DNA sequence encoding a first polypeptide chain of
a ligand-binding domain of a receptor requiring dimerization for
biological activity joined to an immunoglobulin heavy chain
constant region domain, selected from the group C.sub.H1, C.sub.H2,
C.sub.H3, and C.sub.H4; (b) introducing into the host cell a second
DNA construct comprising a transcriptional promoter operatively
linked to a second secretory signal sequence followed downstream by
and in proper reading frame with a second DNA sequence encoding a
second polypeptide chain of the ligand-binding domain of said
receptor joined to an immunoglobulin light chain constant region
domain; (c) growing the host cell in an appropriate growth medium
under physiological conditions to allow the secretion of a receptor
analog encoded by said first and second DNA sequences; and (d)
isolating the receptor analog from the host cell. In one embodiment
the first DNA sequence further encodes an immunoglobulin heavy
chain hinge region domain wherein the hinge region is joined to the
immunoglobulin heavy chain constant region domain.
[0027] Host cells for use in the present invention include cultured
mammalian cells and fungal cells. In a preferred embodiment strains
of the yeast Saccharomyces cerevisiae are used as host cells.
Within another preferred embodiment cultured rodent myeloma cells
are used as host cells.
[0028] Within one embodiment of the present invention, a receptor
analog is a PDGF-R analog comprising the amino acid sequence of
FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29, to
methionine, number 441. Within another embodiment a PDGF-R analog
comprises the amino acid sequence of FIG. 1 (Sequence ID Numbers 1
and 2) from isoleucine, number 29, to lysine, number 531. Within
another embodiment of the invention, a PDGF-R analog comprises the
amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from
glutamine, number 24 to glutamic acid, number 524. is secreted.
Within yet another embodiment of the invention, a PDGF-R analog
comprises the amino acid sequence of FIG. 1 (Sequence ID Numbers 1
and 2) from isoleucine, number 29 to lysine, number 531 and the
amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from
glutamine, number 24 to glutamic acid, number 524 is secreted.
[0029] PDGF-R analogs produced by the above-disclosed methods may
be used, for instance, within a method for determining the presence
of human PDGF or an isoform thereof in a biological sample.
[0030] A method for determining the presence of human PDGF or an
isoform thereof in a biological sample is disclosed and comprises
(a) incubating a polypeptide comprising a PDGF receptor analog
fused to a dimerizing protein with a biological sample suspected of
containing PDGF or an isoform thereof under conditions that allow
the formation of receptor/ligand complexes; and (b) detecting the
presence of receptor/ligand complexes, and therefrom determining
the presence of PDGF or an isoform thereof. Suitable biological
samples in this regard include blood, urine, plasma, serum,
platelet and other cell lysates, platelet releasates, cell
suspensions, cell-conditioned culture media, and chemically or
physically separated portions thereof.
[0031] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 (Sequence ID Numbers 1 and 2) illustrates the
nucleotide sequence of a representative PDGF .beta.-receptor cDNA
and the derived amino acid sequence of the primary translation
product and corresponds to Sequence ID Number 1). Numbers above the
lines refer to the nucleotide sequence; numbers below the lines
refer to the amino acid sequence.
[0033] FIG. 2 illustrates the construction of pBTL10, pBTL11 and
pBTL12.
[0034] FIG. 3 illustrates the construction of pCBS22.
[0035] FIG. 4 illustrates the construction of pBTL13 and
pBTL14.
[0036] FIG. 5 illustrates the construction of pBTL15.
[0037] FIG. 6 illustrates the construction of pBTL22 and
pBTL26.
[0038] FIG. 7 illustrates the construction of pSDL114. Symbols used
are S.S., signal sequence, C.sub.k, immunoglobulin light chain
constant region sequence; .mu. prom, .mu. promoter, .mu. enh; .mu.
enhancer.
[0039] FIG. 8 illustrates the construction of pSDLB113. Symbols
used are S.S., signal sequence; C.sub.H1, C.sub.H2, C.sub.H3,
immunoglobulin heavy chain constant region domain sequences; H,
immunoglobulin heavy chain hinge region sequence; M, immunoglobulin
membrane anchor sequences; C.sub..gamma.1M, immunoglobulin heavy
chain constant region and membrane anchor sequences.
[0040] FIG. 9 illustrates the constructions pBTL115, pBTL114,
p.phi.5V.sub.HHuC.sub..gamma.1M-neo,
plC.phi.5V.sub..kappa.HuC.sub..kappa- .-neo. Symbols used are set
forth in FIGS. 7 and 8, and also include L.sub.H, mouse
immunoglobulin heavy chain signal sequence; V.sub.H, mouse
immunoglobulin heavy chain variable region sequence; E, mouse
immunoglobulin heavy chain enhancer sequence; L.sub..kappa., mouse
immunoglobulin light chain signal sequence; .phi.5V.sub..kappa.,
mouse immunoglobulin light chain variable region sequence;
Neo.sup.R, E coli neomycin resistance gene.
[0041] FIG. 10 illustrates the constructions Zem229R,
p.phi.5V.sub.HFab-neo and pWKI. Symbols used are set forth in FIG.
9.
[0042] FIG. 11 illustrates the sequence of a representative PDGF
.alpha.-receptor cDNA and the deduced amino acid sequence (using
standard one-letter codes) encoded by the cDNA and corresponds to
Sequence ID Numbers 35 and 36. Numbers at the ends of the lines
refer to nucleotide positions. Numbers below the sequence refer to
amino acid positions.
[0043] FIG. 12 illustrates the assembly of a cDNA molecule encoding
a PDGF .alpha.-receptor. Complementary DNA sequences are shown as
lines. Only those portions of the vectors adjacent to the cDNA
inserts are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Prior to setting forth the invention, it may be helpful to
an understanding thereof to set forth definitions of certain terms
to be used hereinafter.
[0045] DNA Construct: A DNA molecule, or a clone of such a
molecule, either single- or double-stranded that has been modified
through human intervention to contain segments of DNA combined and
juxtaposed in a manner that as a whole would not otherwise exist in
nature.
[0046] DNA constructs contain the information necessary to direct
the expression and/or secretion of DNA sequences encoding
polypeptides of interest. DNA constructs will generally include
promoters, enhancers and transcription terminators. DNA constructs
containing the information necessary to direct the secretion of a
polypeptide will also contain at least one secretory signal
sequence.
[0047] Secretory Signal Sequence: A DNA sequence encoding a
secretory peptide. A secretory peptide is an amino acid sequence
that acts to direct the secretion of a mature polypeptide or
protein from a cell. Secretory peptides are characterized by a core
of hydrophobic amino acids and are typically (but not exclusively)
found at the amino termini of newly sythesized proteins. Very often
the secretory peptide is cleaved from the mature protein during
secretion. Such secretory peptides contain processing sites that
allow cleavage of the signal peptides from the mature proteins as
it passes through the secretory pathway. Processing sites may be
encoded within the signal peptide or may be added to the signal
peptide by, for example, in vitro mutagenesis. Certain secretory
peptides may be used in concert to direct the secretion of
polypeptides and proteins. One such secretory peptide that may be
used in combination with other secretory peptides is the third
domain of the yeast Barrier protein.
[0048] Receptor Analog: A non-immunoglobulin polypeptide comprising
a portion of a receptor which is capable of binding ligand and/or
are recognized by anti-receptor antibodies. The amino acid sequence
of the receptor analog may contain additions, substitutions or
deletions as compared to the native receptor sequence. A receptor
analog may be, for example, the ligand-binding domain of a receptor
joined to another protein. Platelet-derived growth factor receptor
(PDGF-R) analogs may, for example, comprise a portion of a PDGF
receptor capable of binding anti-PDGF receptor antibodies, PDGF,
PDGF isoforms, PDGF analogs, or PDGF antagonists.
[0049] Dimerizing Protein: A polypeptide chain having affinity for
a second polypeptide chain, such that the two chains associate
under physiological conditions to form a dimer. The second
polypeptide chain may be the same or a different chain.
[0050] Biological activity: A function or set of activities
performed by a molecule in a biological context (i.e., in an
organism or an in vitro facsimile thereof). Biological activities
may include the induction of extracellular matrix secretion from
responsive cell lines, the induction of hormone secretion, the
induction of chemotaxis, the induction of mitogenesis, the
induction of differentiation, or the inhibition of cell division of
responsive cells. A recombinant protein or peptide is considered to
be biologically active if it exhibits one or more biological
activities of its native counterpart.
[0051] Ligand: A molecule capable of being bound by the
ligand-binding domain of a receptor or by a receptor analog. The
molecule may be chemically synthesized or may occur in nature.
Ligands may be grouped into agonists and antagonists. Agonists are
those molecules whose binding to a receptor induces the response
pathway within a cell. Antagonists are those molecules whose
binding to a receptor blocks the response pathway within a
cell.
[0052] Joined: Two or more DNA coding sequences are said to be
joined when, as a result of in-frame fusions between the DNA coding
sequences or as a result of the removal of intervening sequences by
normal cellular processing, the DNA coding sequences are translated
into a polypeptide fusion.
[0053] As noted above, the present invention provides methods for
producing biologically active dimerized polypeptide fusions and
secreted receptor analogs, which include, for example, PDGF
receptor analogs. Secreted receptor analogs may be used to screen
for new compounds that act as agonists or antagonists when
interacting with cells containing membrane-bound receptors. In
addition, the methods of the present invention provide dimerized
non-immunoglobulin polypeptide fusions of therapeutic value that
are biologically active only as dimers. Moreover, the present
invention provides methods of producing polypeptide dimers that are
biologically active only as non-covalently associated dimers.
Secreted, biologically active dimers that may be produced using the
present invention include nerve growth factor, colony stimulating
factor-1, factor XIII, and transforming growth factor .beta..
[0054] As used herein, the ligand-binding domain of a receptor is
that portion of the receptor that is involved with binding the
natural ligand. While not wishing to be bound by theory, the
binding of a natural ligand to a receptor is believed to induce a
conformational change which elicits a response to the change within
the response pathway of the cell. For membrane-bound receptors, the
ligand-binding domain is generally believed to comprise the
extracellular domain for the receptor. The structure of receptors
may be predicted from the primary translation products using the
hydrophobicity plot function of, for example, P/C Gene or
Intelligenetics Suite (Intelligenetics, Mt. View, Calif.) or may be
predicted according to the methods described, for example, by Kyte
and Doolittle, J. Mol. Biol. 157:105-132, 1982). The ligand-binding
domain of the PDGF .beta.-receptor, for example, has been predicted
to include amino acids 29-531 of the published sequence (Gronwald
et al., ibid.). The ligand-binding domain of the PDGF
.alpha.-receptor has been predicted to include amino acids 25-500
of the published .alpha.-receptor sequence (Matsui et al., ibid.).
As used herein, the ligand-binding domain of the PDGF
.beta.-receptor includes amino acids 29-441 of the sequence of FIG.
1 (Sequence ID Number 1) and C-terminal extensions up to and
including amino acid 531. The ligand-binding domain of the PDGF
.alpha.-receptor is understood to include amino acids 24-524 of
FIG. 11 (Sequence ID Numbers 35 and 36).
[0055] Receptor analogs that may be used in the present invention
include the ligand-binding domains of the epidermal growth factor
receptor (EGF-R) and the insulin receptor. As used herein, a
ligand-binding domain is that portion of the receptor that is
involved in binding ligand and is generally a portion or
essentially all of the extracellular domain that extends from the
plasma membrane into the extracellular space. The ligand-binding
domain of the EGF-R, for example, resides in the extracellular
domain. EGF-R dimers have been found to exhibit higher
ligand-binding affinity than EGF-R monomers (Boni-Schnetzler and
Pilch, Proc. Natl. Acad. Sci. USA 84:7832-7836, 1987). The insulin
receptor (Ullrich et al., Nature 313:756-761, 1985) requires
dimerization for biological activity.
[0056] Another example of a receptor that may be secreted from a
host cell is a platelet-derived growth factor receptor (PDGF-R).
Two classes of PDGF-Rs, which recognized different isoforms of
PDGF, have been identified. (PDGF is a disulfide-bonded, two-chain
molecule, which is made up of an A chain and a B chain. These
chains may be combined as AB heterodimers, AA homodimers or BB
homodimers. These dimeric molecules are referred to herein as
"isoforms".) The .beta.-receptor (PDGF.beta.-R), which recognizes
only the BB isoform of PDGF (PDGF-BB), has been described
(Claesson-Welsh et al., Mol. Cell. Biol. 8:3476-3486, 1988;
Gronwald et al., Proc. Natl. Acad. Sci. USA 85:3435-3439, 1988).
The .alpha.-receptor (PDGF.alpha.-R), which recognizes all three
PDGF isoforms (PDGF-AA, PDGF-AB and PDGF-BB), has been described by
Matsui et al. (Science 243:800-804, 1989) and Kelly and Murray
(pending commonly assigned U.S. patent application Ser. No.
07/355,018, which is incorporated herein by reference). The primary
translation products of these receptors indicate that each includes
an extracellular domain implicated in the ligand-binding process, a
transmembrane domain, and a cytoplasmic domain containing a
tyrosine kinase activity.
[0057] The present invention provides a standardized assay system,
not previously available in the art, for determining the presence
of PDGF, PDGF isoforms, PDGF agonists or PDGF antagonists using a
secreted PDGF receptor analogs. Such an assay system will typically
involve combining the secreted PDGF receptor analog with a
biological sample under physiological conditions which permit the
formation of receptor-ligand complexes, followed by detecting the
presence of the receptor-ligand complexes. The term physiological
conditions is meant to include those conditions found within the
host organism and include, for example, the conditions of
osmolarity, salinity and pH. Detection may be achieved through the
use of a label attached to the PDGF receptor analog or through the
use of a labeled antibody which is reactive with the receptor
analog or the ligand. A wide variety of labels may be utilized,
such as radionuclides, fluorophores, enzymes and luminescers.
Receptor-ligand complexes may also be detected visually, i.e., in
immunoprecipitation assays which do not require the use of a label.
This assay system provides secreted PDGF receptor analogs that may
be utilized in a variety of screening assays for, for example,
screening for analogs of PDGF. The present invention also provides
a methods for measuring the level of PDGF and PDGF isoforms in
biological fluids.
[0058] As noted above, the present invention provides methods for
producing dimerized polypeptide fusions that require dimerization
for biological activity or enhancement of biological activity.
Polypeptides requiring dimerization for biological activity
include, in addition to certain receptors, nerve growth factor,
colony-stimulating factor-1 (CSF-1), transforming growth factor
.beta. (TGF-.beta.), PDGF, and factor XIII. Nerve growth factor is
a non-covalently linked dimer (Harper et al., J. Biol. Chem. 257:
8541-8548, 1982). CSF-1, which specifically stimulates the
proliferation and differentiation of cells of mononuclear
phagocytic lineage, is a disulfide-bonded homodimer (Retternmier et
al., Mol. Cell. Biol. 7: 2378-2387, 1987). TGF-.beta. is
biologically active as a disulfide-bonded dimer (Assoian et al., J.
Biol. Chem. 258: 7155-7160, 1983). Factor XIII is a plasma protein
that exists as a two chain homodimer in its activated form
(Ichinose et al., Biochem. 25: 6900-6906, 1986). PDGF, as noted
above, is a disulfide-bonded, two chain molecule (Murray et al.,
U.S. Pat. 4,766,073).
[0059] The present invention provides methods by which receptor
analogs, including receptor analogs and PDGF-R analogs, requiring
dimerization for activity may be secreted from host cells. The
methods described herein are particularly advantageous in that they
allow the production of large quantities of purified receptors. The
receptors may be used in assays for the screening of potential
ligands, in assays for binding studies, as imaging agents, and as
agonists and antagonists within therapeutic agents.
[0060] A DNA sequence encoding a human PDGF receptor may be
isolated as a cDNA using techniques known in the art (see, for
example, Okayama and Berg, Mol. Cell. Biol. 2: 161-170, 1982; Mol.
Cell. Biol. 3; 280-289, 1983) from a library of human genomic or
cDNA sequences. Such libraries may be prepared by standard
procedures, such as those disclosed by Gubler and Hoffman (Gene
263-269, 1983). It is preferred that the molecule is a cDNA
molecule because cDNA lack introns and are therefore more suited to
manipulation and expression in transfected or transformed cells.
Sources of mRNA for use in the preparation of a cDNA library
include the MG-63 human osteosarcoma cell line (available from ATCC
under accession number CRL 1427), diploid human dermal fibroblasts
and human embryo fibroblast and brain cells (Matsui et al., ibid.).
A cDNA encoding a PDGF.beta.-R has been cloned from a diploid human
dermal fibroblast cDNA library using oligonucleotide probes
complementary to sequences of the mouse PDGF receptor (Gronwald et
al., ibid.). A PDGF.alpha.-R cDNA has been isolated by Matsui et
al. (ibid.) from human embryo fibroblast and brain cells.
Alternatively, a cDNA encoding a PDGF.alpha.-R may be isolated from
a library prepared from MG-63 human osteosarcoma cells using a cDNA
probe containing sequences encoding the transmembrane and
cytoplasmic domains of the PDGF.beta.-R (described by Kelly and
Murray, ibid.). Partial cDNA clones (fragments) can be extended by
re-screening of the library with the cloned cDNA fragment until the
full sequence is obtained. In one embodiment, a ligand-binding
domain of a PDGF receptor is encoded by the sequence of FIG. 1
(Sequence ID Number 1) from amino acid 29 through amino acid 441.
In another embodiment, a ligand-binding domain of a PDGF receptor
is encoded by the sequence of FIG. 1 (Sequence ID Number 1) from
amino acid 29 through amino acid 531. In yet another embodiment, a
ligand-binding domain of a PDGF receptor is encoded by the sequence
of FIG. 11 (Sequence ID Numbers 35 and 36) from amino acid 24
through amino acid 524. One skilled in the art may envision the use
of smaller DNA sequence encoding the ligand-binding domain of a
PDGF receptor containing at least 400 amino acids of the
extracellular domain.
[0061] DNA sequences encoding EGF-R (Ullrich et al., Nature 304:
418-425, 1984), the insulin receptor (Ullrich et al., Nature 313:
756-761, 1985), nerve growth factor (Ullrich et al. Nature 303:
821-825, 1983), colony stimulating factor-1 (Rettenmier et al.,
ibid.), transforming growth factor .beta. (Derynck et al., Nature
316: 701-705, 1985), PDGF (Murray et al., ibid.), and factor XIII
(Ichinose et al., ibid.) may also be used within the present
invention.
[0062] To direct polypeptides requiring dimerization for biological
activity or receptor analogs into the secretory pathway of the host
cell, at least one secretory signal sequence is used in conjunction
with the DNA sequence of interest. Preferred secretory signals
include the alpha factor signal sequence (pre-pro sequence) (Kurjan
and Herkowitz, Cell 30: 933-943, 1982; Kurjan et al., U.S. Pat. No.
4,546,082; Brake, EP 116,201, 1983), the PHO5 signal sequence (Beck
et al., WO 86/00637), the BAR1 secretory signal sequence (MacKay et
al., U.S. Pat. No. 4,613,572; MacKay, WO 87/002670), immunoglobulin
V.sub.H signal sequences (Loh et al., Cell 33: 85-93, 1983; Watson
Nuc. Acids. Res. 12: 5145-5164, 1984) and immunoglobulin
V.sub..kappa. signal sequences (Watson, ibid.). Particularly
preferred signal sequences are the SUC2 signal sequence (Carlson et
al., Mol. Cell. Biol. 3: 439-447, 1983) and PDGF receptor signal
sequences. Alternatively, secretory signal sequences may be
synthesized according to the rules established, for example, by von
Heinje (Eur. J. Biochem. 133: 17-21, 1983; J. Mol. Biol. 184:
99-105, 1985; Nuc. Acids. Res. 14: 4683-3690, 1986).
[0063] Secretory signal sequences may be used singly or may be
combined. For example, a first secretory signal sequence may be
used singly or combined with a sequence encoding the third domain
of Barrier (described in copending commonly assigned U.S. patent
application Ser. No. 07/104,316, which is incorporated by reference
herein in its entirety). The third domain of Barrier may be
positioned in proper reading frame 3' of the DNA sequence of
interest or 5' to the DNA sequence and in proper reading frame with
both the secretory signal sequence and the DNA sequence of
interest.
[0064] In one embodiment of the present invention, a sequence
encoding a dimerizing protein is joined to a sequence encoding a
polypeptide chain of a polypeptide dimer or a receptor analog, and
this fused sequence is joined in proper reading frame to a
secretory signal sequence. As shown herein, the present invention
utilizes such an arrangement to drive the association of the
polypeptide or receptor analog to form a biologically active
molecule upon secretion. Suitable dimerizing proteins include the
S. cerevisiae repressible acid phosphatase (Mizunaga et al., J.
Biochem. (Tokyo) 103: 321-326, 1988), the S. cerevisiae type 1
killer preprotoxin (Sturley et al., EMBO J. 5: 3381-3390, 1986),
the S. calsberaensis alpha galactosidase melibiase (Sumner-Smith et
al., Gene 36: 333-340, 1985), the S. cerevisiae invertase (Carlson
et al., Mol. Cell. Biol. 3: 439-447, 1983), the Neurospora crassa
ornithine decarboxylase (Digangi et al., J. Biol. Chem. 262:
7889-7893, 1987), immunoglobulin heavy chain hinge regions
(Takahashi et al., Cell 29: 671-679, 1982), and other dimerizing
immunoglobulin sequences. In a preferred embodiment, S. cerevisiae
invertase is used to drive the association of polypeptides into
dimers. Portions of dimerizing proteins, such as those mentioned
above, may be used as dimerizing proteins where those portions are
capable of associating as a dimer in a covalent or noncovlent
manner. Such portions may be determined by, for example, altering a
sequence encoding a dimerizing protein through in vitro mutagenesis
to delete portions of the coding sequence. These deletion mutants
may be expressed in the appropriate host to determine which
portions retain the capablity of associating as dimers. Portions of
immunoglobulin gene sequences may be used to drive the association
of non-immunoglobulin polypeptides. These portions correspond to
discrete domains of immunoglobulins. Immunoglobulins comprise
variable and constant regions, which in turn comprise discrete
domains that show similarity in their three-dimensional
conformations. These discrete domains correspond to immunoglobulin
heavy chain constant region domain exons, immunoglobulin heavy
chain variable region domain exons, immunoglobulin light chain
variable region domain exons and immunoglobulin light chain
constant region domain exons in immunoglobulin genes (Hood et al.,
in Immunology, The Benjamin/Cummings Publishing Company, Inc.,
Menlo Park, Calif.; Honjo et al., Cell 18: 559-568, 1979; Takahashi
et al., Cell 29: 671-679, 1982; and Honjo, Ann. Rev. Immun.
1:499-528, 1983)). Particularly preferred portions of
immunoglobulin heavy chains include Fab and Fab' fragments. (An Fab
fragment is a portion of an immunoglobulin heavy chain that
includes a heavy chain variable region domain and a heavy chain
constant region domain. An Fab' fragment is a portion of an
immunoglobulin heavy chain that includes a heavy chain variable
region domain, a heavy chain constant region domain and a heavy
chain hinge region domain.)
[0065] It is preferred to use an immunoglobulin light chain
constant region in association with at least one immunoglobulin
heavy chain constant region domain. In a another embodiment, an
immunoglobulin light chain constant region is associated with at
least one immunoglobulin heavy chain constant region domain joined
to an immunoglobulin hinge region. In one set of embodiments, an
immunoglobulin light chain constant region joined in frame with a
polypeptide chain of a non-immunoglobulin polypeptide dimer or
receptor analog and is associated with at least one heavy chain
constant region. In a preferred set of embodiments a variable
region is joined upstream of and in proper reading frame with at
least one immunoglobulin heavy chain constant region. In another
set of embodiments, an immunoglobulin heavy chain is joined in
frame with a polypeptide chain of a non-immunoglobulin polypeptide
dimer or receptor analog and is associated with an immunoglobulin
light chain constant region. In yet another set of embodiments, a
polypeptide chain of a non-immunoglobulin polypeptide dimer or
receptor analog joined is to at least one immunoglobulin heavy
chain constant region which is joined to an immunoglobulin hinge
region and is associated with an immunoglobulin light chain
constant region. In a preferred set of embodiments an
immunoglobulin variable region is joined upstream of and in proper
reading frame with the immunoglobulin light chain constant
region.
[0066] Immunoglobulin heavy chain constant region domains include
C.sub.H1, C.sub.H2, C.sub.H3, and C.sub.H4 of any class of
immunoglobulin heavy chain including .gamma., .alpha., .epsilon.,
.mu., and .delta. classes (Honjo, ibid., 1983) A particularly
preferred immunoglobulin heavy chain constant region domain is
human C.sub.H1. Immunoglobulin variable regions include V.sub.H,
V.sub..kappa., or V.sub..lambda..
[0067] DNA sequences encoding immunoglobulins may be cloned from a
variety of genomic or cDNA libraries known in the art. The
techniques for isolating such DNA sequences using probe-based
methods are conventional techniques and are well known to those
skilled in the art. Probes for isolating such DNA sequences may be
based on published DNA sequences (see, for example, Hieter et al.,
Cell 22: 197-207, 1980). Alternatively, the polymerase chain
reaction (PCR) method disclosed by Mullis et al. (U.S. Pat. No.
4,683,195) and Mullis (U.S. Pat. No. 4,683,202), incorporated
herein by reference may be used. The choice of library and
selection of probes for the isolation of such DNA sequences is
within the level of ordinary skill in the art.
[0068] Host cells for use in practicing the present invention
include eukaryotic cells capable of being transformed or
transfected with exogenous DNA and grown in culture, such as
cultured mammalian and fungal cells. Fungal cells, including
species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces
spp.), or filamentous fungi (e.g., Aspergillus spp., Neurospora
spp.) may be used as host cells within the present invention.
Strains of the yeast Saccharomyces cerevisiae are particularly
preferred.
[0069] Expression units for use in the present invention will
generally comprise the following elements, operably linked in a 5'
to 3' orientation: a transcriptional promoter, a secretory signal
sequence a DNA sequence encoding nonimmunoglobulin polypeptide
requiring dimerization for biological activity joined to a
dimerizing protein and a transcriptional terminator. The selection
of suitable promoters, signal sequences and terminators will be
determined by the selected host cell and will be evident to one
skilled in the art and are discussed more specifically below.
[0070] Suitable yeast vectors for use in the present invention
include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:
1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979),
pJDB249 and pJDB219 (Beggs, Nature 275:104-108, 1978) and
derivatives thereof. Such vectors will generally include a
selectable marker, which may be one of any number of genes that
exhibit a dominant phenotype for which a phenotypic assay exists to
enable transformants to be selected. Preferred selectable markers
are those that complement host cell auxotrophy, provide antibiotic
resistance or enable a cell to utilize specific carbon sources, and
include LEU2 (Broach et al. ibid.), URA3 (Botstein et al., Gene 8:
17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasaki and Bell,
EP 171,142). Other suitable selectable markers include the CAT
gene, which confers chloramphenicol resistance on yeast cells.
[0071] Preferred promoters for use in yeast include promoters from
yeast glycolytic genes (Hitzeman et al., J Biol. Chem. 255:
12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:
419-434, 1982; Kawasaki, U.S. Pat. No. 4,599,311) or alcohol
dehydrogenase genes (Young et al., in Genetic Engineering of
Microorganisms for Chemicals, Hollaender et al., (eds.), p. 355,
Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101: 192-201,
1983). In this regard, particularly preferred promoters are the
TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311, 1986) and the
ADH2-4.sup.C promoter (Russell et al., Nature 304: 652-654, 1983
and Irani and Kilgore, described in pending, commonly assigned U.S.
patent application Ser. No. 07/183,130, which is incorporated
herein by reference). The expression units may also include a
transcriptional terminator. A preferred transcriptional terminator
is the TPI1 terminator (Alber and Kawasaki, ibid.).
[0072] In addition to yeast, proteins of the present invention can
be expressed in filamentous fungi, for example, strains of the
fungi Aspergillus (McKnight and Upshall, described in commonly
assigned U.S. Pat. No. 4,935,349, which is incorporated herein by
reference). Examples of useful promoters include those derived from
Aspergillus nidulans glycolytic genes, such as the ADH3 promoter
(McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA
promoter. An example of a suitable terminator is the ADH3
terminator (McKnight et al., ibid.). The expression units utilizing
such components are cloned into vectors that are capable of
insertion into the chromosomal DNA of Aspergillus.
[0073] Techniques for transforming fungi are well known in the
literature, and have been described, for instance, by Beggs
(ibid.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933,
1978), Yelton et al., (Proc. Natl. Acad. Sci. USA 81: 1740-1747,
1984), and Russell (Nature 301: 167-169, 1983). The genotype of the
host cell will generally contain a genetic defect that is
complemented by the selectable marker present on the expression
vector. Choice of a particular host and selectable marker is well
within the level of ordinary skill in the art.
[0074] In a preferred embodiment, a Saccharomyces cerevisiae host
cell that contains a genetic deficiency in a gene required for
asparagine-linked glycosylation of glycoproteins is used.
Preferably, the S. cerevisiae host cell contains a genetic
deficiency in the MNN9 gene (described in pending, commonly
assigned U.S. patent application Ser. Nos. 116,095 and 189,547
which are incorporated by reference herein in their entirety). Most
preferably, the S. cerevisiae host cell contains a disruption of
the MNN9 gene. S. cerevisiae host cells having such defects may be
prepared using standard techniques of mutation and selection.
Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) have described
the isolation of mannoprotein biosynthesis mutants that are
defective in genes which affect asparagine-linked glycosylation.
Briefly, mutagenized S. cerevisiae cells were screened using
fluoresceinated antibodies directed against the outer mannose
chains present on wild-type yeast. Mutant cells that did not bind
antibody were further characterized and were found to be defective
in the addition of asparagine-linked oligosaccharide moieties. To
optimize production of the heterologous proteins, it is preferred
that the host strain carries a mutation, such as the S. cerevisiae
pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in
reduced proteolytic activity.
[0075] In addition to fungal cells, cultured mammalian cells may be
used as host cells within the present invention. Preferred cell
lines are rodent myeloma cell lines, which include p3X63Ag8 (ATCC
TIB 9), FO (ATCC CRL 1646), NS-1 (ATCC TIB 18) and 210-RCY-Ag1
(Galfre et al., Nature 277: 131, 1979). A particularly preferred
rodent myeloma cell line is SP2/0-Ag14 (ATCC CRL 1581). In
addition, a number of other cell lines may be used within the
present invention, including COS-1 (ATCC CRL 1650), BHK,
p363.Ag.8.653 (ATCC CRL 1580) Rat Hep I (ATCC CRL 1600), Rat Hep II
(ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1),
Human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), Mouse liver
(ATCC CC 29.1), 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol.
36: 59-72, 1977) and DUKX cells (Urlaub and Chasin, Proc. Natl.
Acad. Sci USA 77: 4216-4220, 1980) A preferred BHX cell line is the
tk.sup.-ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad.
Sci USA 79: 1106-1110, 1982). A preferred BHK cell line is the
tk.sup.-ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad.
Sci. USA 79: 1106-1110, 1982). A tk.sup.- BHK cell line is
available from the American Type Culture Collection, Rockville, Md.
under accession number CRL 1632. A particularly preferred tk.sup.-
BHK cell line is BHK 570 which is available from the American Type
Culture Collection under accession number 10314.
[0076] Mammalian expression vectors for use in carrying out the
present invention will include a promoter capable of directing the
transcription of a cloned gene or cDNA. Preferred promoters include
viral promoters and cellular promoters. Preferred viral promoters
include the major late promoter from adenovirus 2 (Kaufman and
Sharp, Mol Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter
(Subramani et al., Mol. Cell. Biol. 1: 854-864, 1981). Preferred
cellular promoters include the mouse metallothionein 1 promoter
(Palmiter et al., Science 222: 809-814, 1983) and a mouse
V.sub..kappa. promoter (Grant et al., Nuc. Acids Res. 15: 5496,
1987). A particularly preferred promoter is a mouse V.sub.H
promoter (Loh et al., ibid.). Such expression vectors may also
contain a set of RNA splice sites located downstream from the
promoter and upstream from the DNA sequence encoding the peptide or
protein of interest. Preferred RNA splice sites may be obtained
from adenovirus and/or immunoglobulin genes. Also contained in the
expression vectors is a polyadenylation signal located downstream
of the coding sequence of interest. Polyadenylation signals include
the early or late polyadenylation signals from SV40 (Kaufman and
Sharp, ibid.), the polyadenylation signal from the adenovirus 5 E1B
region and the human growth hormone gene terminator (DeNoto et al.,
Nuc. Acids Res. 9: 3719-3730, 1981). A particularly preferred
polyadenylation signal is the V.sub.H gene terminator (Loh et al.,
ibid.). The expression vectors may include a noncoding viral leader
sequence, such as the adenovirus 2 tripartite leader, located
between the promoter and the RNA splice sites. Preferred vectors
may also include enhancer sequences, such as the SV40 enhancer and
the mouse .mu. enhancer (Gillies, Cell 33: 717-728, 1983).
Expression vectors may also include sequences encoding the
adenovirus VA RNAs.
[0077] Cloned DNA sequences may be introduced into cultured
mammalian cells by, for example, calcium phosphate-mediated
transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7: 603, 1981; Graham and Van der Eb,
Virology 52: 456, 1973.) Other techniques for introducing cloned
DNA sequences into mammalian cells, such as electroporation
(Neumann et al., EMBO J. 1: 841-845, 1982), may also be used. In
order to identify cells that have integrated the cloned DNA, a
selectable marker is generally introduced into the cells along with
the gene or cDNA of interest. Preferred selectable markers for use
in cultured mammalian cells include genes that confer resistance to
drugs, such as neomycin, hygromycin, and methotrexate. The
selectable marker may be an amplifiable selectable marker. A
preferred amplifiable selectable marker is the DHFR gene. A
particularly preferred amplifiable marker is the DHFR.sup.r cDNA
(Simonsen and Levinson, Proc. Natl. Adac. Sci. USA 80: 2495-2499,
1983). Selectable markers are reviewed by Thilly (Mammalian Cell
Technology, Butterworth Publishers, Stoneham, Mass.) and the choice
of selectable markers is well within the level of ordinary skill in
the art.
[0078] Selectable markers may be introduced into the cell on a
separate plasmid at the same time as the gene of interest, or they
may be introduced on the same plasmid. If on the same plasmid, the
selectable marker and the gene of interest may be under the control
of different promoters or the same promoter, the latter arrangement
producing a dicistronic message. Constructs of this type are known
in the art (for example, Levinson and Simonsen, U.S. Pat. No.
4,713,339). It may also be advantageous to add additional DNA,
known as "carrier DNA" to the mixture which is introduced into the
cells.
[0079] Transfected mammalian cells are allowed to grow for a period
of time, typically 1-2 days, to begin expressing the DNA
sequence(s) of interest. Drug selection is then applied to select
for growth of cells that are expressing the selectable marker in a
stable fashion. For cells that have been transfected with an
amplifiable selectable marker the drug concentration may be
increased in a stepwise manner to select for increased copy number
of the cloned sequences, thereby increasing expression levels.
[0080] Host cells containing DNA constructs of the present
invention are grown in an appropriate growth medium. As used
herein, the term "appropriate growth medium" means a medium
containing nutrients required for the growth of cells. Nutrients
required for cell growth may include a carbon source, a nitrogen
source, essential amino acids, vitamins, minerals and growth
factors. The growth medium will generally select for cells
containing the DNA construct by, for example, drug selection or
deficiency in an essential nutrient which are complemented by the
selectable marker on the DNA construct or co-transfected with the
DNA construct. Yeast cells, for example, are preferably grown in a
chemically defined medium, comprising a non-amino acid nitrogen
source, inorganic salts, vitamins and essential amino acid
supplements. The pH of the medium is preferably maintained at a pH
greater than 2 and less than 8, preferably at pH 6.5. Methods for
maintaining a stable pH include buffering and constant pH control,
preferably through the addition of sodium hydroxide. Preferred
buffering agents include succinic acid and Bis-Tris (Sigma Chemical
Co., St. Louis, Mo.). Yeast cells having a defect in a gene
required for asparagine-linked glycosylation are preferably grown
in a medium containing an osmotic stabilizer. A preferred osmotic
stabilizer is sorbitol supplemented into the medium at a
concentration between 0.1 M and 1.5 M., preferably at 0.5 M or 1.0
M. Cultured mammalian cells are generally grown in commercially
available serum-containing or serum-free media. Selection of a
medium appropriate for the particular cell line used is within the
level of ordinary skill in the art.
[0081] The culture medium from appropriately grown transformed or
transfected host cells is separated from the cell material, and the
presence of dimerized polypeptide fusions or secreted receptor
analogs is demonstrated. A preferred method of detecting receptor
analogs, for example, is by the binding of the receptors or
portions of receptors to a receptor-specific antibody. An
anti-receptor antibody may be a monoclonal or polyclonal antibody
raised against the receptor in question, for example, an anti-PDGF
receptor monoclonal antibody may be used to assay for the presence
of PDGF-receptor analogs. Another antibody, which may be used for
detecting substance P-tagged peptides and proteins, is a
commercially available rat anti-substance P monoclonal antibody
which may be utilized to visualize peptides or proteins that are
fused to the C-terminal amino acids of substance P. Ligand binding
assays may also be used to detect the presence of receptor analogs.
In the case of PDGF receptor analogs, it is preferable to use fetal
foreskin fibroblasts, which express PDGF receptors, to compete
against the PDGF receptor analogs of the present invention for
labeled PDGF and PDGF isoforms.
[0082] Assays for detection of secreted, biologically active
peptide dimers and receptor analogs may include Western transfer,
protein blot or colony filter. A Western transfer filter may be
prepared using the method described by Towbin et al. (Proc. Natl.
Acad. Sci. USA 76: 4350-4354, 1979). Briefly, samples are
electrophoresed in a sodium dodecylsulfate polyacrylamide gel. The
proteins in the gel are electrophoretically transferred to
nitrocellulose paper. Protein blot filters may be prepared by
filtering supernatant samples or concentrates through
nitrocellulose filters using, for example, a Minifold (Schleicher
& Schuell, Keene, N.H.). Colony filters may be prepared by
growing colonies on a nitrocellulose filter that has been laid
across an appropriate growth medium. In this method, a solid medium
is preferred. The cells are allowed to grow on the filters for at
least 12 hours. The cells are removed from the filters by washing
with an appropriate buffer that does not remove the proteins bound
to the filters. A preferred buffer comprises 25 mM Tris-base, 19 mM
glycine, pH 8.3, 20% methanol.
[0083] The dimerized polypeptide fusions and receptor analogs
present on the Western transfer, protein blot or colony filters may
be visualized by specific antibody binding using methods known in
the art. For example, Towbin et al. (ibid.) describe the
visualization of proteins immobilized on nitrocellulose filters
with a specific antibody followed by a labeled second antibody,
directed against the first antibody. Kits and reagents required for
visualization are commercially available, for example, from Vector
Laboratories, (Burlingame, Calif.), and Sigma Chemical Company (St.
Louis, Mo.)
[0084] Secreted, biologically active dimerized polypeptide fusions
and receptor analogs may be isolated from the medium of host cells
grown under conditions that allow the secretion of the biologically
active dimerized polypeptide fusions and receptor analogs. The cell
material is removed from the culture medium, and the biologically
active dimerized polypeptide fusions and receptor analogs are
isolated using isolation techniques known in the art. Suitable
isolation techniques include precipitation and fractionation by a
variety of chromatographic methods, including gel filtration, ion
exchange chromatography and immunoaffinity chromatography. A
particularly preferred purification method is immunoaffinity
chromatography using an antibody directed against the receptor
analog or dimerized polypeptide fusion. The antibody is preferably
immobilized or attached to a solid support or substrate. A
particularly preferred substrate is CNBr-activated Sepharose
(Pharmacia LKB Technologies, Inc., Piscataway, N.J.). By this
method, the medium is combined with the antibody/substrate under
conditions that will allow binding to occur. The complex may be
washed to remove unbound material, and the receptor analog or
peptide dimer is released or eluted through the use of conditions
unfavorable to complex formation. Particularly useful methods of
elution include changes in pH, wherein the immobilized antibody has
a high affinity for the ligand at a first pH and a reduced affinity
at a second (higher or lower) pH; changes in concentration of
certain chaotropic agents; or through the use of detergents.
[0085] The secreted PDGF receptor analogs of the present invention
can be used within a variety of assays for detecting the presence
of and/or screening for native PDGF, PDGF isoforms or PDGF-like
molecules. These assays will typically involve combining PDGF
receptor analogs, which may be bound to a solid substrate such as
polymeric microtiter plate wells, with a biological sample under
conditions that permit the formation of receptor/ligand complexes.
Screening assays for the detection of PDGF, PDGF isoforms or
PDGF-like molecules will typically involve combining soluble PDGF
receptor analogs with a biological sample and incubating the
mixture with a PDGF isoform or mixture of PDGF isoforms bound to a
solid substrate such as polymeric microtiter plates, under
conditions that permit the formation of receptor/ligand complexes.
Detection may be achieved through the use of a label attached to
the receptor or through the use of a labeled antibody which is
reactive with the receptor. Alternatively, the labeled antibody may
be reactive with the ligand. A wide variety of labels may be
utilized, such as radionuclides, fluorophores, enzymes and
luminescers. Complexes may also be detected visually, i.e., in
immunoprecipitation assays, which do not require the use of a
label.
[0086] Secreted PDGF receptor analogs of the present invention may
also be labeled with a radioisotope or other imaging agent and used
for in vivo diagnostic purposes. Preferred radioisotope imaging
agents include iodine-125 and technetium-99, with technetium-99
being particularly preferred. Methods for producing protein-isotope
conjugates are well known in the art, and are described by, for
example, Eckelman et al. (U.S. Pat. No. 4,652,440), Parker et al.
(WO 87/05030) and Wilber et al. (EP 203,764). Alternatively, the
secreted receptor analogs may be bound to spin label enhancers and
used for magnetic resonance (MR) imaging. Suitable spin label
enhancers include stable, sterically hindered, free radical
compounds such as nitroxides. Methods for labeling ligands for MR
imaging are disclosed by, for example, Coffman et al. (U.S. Pat.
No. 4,656,026). For administration, the labeled receptor analogs
are combined with a pharmaceutically acceptable carrier or diluent,
such as sterile saline or sterile water. Administration is
preferably by bolus injection, preferably intravenously. These
imaging agents are particularly useful in identifying the locations
of atherosclerotic plaques, PDGF-producing tumors, and
receptor-bound PDGF.
[0087] The secreted PDGF receptor analogs of the present invention
may also be utilized within diagnostic kits. Briefly, the subject
receptor analogs are preferably provided in a lyophilized form or
immobilized onto the walls of a suitable container, either alone or
in conjunction with antibodies capable of binding to native PDGF or
selected PDGF isoform(s) or specific ligands. The antibodies, which
may be conjugated to a label or unconjugated, are generally
included in the kits with suitable buffers, such as phosphate,
stabilizers, inert proteins or the like. Generally, these materials
are present in less than about 5% weight based upon the amount of
active receptor analog, and are usually present in an amount of at
least about 0.001% weight. It may also be desirable to include an
inert excipient to dilute the active ingredients. Such an excipient
may be present from about 1% to 99% weight of the total
composition. In addition, the kits will typically include other
standard reagents, instructions and, depending upon the nature of
the label involved, reactants that are required to produce a
detectable product. Where an antibody capable of binding to the
receptor or receptor/ligand complex is employed, this antibody will
usually be provided in a separate vial. The antibody is typically
conjugated to a label and formulated in an analogous manner with
the formulations briefly described above. The diagnostic kits,
including the containers, may be produced and packaged using
conventional kit manufacturing procedures.
[0088] As noted above, the secreted PDGF receptor analogs of the
present invention may be utilized within methods for purifying PDGF
from a variety of samples. Within a preferred method, the secreted
PDGF receptor analogs are immobilized or attached to a substrate or
support material, such as polymeric tubes, beads, polysaccharide
particulates, polysaccharide-containing materials, polyacrylamide
or other water insoluble polymeric materials. Methods for
immobilization are well known in the art (Mosbach et al., U.S. Pat.
No. 4,415,665; Clarke et al., Meth. Enzymology 68: 436-442, 1979).
A common method of immobilization is CNBr activation. Activated
substrates are commercially available from a number of suppliers,
including Pharmacia (Piscataway, N.J.), Pierce Chemical Co.
(Rockford, Ill.) and Bio-Rad Laboratories (Richmond, Calif.). A
preferred substrate is CNBr-activated Sepharose (Pharmacia,
Piscataway, N.J.). Generally, the substrate/receptor analog complex
will be in the form of a column. The sample is then combined with
the immobilized receptor analog under conditions that allow binding
to occur. The substrate with immobilized receptor analog is first
equilibrated with a buffer solution of a composition in which the
receptor analog has been previously found to bind its ligand. The
sample, in the solution used for equilibration, is then applied to
the substrate/receptor analog complex. Where the complex is in the
form of a column, it is preferred that the sample be passed over
the column two or more times to permit full binding of ligand to
receptor analog. The complex is then washed with the same solution
to elute unbound material. In addition, a second wash solution may
be used to minimize nonspecific binding. The bound material may
then be released or eluted through the use of conditions
unfavorable to complex formation. Particularly useful methods
include changes in pH, wherein the immobilized receptor has a high
affinity for PDGF at a first pH and reduced affinity at a second
(higher or lower) pH; changes in concentration of certain
chaotropic agents; or through the use of detergents.
[0089] The secreted PDGF receptor analogs fused to dimerizing
proteins of the present invention may be used in pharmaceutical
compositions for topical or intravenous application. The secreted
PDGF receptor analogs of the present invention may be useful in the
treatment of atherosclerosis by, for example, binding endogenous
PDGF to prevent smooth muscle cell proliferation. The PDGF receptor
analogs fused to dimerizing proteins are used in combination with a
physiologically acceptable carrier or diluent. Preferred carriers
and diluents include saline and sterile water. Pharmaceutical
compositions may also contain stabilizers and adjuvants. The
resulting aqueous solutions may be packaged for use or filtered
under aseptic conditions and lyophilized, the lyophilized
preparation being combined with a sterile aqueous solution prior to
administration.
[0090] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0091] Enzymes, including restriction enzymes, DNA polymerase I
(Klenow fragment), T4 DNA polymerase, T4 DNA ligase and T4
polynucleotide kinase, were obtained from New England Biolabs
(Beverly, Mass.), GIBCO-BRL (Gaithersburg, Md.) and
Boerhinger-Mannheim Biochemicals (Indianapolis, Ind.) and were used
as directed by the manufacturer or as described in Maniatis et al.
(Molecular Cloning: A Laboratorv Manual, Cold Spring Harbor
Laboratory, NY, 1982) and Sambrook et al. (Molecular Cloning: A
Laboratory Manual/Second Edition, Cold Spring Harbor Laboratory,
NY, 1989).
Example 1
Cloning PDGF Receptor cDNAs
[0092] A. Cloning the PDGF .beta.-Receptor
[0093] A cDNA encoding the PDGF .beta.-receptor was cloned as
follows. Complementary DNA (cDNA) libraries were prepared from
poly(A) RNA from diploid human dermal fibroblast cells, prepared by
explant from a normal adult, essentially as described by Hagen et
al. (Proc. Natl. Acad. Sci. USA 83: 2412-2416, 1986). Briefly, the
poly(A) RNA was primed with oligo d(T) and cloned into .lambda.gt11
using Eco RI linkers. The random primed library was screened for
the presence of human PDGF receptor cDNA's using three
oligonucleotide probes complementary to sequences of the mouse PDGF
receptor (Yarden et al., Nature 323: 226-232, 1986). Approximately
one million phage from the random primed human fibroblast cell
library were screened using oligonucleotides ZC904, ZC905 and ZC906
(Table 1; Sequence ID Numbers 5, 6 and 7, respectively). Eight
positive clones were identified and plaque purified. Two clones,
designated RP41 and RP51, were selected for further analysis by
restriction enzyme mapping and DNA sequence analysis. RP51 was
found to contain 356 bp of 5'-noncoding sequence, the ATG
translation initiation codon and 738 bp of the amino terminal
coding sequence. RP41 was found to overlap clone RP51 and contained
2649 bp encoding amino acids 43-925 of the .beta.-receptor
protein.
1TABLE 1 Oligonucleotide Sequences ZC871 (Sequence ID Number 3) 5'
CTC TCT TCC TCA GGT AAA TGA GTG CCA GGG CCG GCA AGC CCC CGC TCC 3'
ZC872 (Sequence ID Number 4) 5' CCG GGG AGC GGG GGC TTG CCG GCC CTG
GCA CTC ATT TAC CTG AGG AAG AGA GAG CT 3' ZC904 (Sequence ID Number
5) 5' CAT GGG CAC GTA ATC TAT AGA TTC ATC CTT GCT CAT ATC CAT GTA
3' ZC905 (Sequence ID Number 6) 5' TCT TGC CAG GGC ACC TGG GAC ATC
TGT TCC CAC ATC ACC GG 3' ZC906 (Sequence ID Number 7) 5' AAG CTG
TCC TCT GCT TCA GCC AGA GGT CCT GGG CAG CC 3' ZC1380 (Sequence ID
Number 8) 5' CAT GGT GGA ATT CCT GCT GAT 3' ZC1447 (Sequence ID
Number 9) 5' TG GTT GTG CAG AGC TGA GGA AGA GAT GGA 3' ZC1453
(Sequence ID Number 10) 5' AAT TCA TTA TGT TGT TGC AAG CCT TCT TGT
TCC TGC TAG CTG GTT TCG CTG TTA A 3' ZC1454 (Sequence ID Number 11)
5' GAT CTT AAC AGC GAA ACC AGC TAG CAG GAA CAA GAA GGC TTG CAA CAA
CAT AAT G 3' ZC1478 (Sequence ID Number 12) 5' ATC GCG AGC ATG CAG
ATC TGA 3' ZC1479 (Sequence ID Number 13) 5' AGC TTC AGA TCT GCA
TGC TGC CGA T 3' ZC1776 (Sequence ID Number 14) 5' AGC TGA GCG CAA
ATG TTG TGT CGA GTG CCC ACC GTG CCC AGC TTA GAA TTC T 3' ZC1777
(Sequence ID Number 15) 5' CTA GAG AAT TCT AAG CTG GGC ACG GTG GGC
ACT CGA CAC AAC ATT TGC GCT C 3' ZC1846 (Sequence ID Number 16) 5'
GAT CGG CCA CTG TCG GTG CGC TGC ACG CTG CGC AAC GCT GTG GGC CAG GAC
ACG CAG GAG GTC ATC GTG GTG CCA CAC TCC TTG CCC TTT AAG CA 3'
ZC1847 (Sequence ID Number 17) 5' AGC TTG CTT AAA GGG CAA GGA GTG
TGG CAC CAC GAT GAC CTC CTG CGT GTC CTG GCC CAC AGC GTT GCG CAG CGT
GCA GCG CAC CGA CAG TGG CC 3' ZC1886 (Sequence ID Number 18) 5' CCA
GTG CCA AGC TTG TCT AGA CTT ACC TTT AAA GGG CAA GGA G 3' ZC1892
(Sequence ID Number 19) 5' AGC TTG AGC GT 3' ZC1893 (Sequence ID
Number 20) 5' CTA GAC GCT CA 3' ZC1894 (Sequence ID Number 21) 5'
AGC TTC CAG TTC TTC GGC CTC ATG TCA GTT CTT CGG CCT CAT GTG AT 3'
ZC1895 (Sequence ID Number 22) 5' CTA CAT CAC ATG AGG CCG AAG AAC
TGA CAT GAG GCC GAA GAA CTG GA 3' ZC2181 (Sequence ID Number 23) 5'
AAT TCG GAT CCA CCA TGG GCA CCA GCC ACC CGG CGT TCC TGG TGT TAG GCT
GCC TGC TGA CCG GCC 3' ZC2182 (Sequence ID Number 24) 5' TGA GCC
TGA TCC TGT GCC AAC TGA GCC TGC CAT CGA TCC TGC CAA ACG AGA ACG AGA
AGG TTG TGC AGC TA 3' ZC2183 (Sequence ID Number 25) 5' AAT TTA GCT
GCA CAA CCT TCT CGT TCT CGT TTG GCA GGA TCG ATG GCA GGC TCA GTT GGC
ACA GGA TCA 3' ZC2184 (Sequence ID Number 26) 5' GGC TCA GGC CGG
TCA GCA GGC AGC CTA ACA CCA GGA ACG CCG GGT GGC TGG TGC CCA TGG TGG
ATC CG 3' ZC2311 (Sequence ID Number 27) 5' TGA TCA CCA TGG CTC AAC
TG 3' ZC2351 (Sequence ID Number 28) 5' CGA ATT CCA C 3' ZC2352
(Sequence ID Number 29) 5' CAT GGT GGA ATT CGA GCT 3' ZC2392
(Sequence ID Number 30) 5' ACG TAA GCT TGT CTA GAC TTA CCT TCA GAA
CGC AGG GTG GG 3'
[0094] The 3'-end of the cDNA was not isolated in the first cloning
and was subsequently isolated by screening 6.times.10.sup.5 phage
of the oligo d(T)-primed cDNA library with a 630 bp Sst I-Eco RI
fragment derived from the 3'-end of clone RP41. One isolate,
designated OT91, was further analyzed by restriction enzyme mapping
and DNA sequencing. This clone was found to comprise the 3'-end of
the receptor coding region and 1986 bp of 3' untranslated
sequence.
[0095] Clones RP51, RP41 and OT91 were ligated together to
construct a full-length cDNA encoding the entire PDGF
.beta.-receptor. RP41 was digested with Acc I and Bam HI to isolate
the 2.12 kb fragment. RP51 was digested with Eco RI and Acc I to
isolate the 982 bp fragment. The 2.12 kb RP41 fragment and the 982
bp RP51 fragment were joined in a three-part ligation, with pUC13,
which had been linearized by digestion with Eco RI and Bam HI. The
resultant plasmid was designated 51/41. Plasmid 51/41 was digested
with Eco RI and Bam HI to isolate the 3 kb fragment comprising the
partial PDGF receptor cDNA. OT91 was digested with Bam HI and Xba I
to isolate the 1.4 kb fragment containing the 3' portion of the
PDGF receptor cDNA. The Eco RI-Bam HI 51/41 fragment, the Bam
HI-Xba I OT91 fragment and the Eco RI-Xba I digested pUC13 were
joined in a three-part ligation. The resultant plasmid was
designated pR-RX1 (FIG. 2).
[0096] B. Cloning the PDGF-.alpha.Receptor
[0097] A cDNA encoding to PDGF .alpha.-receptor was cloned as
follows. RNA was prepared by the method of Chirgwin et al.
(Biochemistry 18: 5294, 1979) and twice purified on oligo d(T)
cellulose to yield poly(A)+ RNA. Complementary DNA was prepared in
.lambda.gt10 phage using a kit purchased from Invitrogen (San
Diego, Calif.) The resulting .lambda. phage DNA was packaged with a
coat particle mixture from Stratagene Cloning Systems (La Jolla,
Calif.) infected into E. coli strain C600 Hfl.sup.- and
titered.
[0098] Approximately 1.4.times.10.sup.6 phage recombinants were
plated to produce plaques for screening. Nitrocellulose filter
lifts were prepared according to standard methods and were
hybridized to a radiolabeled PDGF .beta.-receptor DNA fragment
(Gronwald et al., ibid.) comprising the 1.9 kb Fsp I-Hind III
fragment that encodes the transmembrane and cytoplasmic domains of
the PDGF .beta.-receptor cDNA. Hybridization was performed for 36
hours at 42.degree. C. in a mixture containing 40% formamide,
5.times.SSCP (SSC containing 25 mM phosphate buffer, pH 6.5), 200
.mu.g/ml denatured salmon sperm DNA, 3.times.Denhardt's, and 10%
dextran sulfate. Following hybridization, the filters were washed
extensively at room temperature in 2.times.SSC, then for 15 minutes
at 47-48.degree. C. Following an exposure to X-ray film, the
filters were treated to increasingly stringent wash conditions
followed by film recording until a final wash with 0.1.times.SSC at
65.degree. C. was reached. Film analysis showed that a "family" of
plaques that hybridized at lower wash stringency but not at the
highest stringency. This "family" was selected for further
analysis.
[0099] Two .lambda. phage clones from the "family" obtained from
the initial screening were subcloned into the Not I site of the
pUCtype plasmid vector pBluescript SK.sup.+ (obtained from
Stratagene Cloning Systems, La Jolla,. Calif.) and were analyzed by
restriction mapping and sequence analysis. Restriction enzyme
analysis of a phage clone, designated .alpha.1-1, revealed a
restriction fragment pattern dissimilar from that of the PDGF
.beta.-receptor cDNA with the exception of a common Bgl II-Bgl II
band of approximately 160 bp. The PDGF .beta.-receptor cDNA
contains two similarly spaced Bgl II sites within the region coding
for the second tyrosine kinase domain.
[0100] Restriction analysis of a second plasmid subclone
(designated .alpha.1-7) revealed an overlap of the 5' approximately
1.2 kb of clone .alpha.1-1, and an additional approximately 2.2 kb
of sequence extending in the 5' direction. Sequence analysis
revealed that the 3' end of this clone encodes the second tyrosine
kinase domain, which contains regions of near sequence identity to
the corresponding regions in the PDGF .beta.-receptor. The 5' end
of clone .alpha.1-7 contained non-receptor sequences. Two
additional .alpha.-receptor clones were obtained by probing with
.alpha.1-1 sequences. Clone .alpha.1-1 was digested with Not I and
Spe I, and a 230 bp fragment was recovered. Clone .alpha.1-1 was
also digested with Bam HI and Not I, and a 550 bp fragment was
recovered. A clone that hybridized to the 230 bp probe was
designated .alpha.5-1. This clone contained the 5'-most coding
sequence for the PDGF .alpha.-receptor. Another clone, designated
.alpha.6-3, hybridized to the 550 bp probe and was found to contain
3' coding and non-coding sequences, including the poly(A) tail.
[0101] Clone .alpha.1-1 was radiolabeled (.sup.32P) and used to
probe a northern blot (Thomas, Methods Enzymol. 100: 225-265, 1983)
of the MG-63 poly(A)+ RNA used to prepare the cDNA library. A
single band of approximately 6.6 kb was observed. RNA prepared from
receptor-positive cell lines including the human fibroblast SK4,
WI-38 and 7573 cell lines; the mouse fibroblast line DI 3T3; the
U2-OS human osteosarcoma cell line and baboon aortic smooth muscle
cells, and RNA prepared from receptor-negative lines including A431
(an epithelial cell line) and VA 13 (SV40-transformed WI-38 cells)
were probed by northern format with the .alpha.1-1 cDNA. In all
cases, the amount of the 6.6 kb band detected in these RNA
correlated well with the relative levels of .alpha.-receptor
detected on the respective cell surfaces. The 6.6 kb RNA was not
detected in RNA prepared from any tested cell line of hematopoietic
origin, in agreement with a lack of PDGF .alpha.-receptor protein
detected on these cell types.
[0102] Clones .alpha.1-1 and and .alpha.1-7 were joined at a unique
Pst I site in the region encoding the transmembrane portion of the
receptor. Clone .alpha.1-1 was digested with Xba I and Pst I and
the receptor sequence fragment was recovered. Clone .alpha.1-7 was
digested with Pst I and Bam HI and the receptor fragment was
recovered. The two fragments were ligated with Xba I+Bam
HI-digested pIC19R (Marsh et al. Gene 32: 481-486, 1984) to
construct plasmid p.alpha.17R (FIG. 12).
[0103] The remainder of the 5'-most .alpha.-receptor sequence was
obtained from clone .alpha.5-1 as an Sst I-Cla I fragment. This
fragment was joined to the Eco RI-Sst I receptor fragment of
p.alpha.17R and cloned into Eco RI+Cla I-digested pBluescript SK+
plasmid to construct plasmid p.alpha.17B (FIG. 12). FIG. 11
(Sequence ID Numbers 35 and 36) shows the nucleotide sequence and
deduced amino acid sequence of the cDNA present in p.alpha.17B.
Example 2
Construction of a SUC2 Signal Sequence-PDGF .beta.-Receptor
Fusion
[0104] To direct the PDGF .beta.-receptor into the yeast secretory
pathway, the PDGF .beta.-receptor cDNA was joined to a sequence
encoding the Saccharomyces cerevisiae SUC2 signal sequence.
Oligonucleotides ZC1453 and ZC1454 (Sequence ID Numbers 10 and 11;
Table 1) were designed to form an adapter encoding the SUC2
secretory signal flanked by a 5' Eco RI adhesive end and a 3' Bgl
II adhesive end. ZC1453 and ZC1454 were annealed under conditions
described by Maniatis et al. (ibid.). Plasmid pR-RX1 was digested
with Bgl II and Sst II to isolate the 1.7 kb fragment comprising
the PDGF .beta.-receptor coding sequence from amino acids 28 to
596. Plasmid pR-RX1 was also cut with Sst II and Hind III to
isolate the 1.7 kb fragment comprising the coding sequence from
amino acids 597 through the translation termination codon and 124
bp of 3' untranslated DNA. The two 1.7 kb pR-RX1 fragments and the
ZC1453/ZC1454 adapter were joined with pUC19, which had been
linearized by digestion with Eco RI and Hind III. The resultant
plasmid, comprising the SUC2 signal sequence fused in-frame with
the PDGF .beta.-receptor cDNA, was designated pBTL10 (FIG. 2).
Example 3
Construction of pCBS22
[0105] The BAR1 gene product, Barrier, is an exported protein that
has been shown to have three domains. When used in conjunction with
a first signal sequence, the third domain of Barrier protein has
been shown to aid in the secretion of proteins into the medium
(MacKay et al., U.S. patent application Ser. No. 104,316).
[0106] The portion-of the BAR1 gene encoding the third domain of
Barrier was joined to a sequence encoding the C-terminal portion of
substance P (subP; Munro and Pelham, EMBO J. 3: 3087-3093, 1984).
The presence of the substance P amino acids on the terminus of the
fusion protein allowed the protein to be detected using
commercially available anti-substance P antibodies. Plasmid pZV9
(deposited as a transformant in E. colistrain RR1, ATCC accession
no. 53283), comprising the entire BAR1 coding region and its
associated flanking regions, was cut with Sal I and Bam HI to
isolate the 1.3 kb BAR1 fragment. This fragment was subcloned into
pUC13, which had been cut with Sal I and Bam HI, to generate the
plasmid designated pZV17. Plasmid pZV17 was digested with Eco RI to
remove the 3'-most 0.5 kb of the BAR1 coding region. The
vector-BAR1 fragment was religated to create the plasmid designated
pJH66 (FIG. 3). Plasmid pJH66 was linearized with Eco RI and
blunt-ended with DNA polymerase I (Klenow fragment). Kinased Bam HI
linkers (5' CCG GAT CCG G 3') were added and excess linkers were
removed by digestion with Bam HI before religation. The resultant
plasmid was designated pSW8 (FIG. 3).
[0107] Plasmid pSW81, comprising the TPI1 promoter, the BAR1 coding
region fused to the coding region of the C-terminal portion of
substance P (Munro and Pelham, EMBO J. 3: 3087-3093, 1984) and the
TPI1 terminator, was derived from pSW8. Plasmid pSW8 was cut with
Sal I and Bam HI to isolate the 824 bp fragment encoding amino
acids 252 through 526 of BAR1. Plasmid pPM2, containing the
synthetic oligonucleotide sequence encoding the dimer form of the
C-terminal portion of substance P (subP) in M13mp8, was obtained
from Hugh Pelham (MRC Laboratory of Molecular Biology, Cambridge,
England). Plasmid pPM2 was linearized by digestion with Bam HI and
Sal I and ligated with the 824 bp BAR1 fragment from pSW8. The
resultant plasmid, pSW14, was digested with Sal I and Sma I to
isolate the 871 bp BAR1-substance P fragment. Plasmid pSW16,
comprising a fragment of BAR1 encoding amino acids 1 through 250,
was cut with Xba I and Sal I to isolate the 767 bp BAR1 fragment.
This fragment was ligated with the 871 bp BAR1-substance P fragment
in a three-part ligation with pUC18 cut with Xba I and Sma I. The
resultant plasmid, designated pSW15, was digested with Xba I and
Sma I to isolate the 1.64 kb BAR1-substance P fragment. The ADH1
promoter was obtained from pRL029. Plasmid pRL029, comprising the
ADH1 promoter and the BAR1 5' region encoding amino acids 1 to 33
in pUC18, was digested with Sph I and Xba I to isolate the 0.42 kb
ADH1 promoter fragment. The TPI1 terminator (Alber and Kawasaki,
ibid.) was provided as a linearized fragment containing the TPI1
terminator and pUC18 with a Klenow-filled Xba I end and an Sph I
end. This fragment was ligated with the 0.42 kb ADH1 promoter
fragment and the 1.64 kb BAR1-substance P fragment in a three-part
ligation to produce plasmid pSW22.
[0108] The ADH1 promoter and the coding region of BAR1, from the
translation initiation ATG through the Eco RV site present in
pSW22, were removed by digestion with Hind III and Eco RV. The 3.9
kb vector fragment, comprising the 401 bp between the Eco RV and
the Eco RI sites of the BAR1 gene fused to subP and the TPI1
terminator, was isolated by gel electrophoresis. Oligonucleotide
ZC1478 (Sequence ID Number 12; Table 1) was kinased and annealed
with oligonucleotide ZC1479 (Sequence ID Number 13; Table 1) using
conditions described by Maniatis et al. (ibid.). The annealed
oligonucleotides formed an adapter comprising a Hind III adhesive
end and a polylinker encoding Bgl II, Sph I, Nru I and Eco RV
restriction sites. The ZC1479/ZC1478 adapter was ligated with the
gel-purified pSW22 fragment. The resultant plasmid was designated
pCBS22 (FIG. 3).
Example 4
Construction of pBTL13
[0109] In order to enhance the secretion of the PDGF
.beta.-receptor and to facilitate the identification of the
secreted protein, a sequence encoding the third domain of BAR1
fused to the C-terminal amino acids of substance P was fused in
frame with the 5' 1240 bp of the PDGF .beta.-receptor. Plasmid
pBTL10 (Example 2) was digested with Sph I and Sst I to isolate the
4 kb fragment comprising the SUC2 signal sequence, a portion of the
PDGF .beta.-receptor cDNA and the pUC19 vector sequences. Plasmid
pCBS22 was digested with Sph I and Sst I to isolate the 1.2 kb
fragment comprising the BAR1-subP fusion and the TPI1 terminator.
These two fragments were ligated, and the resultant plasmid was
designated pBTL13 (FIG. 4).
Example 5
Construction of an Expression Vector Encoding the Entire PDGF
.beta.-Receptor
[0110] The entire PDGF .beta.-receptor was directed into the
secretory pathway by fusing a SUC2 signal sequence to the 5' end of
the PDGF .beta.-receptor coding sequence. This fusion was placed
behind the TPI1 promoter and inserted into the vector YEp13 for
expression in yeast.
[0111] The TPI1 promoter was obtained from plasmid pTPIC10 (Alber
and Kawasaki, J. Mol. Appl. Genet. 1: 410-434, 1982), and plasmid
pFATPOT (Kawasaki and Bell, EP 171,142; ATCC 20699). Plasmid
pTPIC10 was cut at the unique Kpn I site, the TPI1 coding region
was removed with Bal-31 exonuclease, and an Eco RI linker
(sequence: GGA ATT CC) was added to the 3' end of the promoter.
Digestion with Bgl II and Eco RI yielded a TPI1 promoter fragment
having Bgl II and Eco RI sticky ends. This fragment was then joined
to plasmid YRp7' (Stinchcomb et al., Nature 282 39-43, 1979) that
had been cut with Bgl II and Eco RI (partial). The resulting
plasmid, TE32, was cleaved with Eco RI (partial) and Bam HI to
remove a portion of the tetracycline resistance gene. The
linearized plasmid was then recircularized by the addition of an
Eco RI-Bam HI linker to produce plasmid TEA32. Plasmid TEA32 was
digested with Bgl II and Eco RI, and the 900 bp partial TPI1
promoter fragment was gel-purified. Plasmid pIC19H (Marsh et al.,
Gene 32:481-486, 1984) was cut with Bgl II and Eco RI and the
vector fragment was gel purified. The TPI1 promoter fragment was
then ligated to the linearized pIC19H and the mixture was used to
transform E. coli RR1. Plasmid DNA was prepared and screened for
the presence of a .about.900 bp Bgl II-Eco RI fragment. A correct
plasmid was selected and designated pICTPIP.
[0112] The TPI1 promoter was then subcloned to place convenient
restriction sites at its ends. Plasmid pIC7 (Marsh et al., ibid.)
was digested with Eco RI, the fragment ends were blunted with DNA
polymerase I (Klenow fragment), and the linear DNA was
recircularized using T4 DNA ligase. The resulting plasmid was used
to transform E. coli RR1. Plasmid DNA was prepared from the
transformants and was screened for the loss of the Eco RI site. A
plasmid having the correct restriction pattern was designated
pIC7RI*. Plasmid pIC7RI* was digested with Hind III and Nar I, and
the 2500 bp fragment was gel-purified. The partial TPI1 promoter
fragment (ca. 900 bp) was removed from pICTPIP using Nar I and Sph
I and was gel-purified. The remainder of the TPI1 promoter was
obtained from plasmid pFATPOT by digesting the plasmid with Sph I
and Hind III, and a 1750 bp fragment, which included a portion of
the TPI1 promoter fragment from pICTPIP, and the fragment from
pFATPOT were then combined in a triple ligation to produce pMVR1
(FIG. 2).
[0113] The TPI1 promoter was then joined to the SUC2-PDGF
.beta.-receptor fusion. Plasmid pBTL10 (Example 2) was digested
with Eco RI and Hind III to isolate the 3.4 kb fragment comprising
the SUC2 signal sequence and the entire PDGF .beta.-receptor coding
region. Plasmid pMVR1 was digested with Bgl II and Eco RI to
isolate the 0.9 kb TPI1 promoter fragment. The TPI1 promoter
fragment and the fragment derived from pBTL10 were joined with
YEp13, which had been linearized by digestion with Bam HI and Hind
III, in a three-part ligation. The resultant plasmid was designated
pBTL12 (FIG. 2).
Example 6
Construction of an Expression Vector Encoding the 5' Extracellular
Portion of the PDGF .beta.-Receptor
[0114] The extracellular portion of the PDGF .beta.-receptor was
directed into the secretory pathway by fusing the coding sequence
to the SUC2 signal sequence. This fusion was placed in an
expression vector behind the TPI1 promoter. Plasmid pBTL10 (Example
2) was digested with Eco RI and Sph I to isolate the approximately
1.3 kb fragment comprising the SUC2 signal sequence and the PDGF
.beta.-receptor extracellular domain coding sequence. Plasmid pMVR1
(Example 5) was digested with Bgl II and Eco RI to isolate the 0.9
kb TPI1 promoter fragment. The TPI1 promoter fragment was joined
with the fragment derived from pBTL10 and YEp13, which had been
linearized by digestion with Bam HI and Sph I, in a three-part
ligation. The resultant plasmid was designated pBTL11 (FIG. 2).
Example 7
Construction of Yeast Expression Vectors pBTL14 and pBTL15, and The
Expression of PDGF .beta.-Receptor-BAR1-subP Fusions
[0115] A. Construction of pBTL14
[0116] The SUC2-PDGF.beta.-R fusion was joined with the third
domain of BAR1 to enhance the secretion of the receptor, and the
expression unit was cloned into a derivative of YEp13 termed pJH50.
YEp13 was modified to destroy the Sal I site near the LEU2 gene.
This was achieved by partially digesting YEp13 with Sal I followed
by a complete digestion with Xho I. The 2.0 kb Xho I-Sal I fragment
comprising the LEU2 gene and the 8.0 kb linear YEp13 vector
fragment were isolated and ligated together. The ligation mixture
was transformed into E. coli strain RR1. DNA was prepared from the
transformants and was analyzed by digestion with Sal I and Xho I. A
clone was isolated which showed a single Sal I site and an inactive
Xho I site indicating that the LEU2 fragment had inserted in the
opposite orientation relative to the parent plasmid YEp13. The
plasmid was designated pJH50.
[0117] Referring to FIG. 4, plasmid pBTL12 (Example 5) was digested
with Sal I and Pst I to isolate the 2.15 kb fragment comprising 270
bp of YEp13 vector sequence, the TPI1 promoter, the SUC2 signal
sequence, and 927 bp of PDGF .beta.-receptor cDNA. Plasmid pBTL13
(Example 4) was digested with Pst I and Hind III to isolate the
1.48 kb fragment comprising 313 bp of PDGF .beta.-receptor cDNA,
the BAR1-subP fusion and the TPI1 terminator. The fragments derived
from pBTL12 and pBTL13 were joined with pJH50, which had been
linearized by digestion with Hind III and Sal I, in a three-part
ligation. The resultant plasmid was designated pBTL14.
[0118] B. Construction of pBTL15
[0119] Referring to FIG. 5, a yeast expression vector was
constructed comprising the TPI1 promoter, the SUC2 signal sequence,
1.45 kb of PDGF .beta.-receptor cDNA sequence fused to the
BAR1-subP fusion and the TPI1 terminator. Plasmid pBTL12 (Example
5) was digested with Sal I and Fsp I to isolate the 2.7 kb fragment
comprising the TPI1 promoter, the SUC2 signal sequence, the
PDGF.beta.-R coding sequence, and 270 bp of YEp13 vector sequence.
Plasmid pBTL13 (Example 4) was digested with Nru I and Hind III to
isolate the 1.4 kb fragment comprising the BAR1-subP fusion, the
TPI1 terminator and 209 bp of 3' PDGF .beta.-receptor cDNA
sequence. The fragments derived from pBTL12 and pBTL13 were joined
in a three-part ligation with pJH50, which had been linearized by
digestion with Hind III and Sal I. The resultant plasmid was
designated pBTL15.
[0120] C. Expression of PDGF.beta.-R-subP Fusions from pBTL14 and
pBTL15
[0121] Yeast expression vectors pBTL14 and pBTL15 were transformed
into Saccharomyces cerevisiae strains ZY100 (MATa leu2-3,112
ade2-101 suc2-.DELTA.9 gal2 pep4::TPI1prom-CAT) and ZY400 (MATa
leu2-3,112 ade2-101 suc2-.DELTA.9 gal2 pep4::TPI1prom-CAT
.DELTA.mnn9::URA3). Transformations were carried out using the
method essentially described by Beggs (ibid.). Transformants were
selected for their ability to grow on -LEUDS (Table 2).
2TABLE 2 Media Recipes -LeuThrTrp Amino Acid Mixture 4 g adenine 3
g L-arginine 5 g L-aspartic acid 2 g L-histidine free base 6 g
L-isoleucine 4 g L-lysine-mono hydrochloride 2 g L-methionine 6 g
L-phenylalanine 5 g L-serine 5 g L-tyrosine 4 g uracil 6 g
L-valine
[0122] Mix all the ingredients and grind with a mortar and pestle
until the mixture is finely ground.
3 -LEUDS 20 g glucose 6.7 g Yeast Nitrogen Base without amino acids
(DIFCO Laboratories Detroit, MI) 0.6 g -LeuThrTrp Amino Acid
Mixture 182.2 g sorbitol 18 g Agar
[0123] Mix all the ingredients in distilled water. Add distilled
water to a final volume of 1 liter. Autoclave 15 minutes. After
autoclaving add 150 mg L-threonine and 40 mg L-tryptophan. Pour
plates and allow to solidify.
4 -LEUDS + sodium succinate, pH 6.5 20 g Yeast Nitrogen Base
without amino acids 0.6 g -LeuTrpThr Amino Acid Mixture 182.2 g
sorbitol 11.8 g succinic acid
[0124] Mix all ingredients in distilled water to a final volume of
1 liter. Adjust the pH of the solution to pH 6.5. Autoclave 15
minutes. After autoclaving add 150 mg L-threonine and 40 mg
L-tryptophan.
5 Fermentation Medium 7 g/l yeast nitrogen base without amino acids
or ammonium sulfate (DIFCO Laboratories) 0.6 g/l ammonium sulfate
0.5 M sorbitol 0.39 g/l adenine sulfate 0.01% polypropylene
glycol
[0125] Mix all ingredients in distilled water. Autoclave 15
minutes. Add 80 ml 50% glucose for each liter of medium.
6 Super Synthetic -LEUD, pH 6.5 (liquid or solid medium) 6.7 g
Yeast Nitrogen Base without amino acids or ammonium sulfate (DIFCO)
6 g ammonium sulfate 160 g adenine 0.6 g -LeuThrTrp Amino Acid
Mixture 20 g glucose 11.8 g succinic acid
[0126] Mix all ingredients and add distilled water to a final
volume of 800 ml. Adjust the pH of the solution to pH 6.4.
Autoclave 15 minutes. For solid medium, add 18 g agar before
autoclaving, autoclave and pour plates.
[0127] Super Synthetic-LEUDS, pH 6.4 (Liquid or Solid Medium)
[0128] Use the same recipe as Super Synthetic -LEUD, pH 6.4, but
add 182.2 g sorbitol before autoclaving.
7 YEPD 20 g glucose 20 g Bacto Peptone (DIFCO Laboratories) 10 g
Bacto Yeast Extract (DIFCO Labloratories) 18 g agar 4 ml adenine 1%
8 ml 1% L-leucine
[0129] Mix all ingredients in distilled water, and bring to a final
volume of 1 liter. Autoclave 25 minutes and pour plates.
[0130] The transformants were assayed for binding to an anti-PDGF
.beta.-receptor monoclonal antibody (PR7212) or an anti-substance P
antibody by protein blot assay. ZY100[pBTL14] and ZY100[pBTL15]
transformants were grown overnight at 30.degree. C. in 5 ml Super
Synthetic -LEUD, pH 6.4 (Table 2). ZY400[pBTL14] and ZY400[pBTL15]
transformants. were grown overnight at 30.degree. C. in 5 ml Super
Synthetic-LEUDS, pH 6.4 (Table 2). The cultures were pelleted by
centrifugation and the supernatants were assayed for the presence
of secreted PDGF .beta.-receptor analogs by protein blot assay
using methods described in Example 18. Results of assays using
PR7212 are shown in Table 3.
8TABLE 3 Results of a protein blot probed with PR7212 Transformant:
ZY100[pBTL14] + ZY400[pBTL14] ++ ZY100[pBTL15] + ZY400[pBTL15]
+
Example 8
Construction of a SUC2-PDGF.beta.-R Fusion Comprising the Complete
PDGF.beta.-R Extracellular Domain
[0131] A. Construction of pBTL22
[0132] The PDGF.beta.-R coding sequence present in pBTL10was
modified to delete the coding region 3' to the extracellular
PDGF.beta.-R domain. As shown in FIG. 6, plasmid pBTL10 was
digested with Sph I and Bam HI and with Sph I and Sst II to isolate
the 4.77 kb fragment and the 466 bp fragment, respectively. The 466
bp fragment was then digested with Sau 3A to isolate the 0.17 kb
fragment. The 0.17 kb fragment and the 4.77 kb were joined by
ligation. The resultant plasmid was designated pBTL21.
[0133] Plasmid pBTL21, containing a Bam HI site that was
regenerated by the ligation of the Bam HI and Sau 3A sites, was
digested with Hind III and Bam HI to isolate the 4.2 kb fragment.
Synthetic oligonucleotides ZC1846 (Sequence ID Number 16; Table 1)
and ZC1847 (Sequence ID Number 17; Table 1) were designed to form
an adapter encoding the PDGF.beta.-R from the Sau 3A site after bp
1856 (FIG. 1; (Sequence ID Number 1)) to the end of the
extracellular domain at 1958 bp (FIG. 1; Sequence ID Number 1),
having a 5' Bam HI adhesive end that destroys the Bam HI site and a
3' Hind III adhesive end. oligonucleotides ZC1846 and ZC1847 were
annealed under conditions described by Maniatis et. al. (ibid.).
The 4.2 pBTL21 fragment and the ZC1846/ZC1847 adapter were joined
by ligation. The resultant plasmid, designated pBTL22, comprises
the SUC2 signal sequence fused in proper reading frame to the
extracellular domain of PDGF.beta.-R in the vector pUC19 (FIG.
6).
[0134] B. Construction of pBTL28
[0135] An in-frame translation stop codon was inserted immediately
after the coding region of the PDGF.beta.-R in pBTL22 using
oligonucleotides ZC1892 (Sequence ID Number 19; Table 1) and ZC1893
(Sequence ID Number 20; Table 1). These oligonucleotides were
designed to form an adapter encoding a stop codon in-frame with the
PDGF.beta.-R coding sequence from pBTL22 flanked by a 5' Hind III
adhesive end and a 3' Xba I adhesive end. Plasmid pBTL22 was
digested with Eco RI and Hind III to isolate the 1.6 kb
SUC2-PDGF.beta.-R fragment. Plasmid pMVR1 was digested with Eco RI
and Xba I to isolate the 3.68 kb fragment comprising the TPI1
promoter, pIC7RI* vector sequences and the TPI1 terminator.
Oligonucleotides ZC1892 and ZC1893 were annealed to form a Hind
III-Xba I adapter. The 1.6 kb SUC2-PDGF.beta.-R fragment, the 3.86
kb pMVR1 fragment and the ZC1892/ZC1893 adapter were joined in a
three-part ligation. The resultant plasmid was designated
pBTL27.
[0136] The expression unit present in pBTL27 was inserted into the
yeast expression vector pJH50 by first digesting pJH50 with Bam HI
and Sal I to isolate the 10.3 kb vector fragment. Plasmid pBTL27
was digested with Bgl II and Eco RI and with Xho I and Eco RI to
isolate the 0.9 kb TPI1 promoter fragment and the 1.65 kb fragment,
respectively. The 10.3 kb pJH50 vector fragment, the 0.9 kb TPI1
promoter fragment and 1.65 kb fragment were joined in a three-part
ligation. The resultant plasmid was designated pBTL28.
[0137] C. Construction of Plasmid pBTL30
[0138] The PDGF.beta.-R coding sequence present in plasmid pBTL22
was modified to encode the twelve C-terminal amino acids of
substance P and an in-frame stop codon. Plasmid pBTL22 was digested
with Eco RI and Hind III to isolate the 1.6 kb SUC2-PDGF.beta.-R
fragment. Plasmid pMVR1 was digested with Eco RI and Xba I to
isolate the 3.68 kb fragment comprising the TPI1 promoter, pIC7RI*
and the TPI1 terminator. Synthetic oligonucleotides ZC1894
(Sequence ID Number 21; Table 1) and ZC1895 (Sequence ID Number 22;
Table 1) were annealed to form an adapter containing the codons for
the twelve C-terminal amino acids of substance P followed by an
in-frame stop codon and flanked on the 5' end with a Hind III
adhesive end and on the 3' end with an Xba I adhesive end. The
ZC1894/ZC1895 adapter, the 1.6 kb SUC2-PDGF.beta.-R fragment and
the pMVR1 fragment were joined in a three-part ligation. The
resultant plasmid, designated pBTL29, was digested with Eco RI and
Xho I to isolate the 1.69 kb SUC2-PDGF.beta.-R-subP-TPI1 terminator
fragment. Plasmid pBTL27 was digested with Bgl II and Eco RI to
isolate the 0.9 kb TPI1 promoter fragment. Plasmid pJH50 was
digested with Bam HI and Sal I to isolate the 10.3 kb vector
fragment. The 1.69 kb pBTL29 fragment, the 0.9 kb TPI1 promoter
fragment and the 10.3 kb vector fragment were joined in a
three-part ligation. The resulting plasmid was designated
pBTL30.
Example 9
Construction and Expression of a SUC2-PDGF.beta.-R-IgG Hinge
Expression Vector
[0139] An expression unit comprising the TPI1 promoter, the SUC2
signal sequence, the PDGF.beta.-R extracellular domain, an
immunoglobulin hinge region and the TPI1 terminator was
constructed. Plasmid pBTL22 was digested with Eco RI and Hind III
to isolate the 1.56 kb fragments Plasmid pMVR1 was digested with
Eco RI and Xba I to isolate the 3.7 kb fragment, comprising the
TPI1 promoter, pIC7RI* vector sequences and the TPI1 terminator.
Oligonucleotides ZC1776 (Sequence ID Number 14; Table 1) and ZC1777
(Sequence ID Number 15; Table 1) were designed to form, when
annealed, an adapter encoding an immunoglobulin hinge region with a
5' Hind III adhesive end and a 3' Xba I adhesive end.
Oligonucleotides ZC1776 and ZC1777 were annealed under conditions
described by Maniatis et al. (ibid.). The 1.56 kb pBTL22 fragment,
the 3.7 kb fragment and the ZC1776/ZC1777 adapter were joined in a
three-part ligation, resulting in plasmid pBTL24.
[0140] The expression unit of pBTL24, comprising the TPI1 promoter,
SUC2 signal sequence, PDGF.beta.-R extracellular domain sequence,
hinge region sequence, and TPI1 terminator, was inserted into
pJH50. Plasmid pBTL24 was digested with Xho I and Hind III to
isolate the 2.4 kb expression unit. Plasmid pJH50 was digested with
Hind III and Sal I to isolate the 9.95 kb fragment. The 2.4 kb
pBTL24 fragment and 9.95 kb pJH50 vector fragment were joined by
ligation. The resultant plasmid was designated pBTL25.
[0141] Plasmid pBTL25 was transformed into Saccharomyces cerevisiae
strain ZY400 using the method essentially described by Beggs
(ibid.). Transformants were selected for their ability to grow on
-LEUDS (Table 2). The transformants were tested for their ability
to bind the anti-PDGF.beta.-R monoclonal antibody PR7212 using the
colony assay method described in Example 18. Plasmid pBTL25
transformants were patched onto nitrocellulose filters that had
been wetted and supported by YEPD solid medium. Antibody PR7212 was
found to bind to the PDGF.beta.-R-IgG hinge fusion secreted by
ZY400[pBTL25] transformants.
Example 10
Construction and Expression of a SUC2 Signal Sequence-PDGF.beta.-R
Extracellular Domain-SUC2 Fusion
[0142] As shown in FIG. 6, an expression unit comprising the TPI1
promoter, SUC2 signal sequence, PDGF.beta.-R extracellular domain
sequence, and SUC2 coding sequence was constructed as follows.
Plasmid pBTL22 was digested with Eco RI and Hind III to isolate the
1.6 kb SUC2-PDGF.beta.-R fragment. Plasmid pMVR1 was digested with
Bgl II and Eco RI to isolate the 0.9 kb TPI1 promoter fragment. The
SUC2 coding region was obtained from pJH40. Plasmid pJH40 was
constructed by inserting the 2.0 kb Hind III-Hind III SUC2 fragment
from pRB58 (Carlson et al., Cell 28: 145-154, 1982) into the Hind
III site of pUC19 followed by the destruction of the Hind III site
3' to the coding region. Plasmid pJH40 was digested with Hind III
and Sal I to isolate the 2.0 kb SUC2 coding sequence. Plasmid pJH50
was digested with Sal I and Bam HI to isolate the 10.3 kb vector
fragment. The 0.9 kb Bgl II-Eco RI TPI1 promoter fragment, the 1.6
kb Eco RI-Hind III SUC2-PDGF.beta.-R, the 2.0 kb Hind III-Sal I
SUC2 fragment and the 10.3 kb Bam HI-Sal I vector fragment were
joined in a four-part ligation. The resultant plasmid was
designated pBTL26 (FIG. 6).
[0143] Plasmid pBTL26 was transformed into Saccharomyces cerevisae
strain ZY400 using the method essentially described by Beggs
(ibid.). Transformants were selected for their ability to grow on
-LEUDS (Table 2). ZY400 transformants (ZY400[pBTL26]) were assayed
by protein blot (Example 18), colony blot (Example 18) and
competition assay.
[0144] Protein blot assays were carried out on ZY400[pBTL26] and
ZY400[pJH50]) (control) transformants that had been grown in
flasks. Two hundred-fifty microliters of a 5 ml overnight cultures
of ZY400[pBTL26] and ZY400 [pJH50] in -LEUDS+sodium succinate, pH
6.5 (Table 2) were inoculated into 50 ml of -LEUDS+sodium
succinate, pH 6.5. The cultures were incubated for 35 hours in an
airbath shaker at 30.degree. C. The culture supernatants were
harvested by centrifugation. The culture supernatants were assayed
as described in Example 18 and were found to bind PR7212
antibody.
[0145] Colony assays were carried out on ZY400[pBTL26]
transformants. ZY400[pBTL26] transformants were patched onto wetted
nitrocellulose filters that were supported on a YEPD plate. The
colony assay carried out as described in Example 8.A showed that
ZY400[pBTL26] antibodies bound PR7212 antibodies.
[0146] Competition binding assays were carried out on ZY400[pBTL26]
and ZY400[pJH50] transformants. The transformants were grown in two
liters of fermentation medium (Table 2) in a New Brunswick Bioflo2
fermentor (New Brunswick, Philadelphia, Pa.) with continuous pH
control at pH 6.4. The cultures were adjusted to pH 7.5 immediately
prior to harvesting. Culture supernatants were concentrated in an
Amicon concentrator (Amicon, San Francisco, Calif.) using an Amicon
10.sup.4 mw spiral filter cartridge. The concentrated supernatants
were further concentrated using Amicon Centriprep 10's. Fifteen
milliliters of the concentrated supernatant samples were added to
the Centripreps, and the. Centripreps were spun in a Beckman GRP
centrifuge (Beckman Instruments Inc., Carlsbad, Calif.) at setting
5 for a total of 60 minutes. The concentrates were removed from the
Centripreps and were assayed in the competition assay.
[0147] The competition binding assay measured the amount of
.sup.125I-PDGF left to bind to fetal foreskin fibroblast cells
after preincubation with the concentrate containing the
PDGF.beta.-R-SUC2 fusion protein. PDGF-AA and PDGF-AB were
iodinated using the Iodopead method (Pierce Chemical).
PDGF-BB.sub.Tyr was iodinated and purified as described in Example
18.F. The concentrate was serially diluted in binding medium (Table
4). The dilutions were mixed with 0.5 ng of iodinated PDGF-AA,
PDGF-BBTyr or PDGF-AB, and the mixtures were incubated for two
hours at room temperature. Three hundred micrograms of unlabeled
PDGF-BB was added to each sample mixture. The sample mixtures were
added to 24-well plates containing confluent fetal foreskin
fibroblast cells (AG1523, available from the Human Genetic Mutant
Cell Repository, Camden, N.J.). The cells were incubated with the
mixture for four hours at 4.degree. C. The supernatants were
aspirated from the wells, and the wells were rinsed three times
with phosphate buffered saline that was held at 4.degree. C. (PBS;
Sigma, St. Louis, Mo.). Five hundred microliters of PBS+1% NP-40
was added to each well, and the plates were shaken on a platform
shaker for five minutes. The cells were harvested and the amount of
iodinated PDGF was determined. The results of the competition
binding assay showed that the PDGF.beta.-R-SUC2 fusion protein was
able to competitively bind all three isoforms of PDGF.
[0148] The PDGF.beta.-R produced from ZY400 [pBTL26] transformants
was tested for cross reactivity to fibroblast growth factor (FGF)
and transforming growth factor-.beta. (TGF-.beta.) using the
competition assay essentially described above. Supernatant
concentrates from ZY400[pBTL26] and ZY400[JH50] (control)
transformants were serially diluted in binding medium (Table 4).
The dilutions were mixed with 7.9 ng of iodinated FGF or 14 ng of
iodinated TGF-.beta., and the mixtures were incubated for two hours
at room temperature. Fourteen micrograms of unlabeled FGF was added
to each mixture containing labeled FGF, and 7 .mu.g of unlabeled
TGF-.beta. was added to each mixture containing labeled TGF-.beta..
The sample mixtures were added to 24-well plates containing
confluent human dermal fibroblast cells. (Human dermal fibroblast
cells express both FGF receptors and TGF.beta. receptors.) The
cells were incubated with the mixtures for four hours at 4.degree.
C. Five hundred microliters of PBS+1% NP-40 was added to each well,
and the plates were shaken on a platform shaker for five minutes.
The cells were harvested and the amount of iodinated FGF or
TGF-.beta. bound to the cells was determined.
[0149] The results of these assays showed that the
PDGF.beta.-R-SUC2 fusion protein did not cross react with FGF or
TGF-.beta..
9TABLE 4 Reagent Recipes Binding Medium 500 ml Ham's F-12 medium 12
ml 1 M HEPES, pH 7.4 5 ml 100x PSN (Penicillin/Streptomycin/
Neomycin, Gibco) 1 g rabbit serum albumin Western Transfer Buffer
25 mM Tris, pH 8.3 19 mM glycine, pH 8.3 20% methanol Western
Buffer A 50 ml 1 M Tris, pH 7.4 20 ml 0.25 mM EDTA, pH 7.0 5 ml 10%
NP-40 37.5 ml 4 M NaCl 2.5 g gelatin
[0150] The Tris, EDTA, NP-40 and NaCl were diluted to a final
volume of one liter with distilled water. The gelatin was added to
300 ml of this solution and the solution was heated in a microwave
until the gelatin was in solution. The gelatin solution was added
back to the remainder of the first solution and stirred at 4
.degree. C. until cool. The buffer was stored at 4 .degree. C.
10 Western Buffer B 50 ml 1 M Tris, pH 7.4 20 ml 0.25 M EDTA, pH
7.0 5 ml 10% NP-40 58.4 g NaCl 2.5 g gelatin 4 g N-lauroyl
sarcosine
[0151] The Tris, EDTA, NP-40, and the NaCl were mixed and diluted
to a final volume of one liter. The gelatin was added to 300 ml of
this solution and heated in a microwave until the gelatin was in
solution. The gelatin solution was added back to the original
solution and the N-lauroyl sarcosine was added. The final mixture
was stirred at 4.degree. C. until the solids were completely
dissolved. This buffer was stored at 4 .degree. C.
11 2x Loading Buffer 36 ml 0.5 M Tris-HCl, pH 6.8 16 ml glycerol 16
ml 20% SDS 4 ml 0.5% Bromphenol Blue in 0.5 M Tris-HCl, pH 6.8
[0152] Mix all ingredients. Immediately before use, add 100 .mu.l
.beta.-mercaptoethanol to each 900 .mu.l dye mix
Example 11
Construction and Expression of PDGF Receptor Analogs From BHK
Cells
[0153] A. Construction of pBTL114 and pBTL115
[0154] The portions of the PDGF .beta.-receptor extracellular
domain present in pBTL14 and pBTL15 were placed in a mammalian
expression vector. Plasmids pBTL14 and pBTL15 were digested with
Eco RI to isolate the 1695 bp and 1905 bp SUC2
signal-PDGF.beta.-R-BAR1 fragments. The 1695 bp fragment and the
1905 bp fragment were each ligated to Zem229R that had been
linearized by digestion with Eco RI.
[0155] The vector Zem229R was constructed as shown in FIG. 10 from
Zem229. Plasmid Zem229 is a pUC18-based expression vector
containing a unique Bam HI site for insertion of cloned DNA between
the mouse metallothionein-1 promoter and SV40 transcription
terminator and an expression unit containing the SV40 early
promoter, mouse dihydrofolate reductase gene, and SV40
transcription terminator. Zem229 was modified to delete the Eco RI
sites flanking the Bam HI cloning site and to replace the Bam HI
site with a single Eco RI cloning site. The plasmid was partially
digested with Eco RI, treated with DNA polymerase I (Klenow
fragment) and dNTPs, and religated. Digestion of the plasmid with
Bam HI followed by ligaion of the linearized plasmid with a Bam
HI-Eco I adapter resulted in a unique Eco RI cloning site. The
resultant plasmid was designated Zem229R.
[0156] The ligation mixtures were transformed into E. coli strain
RR1. Plasmid DNA was prepared and the plasmids were subjected to
restriction enzyme analysis. A plasmid having the 1695 bp pBTL14
fragment inserted into Zem229R in the correct orientation was
designated pBTL114 (FIG. 9). A plasmid having the 1905 bp pBTL15
fragment inserted into Zem229R in the correct orientation was
designated pBTL115 (FIG. 9).
[0157] B. Expression of Secreted PDGF .beta.-receptor Analogs in
tk.sup.- ts13 BHK Cells
[0158] Plasmids pBTL114 and pBTL115 were each transfected into
tk.sup.-ts13 cells using calcium phosphate precipitation
(essentially as described by Graham and van der Eb, J. Gen. Virol.
36: 59-72, 1977). The transfected cells were grown in Dulbecco's
modified Eagle's medium (DMEM) containing 10% fetal calf serum,
1.times.PSN antibiotic mix (Gibco 600-5640), 2.0 mM L-glutamine.
The cells were selected in 250 nM methotrexate (MTX) for 14 days,
and the resulting colonies were screened by the immunofilter assay
(McCracken and Brown, Biotechniques, 82-87, March/April 1984).
Plates were rinsed with PBS or No Serum medium (DMEM plus
1.times.PSN antibiotic mix). Teflon.RTM. mesh (Spectrum Medical
Industries, Los Angeles, Calif. ) was then placed over the cells.
Nitrocellulose filters were wetted with PBS or No Serum medium, as
appropriate, and placed over the mesh. After six hours incubation
at 37.degree. C., filters were removed and placed in Wester buffer
A (Table 4) overnight at room temperature. The filters were
developed using the antibody PR7212 and the procedure described in
Example 8. The filters showed that conditioned media from
pBTL114-transfected and pBTL115-transfected BHK cells bound the
PR7212 antibody indicating the presence of biologically active
secreted PDGF.beta.-R.
Example 12
Expression of PDGF .beta.-Receptor Analogs in Cultured Mouse
Myeloma Cells
[0159] A. Construction of pIC.mu.PRE8
[0160] The immunoglobulin .mu. heavy chain promoter and enhancer
were sublconed into pIC19H to provide a unique Hind III site 3' to
the promoter. Plasmid p.mu. (Grosschedl and Baltimore, Cell 41:
885-897, 1985) was digested with Sal I and Eco RI to isolate the
3.1 kb fragment comprising the .mu. promoter. Plasmid pIC19H was
linearized by digestion with Eco RI and Xho I. The .mu. promoter
fragment and the linearized pIC19H vector fragment were joined by
ligation. The resultant plasmid, designated pIC.mu.3, was digested
with Ava II to isolate the 700 bp .mu. promoter fragment. The 700
bp fragment was blunt-ended by treatment with DNA polymerase I
(Klenow fragment) and deoxynucleotide triphosphates. Plasmid pIC19H
was linearized by digestion with Xho I, and the adhesive ends were
filled in by treatment with DNA polymerase I (Klenow fragement) and
deoxynucleotide triphosphates. The blunt-ended Ava II fragment was
ligated with the blunt-ended, linearized pIC19H, and the ligation
mixture was transformed into E coli JM83. Plasmid DNA was prepared
from the transformants and was analyzed by restriction digest. A
plasmid with a Bgl II site 5' to the promoter was designated
PIC.mu.PR1(-). Plasmid pIC.mu.PR1(-) was digested with Hind III and
Bgl II to isolate the 700 bp .mu. promoter fragment. Plasmid pIC19R
was linearized by digestion with Hind III and Bam HI. The 700 bp
promoter fragment was joined with the linearized pIC19R by
ligation. The resultant plasmid, designated pIC.mu.PR7, comprised
the .mu. promoter with an unique Sma I site 5' to the promoter and
a unique Hind III site 3' to the promoter.
[0161] The immunoglobulin heavy chain .mu. enhancer (Gillies et
al., Cell 33: 717-728, 1983) was inserted into the unique Sma I
site to generate plasmid pIC.mu.PRE8. Plasmid pJ4 (obtained from F.
Blattner, Univ. Wisconsin, Madison, Wis.), comprising the 1.5 kb
Hind III-Eco RI .mu. enhancer fragment in the vector pAT153
(Amersham, Arlington Heights, Ill.), was digested with Hind III and
Eco RI to isolate the 1.5 kb enhancer fragment. The adhesive ends
of the enhancer fragment were filled in by treatment with T4 DNA
polymerase and deoxynucleotide triphosphates. The blunt-ended
fragment and pIC.mu.PR7, which had been linearized by digestion
with Sma I, were joined by ligation. The ligation mixture was
transformed into E. coli RR1. Plasmid DNA was prepared from the
transformants, and the plasmids were analyzed by restriction
digests. A plasmid comprising the .mu. enhancer and the .mu.
promoter was designated pIC.mu.PRE8 (FIG. 7).
[0162] B. Construction of pSDL114
[0163] The DNA sequence encoding the extracellular domain of the
PDGF .beta.-receptor was joined with the DNA sequence encoding the
human immunoglobulin light chain constant region. The PDGF
.beta.-receptor extracellular domain was obtained from mpBTL22,
which comprised the Eco RI-Hind III fragment from pBTL22 (Example
8.A.) cloned into Eco RI-Hind III cut M13mp18. Single stranded DNA
was prepared from a mpBTL22 phage clone, and the DNA was subjected
to in vitro mutagenesis using the oligonucleotide ZC1886 (Table 1)
and the method described by Kunkel (Proc. Natl. Acad. Sci. USA 82:
488-492, 1985). A phage clone comprising the mutagenized
PDGF.beta.-R with a donor splice site (5' splice site) at the 3'
end of the PDGF.beta.-R extracellular domain was designated
pBTLR-HX (FIG. 7).
[0164] The native PDGF.beta.-R signal sequence was obtained from
pPR5. Plasmid pPR5, comprising 738 bp of 5' coding sequence with an
Eco RI site immediately 5' to the translation initiation codon, was
constructed by in vitro mutagenesis of the PDGF.beta.-R cDNA
fragment from RP51 (Example 1). Replicative form DNA of RP51 was
digested with Eco RI to isolate the 1.09 kb PDGF.beta.-R fragment.
The PDGF.beta.-R fragment was cloned into the Eco RI site of
M13mp18. Single stranded template DNA was prepared from a phage
clone containing the PDGF.beta.-R fragment in the proper
orientation. The template DNA was subjected to in vitro mutagenesis
using oligonucleotide ZC1380 (Sequence ID Number 8; Table 1) and
the method described by Zoller and Smith (Meth. Enzymol. 100:
468-500, 1983). The mutagenesis resulted in the placement of an Eco
RI site immediately 5' to the translation initiation codon.
Mutagenized phage clones were analyzed by dideoxy sequence
analysis. A phage clone containing the ZC1380 mutation was
selected, and replicative form (Rf) DNA was prepared from the phage
clone. The Rf DNA was digested with Eco RI and Acc I to isolate the
0.63 kb fragment. Plasmid pR-RXI (Example 1) was digested with Acc
I and Eco RI to isolate the 3.7 kb fragment. The 0.63 kb fragment
and the 3.7 kb fragment were joined by ligation resulting in
plasmid pPR5 (FIG. 7).
[0165] As shown in FIG. 7, the PDGF.beta.-R signal peptide and part
of the extracellular domain were obtained from plasmid pPR5 as a
1.4 kb Eco RI-Sph I fragment. Replicative form DNA from phage clone
pBTLR-HX was digested with Sph I and Hind III to isolate the
approximately 0.25 kb PDGF.beta.-R fragment. Plasmid pUC19 was
linearized by digestion with Eco RI and Hind III. The 1.4 kb Eco
RI-Sph I PDGF.beta.-R fragment, the 0.25 kb Sph I-Hind III fragment
from pBTLR-HX and the Eco RI-Hind III cut pUC19 were joined in a
three-part ligation. The resultant plasmid, pSDL110, was digested
with Eco RI and Hind III to isolate the 1.65 kb PDGF.beta.-R
fragment.
[0166] Plasmid pICHuC.kappa.3.9.11 was used as the source of the
human immunoglobulin light chain gene (FIG. 7). The human
immunoglobulin light chain gene was isolated from a human genomic
library using an oligonucleotide probe (5' TGT GAC ACT CTC CTG GGA
GTT A 3'; Sequence ID Number 32), which was based on a published
human kappa C gene sequence (Hieter et al., Cell 22: 197-207,
1980). The human light chain (kappa) constant region was subcloned
as a 1.1 kb Sph I-Hinf I genomic fragment of the human kappa gene,
which has been treated with DNA polymerase DNA I (Klenow Fragment)
to fill in the Hinf I adhesive end, into Sph I-Hinc II cut pUC19.
The 1.1 kb human kappa constant region was susbsequently isolated
as a 1.1 kb Sph I-Bam HI fragment that was subcloned into Sph I-Bgl
II cut pIC19R (Marsh et al., ibid.). The resultant plasmid was
designated pICHuC.lambda.3.9.11. Plasmid pICHuC.sub..kappa.3.9.11
was digested with Hind III and Eco RI to isolate the 1.1 kb kappa
constant region gene. Plasmid pIC19H was linearized by digestion
with Eco RI. The 1.65 kb PDGF.beta.-R fragment, the 1.1 kb human
kappa constant region fragment and the linearized pIC19H were
joined in a three part ligation. The resultant plasmid, pSDL112,
was digested with Bam HI and Cla I to isolate the 2.75 kb fragment.
Plasmid p.mu.PRE8 was linearized with Bgl II and Cla I. The 2.75 kb
fragment and the linearized p.mu.PRE8 were joined by ligation. The
resultant plasmid was designated pSDL114 (FIG. 7).
[0167] Plasmid pSDL114 was linearized by digestion with Cla I and
was cotransfected with Pvu I-digested p416 into SP2/0-Ag14 (ATCC
CRL 1581) by electroporation using the method essentially described
by Neumann et al. (EMBO J. 1: 841-845, 1982). (Plasmid p416
comprises the Adenovirus 5 ori, SV40 enhancer, Adenovirus 2 major
late promoter, Adenovirus 2 tripartite leader, 5' and 3' splice
sites, the DHFR.sup.r cDNA, the SV40 polyadenylation signal and
pML-1 (Lusky and Botchan, Nature 293:79-81, 1981) vector
sequences.) Transfectants were selected in growth medium containing
methotrexate.
[0168] Media from drug resistant clones were tested for the
presence of secreted PDGF .beta.-receptor analogs by enzyme-linked
immunosorbant assay (ELISA). Ninety-six well assay plates were
prepared by incubating 100 .mu.l of 1 .mu.g/ml polyclonal goat
anti-human kappa chain (Cappel Laboratories, Melvern, Pa.) diluted
in phosphate buffered saline (PBS; Sigma) overnight at 40.degree.
C. Excess antibody was removed by three washes with 0.5% Tween 20
in PBS. One hundred microliters of spent media was added to each
well, and the well were incubated for one hour at 4.degree. C.
Unbound proteins were removed by eight washes with 0.5% Tween 20 in
PBS. One hundred microliters of peroxidase-conjugated goat
anti-human kappa antibody (diluted 1:1000 in a solution containing
5% chicken serum (GIBCO-BRL)+0.5% Tween 20 in PBS) was added to
each well and the wells were incubated for one hour at 4.degree. C.
One hundred microliters of chromophore (100 .mu.l ABTS
(2,2'-Azinobis(3-ethylbenz-thi- azoline sulfonic acid) diammonium
salt; Sigma)+1 .mu.l 30% H.sub.2O.sub.2+12.5 ml citrate/phosphate
buffer (9.04 g/l citric acid, 10.16 g/l Na.sub.2HPO.sub.4)) was
added to each well, and the wells were incubated to thirty minutes
at room temperature. The samples were measured at 405 nm. The
results of the assay showed that the PDGF.beta.-R analog secreted
by the transfectants contained an immunoglobulin light chain.
[0169] Spent media from drug resistant clones was also tested for
the presence of secreted PDGF .beta.-receptor analogs by
immunoprecipitation. Approximately one million drug resistant
transfectants were metabolically labeled by growth in DMEM medium
lacking cysteine+2% calf serum for 18 hours at 37.degree. C. in the
presence of 50 .mu.CI .sup.35S-cysteine. Media was harvested from
the labeled cells and 250 .mu.l of the spent media was assayed by
immunoprecipitation with the anti-PDGF .beta.-receptor antibody
PR7212 to detect the presence of metabolically labeled PDGF
.beta.-receptor analogs. PR7212, diluted in PBS, was added to the
media to a final concentration of 2.5 .mu.g per 250 .mu.l spent
media. Five microliters of rabbit anti-mouse Ig diluted in PBS was
added to the PR7212/media mixtures. The immunocomplexes were
precipitated by the addition of 50 .mu.l 10% fixed Staph A
(weight/volume in PBS). The immunocomplexes were analyzed on 8%
SDS-polyacrylamide gels followed by autoradiography overnight at
-70.degree. C. The results of the immunoprecipitation showed that
the PDGF .beta.-receptor analog secreted by the transfectants was
bound by the anti-PDGF .beta.-receptor antibody. The combined
results of the ELISA and immunoprecipitation assays showed that the
PDGF .beta.-receptor analog secreted by the transfectants contained
both the PDGF .beta.-receptor ligand-binding domain and the human
light chain constant region.
[0170] C. Cotransfection of pSDL114 with an Immunoglobulin Heavy
Chain
[0171] Plasmid pSDL114 was cotransfected with
p.phi.5V.sub.HhuC.sub..gamma- .1M-neo, which encodes a neomycin
resistance gene expression unit and a complete mouse/human chimeric
immunoglobulin heavy chain gene expression unit.
[0172] Plasmid p.phi.5V.sub.HhuC.sub..gamma.1M-neo was constructed
as follows. The mouse immunoglobulin heavy chain gene was isolated
from a lambda genomic DNA library constructed from the murine
hybridoma cell line NR-ML-05 (Serafini et al., Eur. J. Nucl. Med.
14: 232, 1988) using an oligonucleotide probe designed to span the
V.sub.H/D/J.sub.H junction (5' GCA TAG TAG TTA CCA TAT CCT CTT GCA
CAG 3'; Sequence ID Number 33). The human immunoglobulin gamma-1 C
gene was isolated from a human genomic library using a cloned human
gamma-4 constant region gene (Ellison et al., DNA 1: 11-18, 1981).
The mouse immunoglobulin variable region was isolated as a 5.3 kb
Sst I-Hind III fragment from the original phage clone and the human
gamma-1 C gene was obtained from the original phage clone as a 6.0
kb Hind III-Xho I fragment. The chimeric gamma-1 C gene was created
by joining the V.sub.H and C.sub.H fragments via the common Hind
III site and incorporating them with the E. coli neomycin
resistance gene expression unit into pIC19H to yield
p.phi.5V.sub.HhuC.sub..gamma.1M- -neo.
[0173] Plasmid pSDL114 was linearized by digestion with Cla I and
was co-transfected into SP2/O-Ag14 cells with Asp 718 linearized
p.phi.5V.sub.HhuC.sub..gamma.1M-neo. The transfectants were
selected in growth medium containing methotrexate and neomycin.
Media from drug-resistant clones were tested for their ability to
bind PDGF in a competition binding assay.
[0174] The competition binding assay measured the amount of
.sup.125I-PDGF left to bind to human dermal fibroblast cells after
preincubation with the spent media from
pSDL114-p.phi.5V.sub.HhuC.sub..gamma.1M-neo transfected cells. The
media were serially diluted in binding medium (Table 4). The
dilutions were mixed with 0.5 ng of iodinated PDGF-BB or iodinated
PDGF-AA, and the mixtures were incubated for two hours at room
temperature. Three hundred micrograms of unlabeled PDGF-BB or
unlabeled PDGF-AA was added to one tube from each series. The
sample mixtures were added to 24 well plates containing confluent
human dermal fibroblast cells. The cells were incubated with the
mixture for four hours at 4.degree. C. The supernatants were
aspirated from the wells, and the wells were rinsed three times
with phosphate buffered saline that was held a 4.degree. C. (PBS;
Sigma, St. Louis, Mo.). Five hundred microliters of PBS+1% NP-40
was added to each well, and the plates were shaken on a platform
shaker for five minutes. The cells were harvested and the amount of
iodinated PDGF was determined. The results of the competition
binding assay showed that the protein produced from
pSDL114-p.phi.5V.sub.HhuC.sub..gamma.1M-neo transfected cells was
able to competitively bind PDGF-BB but did not bind PDGF-AA.
[0175] The PDGF .beta.-receptor analog produced from a
pSDL114-p.phi.5V.sub.HhuC.sub..gamma.1M-neo transfectant was
assayed to determine if the receptor analog was able to bind
PDGF-BB with high affinity. Eight and one half milliliters of spent
media containing the PDGF.beta.-R analogs from a
pSDL114-p.phi.5V.sub.HhuC.sub..gamma.1M-neo transfectant was added
to 425 .mu.l of Sepharose Cl-4B-Protein A beads (Sigma, St. Louis,
Mo.), and the mixture was incubated for 10 minutes at 4.degree. C.
The beads were pelleted by centrifugation and washed with binding
medium (Table 4). Following the wash the beads were resuspended in
8.5 ml of binding media, and 0.25 ml aliquots were dispensed to 1.5
ml tubes. Binding reactions were prepared by adding iodinated
PDGF-BB.sub.Tyr (Example 18.F.) diluted in DMEM+10% fetal calf
serum to the identical aliquots of receptor-bound beads to final
PDGF-BB.sub.Tyr concentrations of between 4.12 pM and 264 pM.
Nonspecific binding was determined by adding a 100 fold excess of
unlabeled BB to an identical set of binding reactions. Mixtures
were incubated overnight at 4.degree. C.
[0176] The beads were pelleted by centrifugation, and unbound
PDGF-BB was removed with three washes in PBS. The beads were
resuspended in 100 .mu.l of PBS and were counted. Results of the
assay showed that the PDGF.beta.-R analog was able to bind PDGF-BB
with high affinity.
[0177] D. Construction of pSDL113
[0178] As shown in FIG. 8, the DNA sequence encoding the
extracellular domain of the PDGF .beta.-receptor was joined with
the DNA sequence encoding a human immunoglobulin heavy chain
constant region joined to a hinge sequence. Plasmid pSDL110 was
digested with Eco RI and Hind III to isolate the 1.65 kb
PDGF.beta.-R fragment. Plasmid pICHu.sub..gamma.-1M was used as the
source of the heavy chain constant region and hinge region. Plasmid
pICHu.sub..gamma.-1M comprises the approximately 6 kb Hind III-Xho
I fragment of a human gamma-1 C gene subcloned into pUC19 that had
been linearized by digestion with Hind III and Sal I. Plasmid
pICHu.sub..gamma.-1M was digested with Hind III and Eco RI to
isolate the 6 kb fragment encoding the human heavy chain constant
region. Plasmid pIC19H was linearized by digestion with Eco RI. The
1.65 kb PDGF.beta.-R fragment, the 6 kb heavy chain constant region
fragment and the linearized pIC19H were joined in a three part
ligation. The resultant plasmid, pSDL111, was digested with Bam HI
to isolate the 7.7 kb fragment. Plasmid p.mu.PRE8 was linearized
with Bgl II and was treated with calf intestinal phosphatase to
prevent self-ligation. The 7.7 kb fragment and the linearized
p.mu.PRE8 were joined by ligation. A plasmid containing the insert
in the proper orientation was designated pSDL113 (FIG. 8).
[0179] Plasmid pSDL113 is linearized by digestion with Cla I and is
cotransfected with Pvu I-digested p416 into SP2/0-Ag14 by
electroporation. Transfectants are selected in growth medium
containing methotrexate.
[0180] Media from drug resistant clones are tested for the presence
of secreted PDGF.beta.-R analogs by immunoprecipitation using the
method described in Example 12.B.
[0181] E. Cotransfection of pSDL113 with an Immunoglobulin Light
Chain Gene
[0182] Plasmid pSDL113 is linearized by digestion with Cla I and
was cotransfected with pIC.phi.5V.sub..kappa.HuC.sub..kappa.-Neo,
which encodes a neomycin resistance gene and a mouse/human chimeric
immunoglobulin light chain gene. The mouse immunoglobulin light
chain gene was isolated from a lambda genomic DNA library
constructed from the murine hybridoma cell line NR-ML-05 (Woodhouse
et al., ibid.) using an oligonucleotide probe designed to span the
V.sub..kappa./J.sub..kappa. junction (5' ACC GAA CGT GAG AGG AGT
GCT ATA A 3'; Sequence ID Number 34). The human immunoglobulin
light chain constant region gene was isolated as described in
Example 12.B. The mouse NR-ML-05 immunoglobulin light chain
variable region gene was subcloned from the original mouse genomic
phage clone into pIC19R as a 3 kb Xba I-Hinc II fragment. The human
kappa C gene was subcloned from the original human genomic phage
clone into pUC19 as a 2.0 kb Hind III-Eco RI fragment. The chimeric
kappa gene was created by joining the NR-ML-05 light chain variable
region gene and human light chain constant region gene via the
common Sph I site and incorporating them with the E. coli neomycin
resistance gene into pIC19H to yield
pIC.phi.5V.sub..kappa.HuC.sub..kappa.-Neo (FIG. 9).
[0183] The linearized pSDL113 and
pIC.phi.5V.sub..kappa.HuC.sub..kappa.-Ne- o are transfected into
SP2/0-Ag14 cells by electroporation. The transfectants are selected
in growth medium containing methotrexate and neomycin.
[0184] F. Cotransfection of pSDL113 and pSDL114
[0185] A clone of SP2/0-Ag14 stably transfected with pSDL114 and
p416 was co-transfected with Cla I-digested pSDL113 and Bam
HI-digested pICneo by electroporation. (Plasmid pICneo comprises
the SV40 promoter operatively linked to the E. coli neomycin
resistance gene and pIC19H vector sequences.) Transfected cells
were selected in growth medium containing methotrexate and G418.
Media from drug-resistant clones were tested for their ability to
bind PDGF-BB or PDGF-AA in a competition binding assay as described
in Example 12.C. The results of the assay showed that the
transfectants secreted a PDGF .beta.-receptor analog which was
capable of competitively binding PDGF-BB but did not detectably
bind to PDGF-AA.
[0186] G. Cotransfection of pSDL114 with Fab
[0187] A clone of SP2/0-AG14 stably transfected with pSDL114 and
p416 was transfected with the Fab region of the human gamma-4 gene
(.gamma..sub.4) in plasmid p.phi.5V.sub.HFab-neo.
[0188] Plasmid p.phi.5V.sub.HFab-neo was constructed by first
digesting plasmid p24BRH (Ellison et al., DNA 1: 11, 1988) was
digested with Xma I and Eco RI to isolate the 0.2 kb fragment
comprising the immunoglobulin 3' untranslated region. Synthetic
oligonucleotides ZC871 (Sequence ID Number 3; Table 1) and ZC872
(Sequence ID Number 4; Table 1) were kinased and annealed using
essentially the methods described by Maniatis et al. (ibid.). The
annealed oligonucleotides ZC871/ZC872 formed an Set I-Xma I
adapter. The ZC871/ZC872 adapter, the 0.2 kb p24BRH fragment and
Sst I-Eco RI linearized pUC19 were joined in a three-part ligation
to form plasmid p.gamma..sub.43'. Plasmid p.gamma..sub.43' was
linearized by digestion with Bam HI and Hind III. Plasmid p24BRH
was cut with Hind III and Bgl II to isolate the 0.85 kb fragment
comprising the C.sub.H1 region. The p.gamma..sub.43' fragment and
the Hind III-Bgl II p24BRH fragment were joined by ligation to form
plasmid p.gamma..sub.4Fab. Plasmid p.gamma..sub.4Fab was digested
with Hind III and Eco RI to isolate the 1.2 kb fragment comprising
.gamma..sub.4Fab. Plasmid pICneo, comprising the SV40 promoter
operatively linked to the E. coli neomycin resistance gene and
pIC19H vector sequences, was linearized by digestion with Sst I and
Eco RI. Plasmid p.phi.5V.sub.H, comprising the mouse immunoglobulin
heavy chain gene variable region and pUC18 vector sequences, was
digested with Sst I and Hind III to isolate the 5.3 kb V.sub.H
fragment. The linearized pICneo was joined with the 5.3 kb Sst
I-Hind III fragment and the 1.2 kb Hind III-Eco RI fragment in a
three-part ligation. The resultant plasmid was designated
p.phi.5V.sub.HFab-neo (FIG. 10).
[0189] A pSDL114/p416-transfected SP2/0-AG14 clone was transfected
with Sca I-linearized p.phi.5V.sub.HFab-neo. Transfected cells were
selected in growth medium containing methotrexate and G418. Media
from drug-resistant clones were tested for their ability to bind
PDGF in a competition binding assay as described in Example 12.C.
The results of the assay showed that the PDGF .beta.-receptor
analog secreted from the transfectants was capable of competitively
binding PDGF-BB.
[0190] H. Cotransfection of pSDL114 with Fab'
[0191] A stably transfected SP2/0-AG14 isolate containing pSDL114
and p416 was transfected with plasmid pWKI, which contained the
Fab' portion of an immunoglobulin heavy chain gene. Plasmid pWKI
was constructed as follows.
[0192] The immunoglobulin gamma-1 Fab' sequence, comprising the
C.sub.H1 and hinge regions sequences, was derived from the gamma-1
gene clone described in Example 12.C. The gamma-1 gene clone was
digested with Hind III and Eco RI to isolate the 3.0 kb fragment,
which was subcloned into Hind III-Eco RI linearized M13mp19.
Single-stranded template DNA from the resultant phage was subjected
to site-directed mutagenesis using oligonucleotide ZC1447 (Sequence
ID Number 9; Table 1) and essentially the method of Zoller and
Smith (ibid.). A phage clone was identified having a ZC1447 induced
deletion resulting in the fusion of the hinge region to a DNA
sequence encoding the amino acids Ala-Leu-His-Asn-His-Tyr-
-Thr-Glu-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly-Lys (Sequence ID Number
31) followed in-frame by a stop codon. Replicative form DNA from a
positive phage clone was digested with Hind III and Eco RI to
isolate the 1.9 kb fragment comprising the C.sub.H1 and hinge
regions. Plasmid p.phi.5V.sub.H was digested with Sst I and Hind
III to isolate the 5.3 kb fragment comprising the mouse
immunoglobulin heavy chain gene variable region. Plasmid picneo was
linearized by digestion with Sst I and Eco RI. The linearized
picneo was joined with the 5.3 kb Hind III-Sst I fragment and the
1.9 kb Hind III-Eco RI fragment in a three-part ligation. The
resultant plasmid was designated pWKI (FIG. 10).
[0193] An SP2/0-AG14 clone stably transfected with pSDL114 and p416
was transfected with Asp 718-linearized pWKI. Transfected cells
were selected by growth in medium containing methotrexate and G418.
Media samples from transfected cells were assayed using the
competition assay described in Example 12.C. Results from the
assays showed that the transfected cells produced a PDGF
.beta.-receptor analog capable of competitively binding
PDGF-BB.
Example 13
Purification and Characterization of PDGF .beta.-Receptor Analogs
from Mammalian Cells Co-transfected with pSDL113 and pSDL114
[0194] A. Purification of PDGF .beta.-Receptor Analogs
[0195] The PDGF .beta.-receptor analog was purified from
conditioned culture media from a clone of transfected cells grown
in a hollow fiber system. The media was passed over a protein-A
sepharose column, and the column was washed sequentially with
phosphate buffered saline, pH 7.2 (PBS; Sigma, St. Louis, Mo.) and
0.1 M citrate, pH 5.0. The PDGF .beta.-receptor analog was eluted
from the protein-A column with 0.1 M citrate pH 2.5 and immediately
neutralized by the addition of Tris-base, pH 7.4. The eluate
fractions containing PDGF .beta.-receptor analog, as determined by
silver stain, were pooled and chromatographed over an S-200 column
(Pharmacia LKB Technologies, Inc., Piscataway, N.J.) equilibrated
with PBS. The peak fractions from the S-200 column were pooled and
concentrated on a centriprep-10 concentrator (Amicon). Glycerol
(10% final volume) was added to the preparation and the sample
frozen at -80.degree. C. PDGF .beta.-receptor analogs purified from
pSDL114+pSDL113 co-transfected cells were termed "tetrameric PDGF
.alpha.-receptors".
[0196] B. Measurement of The Relative Binding Affinity of
Tetrameric PDGF .beta.-Receptor Analog by Soluble Receptor
Assay
[0197] Purified tetrameric PDGF .beta.-receptor analog was compared
to detergent solubilized extracts of human dermal fibroblasts for
.sup.125I-labeled PDGF-BB binding activity in a soluble receptor
assay essentially as described by Hart et al. (J. Biol. Chem. 262:
10780-10785, 1987). Human dermal fibroblast cells were extracted at
20.times.10.sup.6 cell equivalents per ml in TNEN extraction buffer
(20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40,
1 mM PMSF, 10% glycerol). Two hundred and fifty thousand PDGF
.beta.-receptor-subunits per cell was used to calculate the
tetrameric PDGF .beta.-receptor analog number per volume of
extract. This value has been previously published by Seifert et al.
(J. Biol Chem. 264: 8771-8778, 1989). The PDGF .beta.-receptor
analog number was determined from the protein concentration of the
PDGF .beta.-receptor analog assuming an average molecular weight of
140 kDa for each immunoglobulin-PDGF .beta.-receptor monomer, and
four monomers per tetramer. Thus, each tetrameric molecule contains
four receptor molecules.
[0198] Increasing amounts of either detergent solubilized extracts
of human dermal fibroblast cells or purified PDGF .beta.-receptor
analog were incubated with 1 ng of .sup.125I-labeled PDGF-BB for
one hour at 37.degree. C. The sample was then diluted with 1 ml
binding media and was added to monolayers of human dermal
fibroblast cells grown in 24-well culture dishes. The samples were
incubated for two hours at 4.degree. C. The wells were washed to
remove unbound, .sup.125I-labeled PDGF-BB. On half of a milliliter
of extraction buffer (PBS+1% Nonidet P-40) was added to each well
followed by a 5 minute incubation. The extraction mixtures were
harvested and counted in a gamma counter.
[0199] The results showed that the PDGF .beta.-receptor analog had
the same relative binding affinity as solubilized PDGF
.beta.-receptor-subunit from mammalian cells in a solution phase
binding assay.
[0200] C. Determination of the Binding Affinity of the PDGF
.beta.-Receptor Analog in a Solid Phase Format
[0201] The apparent dissociation constant K.sub.D(app) of the PDGF
.beta.-receptor analog was determined essentially as described by
Bowen-Pope and Ross (Methods in Enzymology 109: 69-100, 1985),
using the concentration of .sup.125I-labeled PDGF-BB giving
half-maximal specific .sup.125I-labeled PDGF-BB binding. Saturation
binding assays to determine the concentration of .sup.125I-labeled
PDGF-BB that gave half-maximal binding to immobilized PDGF
.beta.-receptor analog were conducted as follows.
[0202] Affinity purified goat anti-human IgG, R- and L-chain
(Commercially available from Cappel Labs) was diluted into 0.1 M
Na.sub.2HCO.sub.3, pH 9.6 to a concentration of 2 .mu.g/ml. One
hundred microliters of the antibody solution was coated onto each
well of 96-well microtiter plates for 18 hours at 4.degree. C. The
wells were washed once with ELISA C buffer (PBS+0.05% Tween-20)
followed by an incubation with 175 .mu.l/well of ELISA B buffer
(PBS+1% BSA+0.05% Tween-20) to block the wells. The wells were
washed once with ELISA B buffer. One hundred microliters of 12.1
ng/ml or 24.3 ng/ml of tetrameric PDGF .beta.-receptor analog
protein diluted in ELISA B was added to each well and the plates
were incubated for 2 hours at 37.degree. C. Unbound protein was
removed from the wells by two washes with ELISA C.
.sup.125I-labeled PDGF-BB.sub.Tyr (Example 18.F.) was serially
diluted into binding media (25 mM HEPES, pH 7.2, 0.25% rabbit serum
albumin diluted in HAMs F-12 medium (GIBCO-BRL)), and 100 .mu.l of
the dilutions were added to the wells. The plates were incubated
for two hours at room temperature. The unbound .sup.125I-labeled
PDGF-BB was removed, and the wells were washed three times with
binding media. Following the last wash, 100 .mu.l of 0.1 M citrate,
pH 2.5 was added to each well. After five minutes, the citrate
buffer was removed, transferred to a tube and counted in a gamma
counter. The counts reflect counts of .sup.125I-labeled
PDGF-BB.sub.Tyr bound by the receptor analog. Nonspecific binding
for each concentration of .sup.125I-labeled PDGF-BB.sub.Tyr was
determined by a parallel assay wherein separate wells coated only
with goat anti-human IgG were incubated with the .sup.125I-labeled
PDGF-BB concentrations. Nonspecific binding was determined to be
2.8% of the total input counts per well and averaged 6% of the
total counts bound.
[0203] Saturation binding assay on 12.1 and 24.3 ng/ml of
tetrameric PDGF .beta.-receptor analog gave half-maximal binding at
0.8 and 0.82 ng/ml .sup.125I-labeled PDGF-BB.sub.Tyr, respectively.
By Scatchard analysis (Scatchard, Ann. NY Acad. Sci 51: 660-667,
1949) these values were shown to correspond to a K.sub.D(app) of
2.7.times.10.sup.-11 which agree with the published values for PDGF
receptors on mammalian cells.
Example 14
Solid Phase Ligand Binding Assay Using the PDGF .beta.-Receptor
Analog
[0204] A. Solid Phase Radioreceptor Competition Binding Assay
[0205] In a solid phase radioreceptor competition binding assay
(RRA), the wells of 96-well microtiter plates were coated with 100
.mu.l of 2 .mu.g/ml affinity purified goat anti-human IgG (Cappel
Labs) diluted in 0.1 M Na.sub.2HCO.sub.3, pH 9.6. After an eighteen
hour incubation at 4.degree. C., the wells were washed once with
ELISA C. The wells were blocked by incubation for 2 hours at
37.degree. C. with 175 .mu.l/well ELISA B. The wells were washed
once with ELISA B then incubated for 2 hours at 37.degree. C. with
50 ng/ml tetrameric PDGF .beta.-receptor analog diluted in ELISA B.
The unbound receptor was removed, and the test wells were incubated
with increasing concentrations of serially diluted, unlabeled
PDGF-BB (diluted in binding media. Following a two hour incubation
at room temperature, the wells were washed three times with binding
media. One hundred microliters of 5 ng/ml .sup.125I-labeled
PDGF-BB.sub.Tyr (Example 18.F.) was added to each well, and the
plates were incubated for an additional two hours at room
temperature. The wells were washed three times with binding media
followed by a 5 minute incubation with 100 .mu.l/well of 0.1 M
citrate, pH 2.5. The samples were harvested and counted in a gamma
counter.
[0206] Radioreceptor assay (RRA) competition binding curves were
generated for PDGF .beta.-receptor analog protein plated at 48.6
ng/ml. The sensitivity of the assays is 1 ng/ml of PDGF-BB, with 8
ng/ml giving 50% inhibition in .sup.125I-PDGF-BB binding, and a
working range between 1 and 32 ng/ml of PDGF-BB. The values were
similar to those obtained using monolayers of SK-5 cells in an
RRA.
[0207] B. Use of Tetrameric PDGF .beta.-Receptor Analogs As
Antagonists for PDGF-Stimulated Mitogenesis.
[0208] A tetrameric PDGF .beta.-receptor analog, purified as
described in Example 13, was analyzed for the ability to neutralize
PDGF-stimulated mitogenesis in mouse 3T3 cells. Increasing amounts
of the purified tetrameric PDGF .beta.-receptor analog were mixed
with 5 ng of PDGF. The mixtures were then added to cultures of
mouse 3T3 cells. The ability of the PDGF to stimulate a mitogenic
response, as measured by the incorporation of .sup.3H-thymidine,
was determined essentially as described (Raines and Ross, Methods
in Enzymology 109: 749-773, 1985, which is incorporated by
reference herein). The tetrameric PDGF .beta.-receptor analog
demonstrated a dose response inhibition of PDGF-BB-stimulated
.sup.3H-thymidine incorporation, while having essentially no effect
on PDGF-AA- and PDGF-AB-stimulated .sup.3H-thymidine
incorporation.
[0209] C. Binding of Tetrameric PDGF .beta.-receptor Analog to
Immobilized PDGF.
[0210] A tetrameric PDGF .beta.-receptor analog, purified as
described in Example 13, was analyzed for its ability to bind to
immobilized PDGF. PDGF-BB (100 ng/ml) was coated onto wells a
96-well microtiter plate, and the plates were incubated 18 hours at
4.degree. C. followed by one wash with ELISA C buffer. The wells
were incubated for 2 hours 37.degree. C. with ELISA B buffer to
block the wells. Increasing concentrations of .sup.125I-labeled
tetrameric PDGF .beta.-receptor analog, diluted in binding media,
was added to the wells for two hours at room temperature. The wells
were washed four times with ELISA C buffer to remove unbound
receptor analog. One hundred microliters of 1 M H.sub.2SO.sub.4 was
added to each well and the plates were incubated for five minutes
at room temperature. The solution was then harvested and
transferred to tubes to be counted in a gamma counter. Nonspecific
binding was determined to be less than 10% of the total counts
bound.
[0211] A receptor competition binding assay was developed using
this assay format. The assay was carried out as described above,
and simultaneous to the addition of the .sup.125I-labeled
tetrameric PDGF .beta.-receptor analog, increasing amounts of
PDGF-AA, AB or BB were added to the PDGF-BB coated wells. Under
these condtions, only PDGF-BB was found to significantly block the
binding of the labeled PDGF .beta.-receptor analog to the
immobilized PDGF-BB.
Example 15
Construction and Expression of PDGF.alpha.-R Analogs in Cultured
Mouse Myeloma Cells
[0212] A. Construction of an Optimized PDGF.alpha.-R cDNA
[0213] The PDGF .alpha.-receptor coding region was optimized for
expression in mammalian cells as follows. The 5' end of the cDNA
was modified to include an optimized Kozak consensus translation
initiation sequence (Kozak, Nuc, Acids Res. 12: 857-872, 1984) and
Eco RI and Bam HI sites just 5' of the initiation methionine codon.
Oligonucleotides ZC2181, ZC2182, ZC2183 and ZC2184 (Sequence ID
Numbers 23, 24, 25 and 26, respectively; Table 1) were designed to
form, when annealed, an adapter having an Eco RI adhesive end, a
Bam HI restriction site, a sequence encoding a Kozak consensus
sequence 5' to the initiating methionine codon, a mammalian codon
optimized sequence encoding amino acids 1-42 of FIG. 11, and an Eco
RI adhesive end that destroys the Eco RI site within the
PDGF.alpha.-R coding sequence. The adapter also introduced a
diagnostic Cla I site 3' to the initiation methionine codon.
Oligonucleotides ZC2181, ZC2182, ZC2183 and ZC2184 were kinased,
annealed and ligated. Plasmid p.alpha.17B was linearized by partial
digestion with Eco RI. The linearized p.alpha.17B was ligated with
the ZC2181/ZC2182/ZC2183/ZC2184 oligonucleotide adapter, and the
ligation mixture was transformed into E. coli Plasmid DNA prepared
from the transformants was analyzed by restriction analysis and a
positive clone having the oligonucleotide adapter in the correct
orientation was digested with Eco RI and Pst I to isolate the 1.6
kb fragment. This fragment was subcloned into EcoRI+Pst
I-linearized M13mp19. The resultant phage clone was designated
792-8. Single-stranded 792-8 DNA was sequenced to confirm the
orientation of the adapter.
[0214] A fragment encoding the ligand-binding domain of the PDGF
.alpha.-receptor (PDGF.alpha.-R) was then generated as follows.
Restriction sites and a splice donor sequence were introduced at
the 3' end of the PDGF.alpha.-R extracellular domain by PCR
amplification of the 792-8 DNA and oligonucleotides ZC2311 and
ZC2392 (Sequence ID Numbers 27 and 30, Table 1). Oligonucleotide
ZC2311 is a sense primer encoding nucleotides 1470 to 1489 of FIG.
11. Oligonucleotide ZC2392 is an antisense primer that encodes
nucleotides 1759 to 1776 of FIG. 11 followed by a splice donor and
Xba I and Hind III restriction sites. The 792-8 DNA was amplified
using manufacturer recommended (Perkin Elmer Cetus, Norwalk, Conn.)
conditions and the GeneAmp.TM. DNA amplification reagent kit
(Perkin Elmer Cetus), and blunt-ended 329 bp fragment was isolated.
The blunt-end fragment was digested with Nco I and Hind III and
ligated with Sma I-digested pUC18. A plasmid having an insert with
the Nco I site distal to the Hind III site present in the pUC18
polylinker was designated pUC18 Sma-PCR Nco HIII #13. The Hind III
site present in the insert was not regenerated upon ligation with
the linearized pUC18. Plasmid pUC18 Sma-PCR Nco HIII #13 was
digested with Nco I and Hind III to isolate the 355 bp
PDGF.alpha.-R containing fragment encoding PDGF.alpha.R.
Oligonucleotides ZC2351 and ZC2352 (Table 1; Sequence ID Numbers 28
and 29) were kinased and annealed to form an Sst I-Nco I adapter
encoding an internal Eco RI site and a Kozak consensus translation
initiation site. The 355 bp Nco I-Hind III fragment, the
ZC2351/ZC2352 adapter and a 1273 bp Nco I fragment comprising the
extracellular domain of of PDGF .alpha.-R derived from 792-8 were
ligated with Hind III+SstI-digested pUC18 and tranformed into E.
coli. Plasmid DNA was isolated from the transformants and analyzed
by restriction analysis. None of the isolates contained the 1273 bp
Nco I fragment. A plasmid containing the Nco I-Hind III fragment
and the ZC2351/ZC2352 adapter was desginated pUC18 Hin Sst .DELTA.
Nco #46. Plasmid pUC18 Hin Sst .DELTA.Nco #46 was linearized by
digestion and joined by ligation with the 1273 bp Nco I fragment
comprising the extracellular domain of the PDGF.alpha.-R from clone
.alpha.18 R-19. The ligations were transformed into E. coli, and
plasmid DNA was isolated from the transformants. Analysis of the
plasmid DNA showed that only clones with the Nco I fragment in the
wrong orientation were isolated. A clone having the Nco I fragment
in the wrong orientation was digested with Nco I, religated and
transformed into E. coli. Plasmid DNA was isolated from the
transformants and was analyzed by restriction analysis. A plasmid
having the Nco I insert in the correct orientation was digested to
completion with Hind III and partially digested with Sst I to
isolate the 1.6 kb fragment comprising the extracellular domain of
the PDGF.alpha.-R preceded by a consensus initiation sequence
(Kozak, ibid.) and followed by a splice donor site.
[0215] B. Construction of pPAB7
[0216] The DNA sequence encoding the extracellular domain of the
PDGF.alpha.-R was joined to the immunoglobulin .mu.
enhancer-promoter and to a DNA sequence encoding an immunoglobulin
light chain constant region. The immunoglobulin .mu.
enhancer-promoter was obtained from plasmid pJH1 which was derived
from plasmid PIC.mu.PRE1 (Example 12.A.) by digestion with Eco RI
and Sst I to isolate the 2.2 kb fragment comprising the
immunoglobulin enhancer and heavy chain variable region promoter.
The 2.2 kb Sst I-Eco RI fragment was ligated with Sst I+Eco
RI-linearized pUC19. The resulting plasmid, designated pJH1,
contained the immunoglobulin enhancer and heavy chain variable
region promoter immediately 5' to the pUC19 linker sequences.
Plasmid pH1 was linearized by digestion with Sst I and Hind III and
joined with the 1.6 kb partial Sst I-Hind III fragment containing
the PDGF.alpha.-R extracellular domain sequences. The resulting
plasmid having the immunoglobulin .mu. enhancer-promoter joined to
the PDGF.alpha.-R extracellular domain was designated pPAB6.
Plasmid pSDL112 was digested with Hind III to isolate the 1.2 kb
fragment encoding the immunoglobulin light chain constant region
(C.kappa.). The 1.2 kb Hind III fragment was ligated with Hind
III-linearized pPAB6. A plasmid having the C.sub..kappa. sequence
in the correct orientation was desginated pPAB7.
[0217] C. Construction of pPAB9
[0218] The partial Sst I-Hind III fragment encoding the
extracellular domain of the PDGF.alpha.-R was joined to the
immunoglobulin heavy chain constant region. For convenience, the
internal Xba I site in plasmid pJH1 was removed by digestion with
Xba I, blunt-ending with T4 DNA polymerase, and religation. A
plasmid which did not contain the internal Xba I site, but retained
the Xba I site in the polylinker was desginated 11.28.3.6. Plasmid
11.28.3.6 was linearized by digestion with Sst I and Xba I. Plasmid
pPAB6 was digested to completion with Hind III and partially
digested with Sst I to isolate the 1.6 kb Sst I-Hind III fragment
containing the PDGF.alpha.-R extracellular domain. Plasmid
p.phi.5V.sub.HhuC.sub..gamma.1M-neo (Example 12.C.) was digested
wtih Hind III and Xba I to isolate the 6.0 kb fragment encoding the
immunoglobulin heavy chain constant region (huC.sub..gamma.1M). The
Sst I-Hind III-linearized 11.28.3.6, the 1.6 kb Sst I-Hind III
PDGF.alpha.-R fragment and the 6.0 kb Hind III-Xba I
huC.sub..gamma.1M fragment were ligated to form plasmid pPAB9.
[0219] D. Expression of pPAB9 in Mammalian Cells
[0220] Bg1 II-linearized pPAB7 and Pvu I-linearized pPAB9 were
cotransfected with Pvu I-linearized p416 into SP2/0-Ag14 cells by
electroporation. Transfected cells were initially selected in
growth medium containing 50 nM methotrexate and were subsequently
amplified in a growth medium containing 100 .mu.M methotrexate.
Media from drug resistant clones were tested for the presence of
secreted PDGF .alpha.-receptor analogs by enzyme-linked
immunosorbant assay (ELISA). Ninety-six well assay plates were
prepared by incubating 100 .mu.l of 1 .mu.g/ml monoclonal antibody
292.1.8 which is specific for the PDGF .beta.-receptor diluted in
phosphate buffered saline (PBS; Sigma] overnight at 4.degree. C.
Excess antibody was removed by three washes with 0.5% Tween 20 in
PBS. One hundred microliters of spent media was added to each well,
and the plates were incubated for one hour at 4.degree. C. Unbound
proteins were removed by eight washes with 0.5% Tween 20 in PBS.
One hundred microliters of peroxidase-conjugated goat anti-human
IgG heavy chain antibody (diluted 1:1000 in a solution containing
5% chicken serum (GIBCO-BRL)+0.5% Tween 20 in PBS) was added to
each well, and the plates were incubated for one hour at 4.degree.
C. One hundred microliters of chromophore (100 .mu.l ABTS
[2,2'-Azinobis(3-ethylbenz-thiazoline sulfonic acid] diammonium
salt; Sigma]+1 .mu.l 30% H.sub.2O.sub.2+12.5 ml citrate/phosphate
buffer [9.04 g/l citric acid, 10.16 g/l Na.sub.2HPO.sub.4]) was
added to each well, and the wells were incubated for 30 minutes at
room temperature. The samples were measured at 405 nm. The results
of the assay showed that the PDGF .alpha.-receptor analogs secreted
by the transfectants contained an immunoglobulin heavy chain.
[0221] Analysis of spent media from transfected cells by Northern
analysis, Western analysis and by radioimmunoprecipitation showed
that the transfectants did not express a PDCF .alpha.-receptor
analog from the pPAB7 construction. Transfectants were subsequently
treated as containing only pPAB9.
[0222] Drug resistant clones was also tested for the presence of
secreted PDGF .alpha.-receptor analogs by immunoprecipitation. For
each clone, approximately one million drug resistant transfectants
were grown in DMEM lacking cysteine+2% calf serum for 18 hours at
37.degree. C. in the presence of 50 .mu.Ci .sup.35S-cysteine. The
spent media was harvested from the labeled cells and 250 .mu.l of
medium from each clone was assayed for binding to the anti-PDGF
.alpha.-receptor antibody 292.18. Monoclonal antibody 292.18
diluted in PBS was added to each sample to a final concentration of
2.5 .mu.g per 250 .mu.l spent media. Five microliters of rabbit
anti-mouse Ig diluted in PBS was added to each sample, and the
immunocomplexes were precipitated by the addition of 50 .mu.l 10%
fixed Staph A (weight/volume in PBS). The immunocomplexes were
analyzed on 8% SDS-polyacrylamide gels followed by autoradiography
overnight at -70.degree. C. The results of the immunoprecipitation
showed that the PDGF .alpha.-receptor analog secreted by the
transfectants was bound by the anti-PDGF .alpha.-receptor antibody.
The combined results of the ELISA and immunoprecipitation assays
showed that the PDGF .alpha.-receptor analog secreted by the
transfectants contained both the PDGF .alpha.-receptor
ligand-binding domain and the human heavy chain.
[0223] Spent medium from drug-resistant clones were tested for
their ability to bind PDGF in a competition binding assay
essentially as described in Example 12.C. The results of the assay
showed that the transfectants secreted a PDGF .alpha.-receptor
analog capable of binding PDGF-AA. A clone containing the pPAB9 was
desginated 3.17.1.57.
[0224] E. Co-expression of pPAB7 and pPAB9 in Mammalian Cells
[0225] Bgl II-linearized pPAB7 and Bam HI-linearized pICneo were
cotransfected into clone 3.17.1.57, and transfected cells were
selected in the presence of neomycin. Media from drug resistant
cells were assayed for the presence of immunoglobulin heavy chain,
immunoglobulin light chain and the PDGF .alpha.-receptor
ligand-binding domain by ELISA essentially as described above.
Briefly, ninety-six well assay plates were prepared by incubating
100 .mu.l of 1 .mu.g/ml goat anti-human IgG Fc antibody (Sigma) or
100 .mu.l of 1 .mu.g/ml 292.18 overnight at 4.degree. C. Excess
antibody was removed by three washes with 0.5% Tween 20 in PBS. One
hundred microliters of spent media was added to each well of each
plate, and the plates were incubated for one hour at 4.degree. C.
Unbound proteins were removed by eight washes with 0.5% Tween 20 in
PBS. One hundred microliters of peroxidase-conjugated goat
anti-human IgG antibody (diluted 1:1000 in a solution containing 5%
chicken serum (GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each
well of the plate coated with the anti-Fc antibody, and 100 .mu.l
of peroxidase-conjugated goat anti human kappa antibody (diluted
1:1000 in a solution containing 5% chicken serum (GIBCO-BRL)+0.5%
Tween 20 in PBS) was added to each well of the plate coated with
292.18. The plates were incubated for one hour at 4.degree. C. One
hundred microliters of chromophore (100 .mu.l ABTS
[2,2'-Azinobis(3-ethylbenz-thiazoline sulfonic acid) diammonium
salt; Sigma]+1 .mu.l 30% H.sub.2O.sub.2+12.5 ml citrate/phosphate
buffer [9.04 g/l citric acid, 10.16 g/l Na.sub.2HPO.sub.4]) was
added to each well of each plate, and the plates were incubated to
30 minutes at room temperature. The samples were measured at 405
nm, the wavelength giving maximal absorbance of the chromogenic
substrate, to identify clones having absorbances higher than
background indicating the presence of immunoglobulin heavy chain.
Clones that gave positive results in both ELISA assays (showing
that the clones produced proteins containing heavy chain regions,
light chain constant regions and the PDGF .alpha.-receptor
ligand-binding region) were selected for further
characterization.
[0226] Drug resistant clones that were positive for both ELISA
assays were subsequently tested for the presence of secreted PDGF
.alpha.-receptor analogs by immunoprecipitation. For each positive
clone, approximately one million drug resistant transfectants were
grown in DMEM lacking cysteine+2% calf serum for 18 hours at
37.degree. C. in the presence of 50 .mu.CI .sup.35S-cysteine. The
spent media was harvested from the labeled cells and 250 .mu.l of
medium from each clone was assayed for binding to monoclonal
antibody 292.18. Monoclonal antibody 292.18 diluted in PBS was
added to each sample to a final concentration of 2.5 .mu.g. Five
microliters of rabbit anti-mouse Ig diluted in PBS was added to
each sample and the immunocomplexes were precipitated by the
addition of 50 .mu.l 10% fixed Staph A (weight/volume in PBS). The
immunocomplexes were analyzed on 8% SDS-polyacrylamide gels
followed by autoradiography overnight at -70.degree. C. The results
of the immunoprecipitation showed that the PDGF .alpha.-receptor
analog secreted by the transfectants was bound by the anti-PDGF
.alpha.-receptor antibody. The combined results of the ELISA and
immunoprecipitation assays showed that the PDGF .alpha.-receptor
analog secreted by the transfectants contained the PDGF
.alpha.-receptor ligand-binding domain, the human heavy chain and
the human light chain constant region. A clone that secreted a PDGF
.alpha.-receptor analog that was positive for both the
above-described ELISA assays and the immunoprecipitation assay was
designated 5.6.2.1.
Example 16
Purification and Characterization of PDGF .alpha.-Receptor
Analogs
[0227] A. Purification of PDGF .alpha.-Receptor Analogs From Clone
3.17.1.57
[0228] The PDGF .alpha.-Receptor analog was purified from the
conditioned culture media of clone 3.17.1.57 by cycling
cell-conditioned medium over an immunoaffinity column composed of
monoclonal antibody 292.18 bound to a CNBr-activated Sepharose 4B
resin, which is specific for the PDGF .alpha.-receptor. The column
was washed with PBA, then eluted with 0.1 M citrate, pH 3.0. The
peak column fractions containing the .alpha.-receptor were pooled,
neutralized to pH 7.2 by the addition of 2 M Tris, pH 7.4, then
passed over a protein-A Sepharose column. This column was washed
sequentially with PBS, then with 0.1 M citrate, pH 5.0. The PDGF
.alpha.-receptor analog was then eluted with 0.1 M citrate, pH 3.0.
The peak eluate fractions were pooled, and glycerol was added to a
final concentration of 10%. The sample was concentrated on a
centriprep 10 concentrator (Amicon). The PDGF .alpha.-receptor
analog purified from clone 3.17.1.57 was termed a "dimeric PDGF
.alpha.-receptor analog".
[0229] B. Purification of PDGF .alpha.-Receptor Analogs From Clone
5.6.2.1
[0230] The PDGF .alpha.-receptor analog was purified from the
conditioned culture media of clone 5.6.2.1 by cycling
cell-conditioned medium over the immunoaffinity column described
above. The column was washed with PBS then eluted with 0.1 M
citrate, pH 3.0. The peak column fractions containing the
.alpha.-receptor were pooled, neutralized to pH 7.2 by the addition
of 2 M Tris (what pH 7.4), then passed over a protein-A sepharose
column. This column was washed sequentially with PBS then with 0.1
M citrate, pH 5.0. The PDGF .alpha.-receptor analog was then eluted
with 0.M citrate, pH 3.0. The peak eluate fractions were pooled and
glycerol was added to a final concentration of 10%. The sample was
concentrated on a centriprep 10 concentrator. The PDGF
.alpha.-receptor analogs purified from clone 5.6.2.1 was termed a
"tetrameric PDGF .alpha.-receptor analog".
Example 17
[0231] A. Use of the PDGF .alpha.-receptor Analogs in Ligand
Binding Studies
[0232] Purified tetrameric PDGF .alpha.-receptor analog and
purified dimeric PDGF .alpha.-receptor analog were compared to
monolayers of a control cell line of canine kidney epithelial
cells, which do not naturally express the PDGF .alpha.-receptor,
transfected with the human PDGF .alpha.-receptor cDNA for ligand
binding activity. The dissociation constant (Kd) of the receptor
preparations was determined by saturation binding and subsequent
Scatchard analysis.
[0233] Ligand binding of the purified PDGF .alpha.-receptor analogs
was determined using a solid phase binding assay. Affinity-purified
goat anti-human IgG was diluted to a concentration of 2 .mu.g/ml in
0.1 M Na.sub.2HCO.sub.3, pH 9.6 and 100 .mu.l/well of the solution
was used to coat 96-well microtiter plates for 18 hours at
4.degree. C. Excess antibody was removed from the wells with one
wash with ELISA C buffer (PBS, 0.05% Tween-20). The plates were
incubated with 175 .mu.l/well of ELISA B buffer (PBS, 1% BSA, 0.05%
Tween-20) to block the wells, followed by two washes with ELISA C
buffer. One hundred microliters of 50 ng/ml PDGF .alpha.-receptor
analog (dimeric or tetrameric) diluted in ELISA buffer B was added
to each well and the plates were incubated over night at 4.degree.
C. Unbound protein was removed from the wells with two washes with
ELISA buffer B. .sup.125I-labeled PDGF-AA was serially diluted in
binding media (Hams F-12, 25 mM HEPES pH 7.2, 0.25% rabbit serum
albumin), and 100 .mu.l of each dilution was added to the wells.
The samples were incubated for two hours at room temperature.
Unbound .sup.125I-labeled PDGF-AA was removed with three washes
with binding media one hundred microliters of 0.1 M citrate, pH 2.5
was added to each well, and the plates were incubated for five
minutes. After the incubation, the citrate buffer was removed and
transferred to a tube for counting in a gamma counter. Nonspecific
binding for each concentration of .sup.125I-labeled PDGF-AA was
determined by a parallel assay wherein separate wells coated only
with goat anti-human IgG were incubated with the .sup.125I-labeled
PDGF-AA samples.
[0234] A saturation binding assay was performed on alpha T-7 cells
transfected with the PDGF .alpha.-receptor. The cells were grown to
confluency in 24-well culture plates. The cells were washed one
time with binding media. Iodinated PDGF-AA was serially diluted in
binding media. One milliliter of each dilution was added to the
wells, and the plates were incubated for 3 hours at 4.degree. C.
Unbound .sup.125I-labeled PDGF-AA was removed and the cells were
washed three times with binding media. PBS containing 1% Triton
X-100 was added to the cells for 5 minutes. The extracts were
harvested and counted in a gamma counter. Nonspecific binding was
determined at a single concentration of .sup.125I-labeled PDGF-AA
using a 500-fold excess PDGF-BB.
[0235] The dissociation constants determined by Scatchard analysis
(ibid.) of the saturation binding assays for the tetrameric PDGF
.alpha.-receptor analog, dimeric PDGF .alpha.-receptor analog and
the control cells (Table 5).
12TABLE 5 Dissociation Constants for the Tetrameric PDGF
.alpha.-Receptor, the Dimeric PDGF .alpha.-receptor and control
cells Transfected with the PDGF .alpha.-receptor Receptor kD
Tetrameric PDGF .alpha.-receptor analog 1.6 .times. 10.sup.-11
Dimeric PDGF .alpha.-receptor analog 8.51 .times. 10.sup.-11
Control cells [PDGF .alpha.-receptor] 3.7 .times. 10.sup.-11
[0236] A solid-phase competition binding assay was established
using the tetrameric PDGF .alpha.-receptor analog. Ninety six-well
microtiter plates were coated with goat anti-human IgG (2
.mu.g/ml), the wells blocked with ELISA B buffer, 50 ng/ml of
purified tetrameric PDGF .alpha.-receptor analog diluted in binding
media was added, and the plates were incubated two hours at room
temperature. Unbound receptor was removed and the wells were washed
with binding media. The plates were incubated for two hours at room
temperature with increasing concentrations of either PDGF-AA or
PDGF-BB diluted in binding media. The wells were washed, then
incubated for two hours at room temperature with 3 ng/ml
.sup.125I-labeled PDGF-AA diluted in binding media. Unbound labeled
PDGF-AA was removed, the wells were subsequently washed with
binding media, and the bound, labeled PDGF-AA was harvested by the
addition of 0.1 M citrate, pH 2.5, as described for the saturation
binding studies. PDGF-AB, PDGF-AA and PDGF-BB were found to compete
for receptor binding with .sup.125I-PDGF-AA.
[0237] B. Use of Tetrameric PDGF .alpha.-Receptor Analogs As
Antagonists for PDGF-Stimulated Mitogenesis.
[0238] A dimeric PDGF .alpha.-receptor analog, purified as
described in Example 16.B., was analyzed for the ability to
neutralize PDGF-stimulated mitogenesis in mouse 3T3 cells.
Increasing amounts of the purified tetrameric PDGF .alpha.-receptor
analog were mixed with PDGF-AA, -AB or -BB ranging 0.6 to 5 ng. The
mixtures were then added to cultures of confluent mouse 3T3 cells.
The ability of the PDGF to stimulate a mitogenic response, as
measured by the incorporation of .sup.3-thymidine, was determined
essentially as described (Raines and Ross, Methods in Enzymology
109: 749-773, 1985, which is incorporated by reference herein). The
dimeric PDGF .alpha.-receptor analog demonstrated a dose response
inhibition of PDGF-stimulated .sup.3H-thymidine incorporation for
all three isoforms of PDGF.
[0239] C. Inverse Ligand-Receptor Radioreceptor Assay
[0240] An inverse ligand-receptor radioreceptor assay was designed
to screen for the presence of PDGF-BB, PDGF-BB binding proteins,
PDGF-BB related molecules, and PDGF-.beta.receptor antagonists in
test samples. PDGF-BB (100 ng/ml) was coated onto the walls of
96-well microtiter plates, and the plates were incubated at
4.degree. C. for 16 hours. The wells were washed once with ELISA C
buffer and then incubated with ELISA B buffer to block the
nonspecific binding sites. To the wells were added 50 .mu.l of
either PDGF standard or a test sample and 50 .mu.l of
.sup.125I-labeled tetrameric PDGF .beta.-receptor analog. The
samples were incubated for one hour at room temperature. The wells
were washed once with ELISA C buffer, and 0.1 M citrate, pH 2.5
containing 1% NP-40 was added to each well to disrupt the
ligand-receptor analog bond and elute the bound receptor analog.
The acid wash was collected and counted in a gamma counter. The
presence of PDGF or a molecule which mimics PDGF or otherwise
interferes with the binding of the well-bound PDGF-BB with its
receptor will cause a decrease in the binding of the radiolabeled
tetrameric PDGF .beta.-receptor. Using this assay, PDGF-BB was
found to inhibit receptor binding while PDGF-AA and PDGF-AB caused
no significant decrease in receptor binding.
Example 18
Assay Methods
[0241] A. Preparation of Nitrocellulose Filters for Colony
Assay
[0242] Colonies of transformants were tested for secretion of PDGF
.beta.-receptor analogs by first growing the cells on
nitrocellulose filters that had been laid on top of solid growth
medium. Nitrocellulose filters (Schleicher & Schuell, Keene,
N.H.) were placed on top of solid growth medium and were allowed to
be completely wetted. Test colonies were patched onto the wetted
filters and were grown at 30.degree. C. for approximately 40 hours.
The filters were then removed from the solid medium, and the cells
were removed by four successive rinses with Western Transfer Buffer
(Table 4). The nitrocellulose filters were soaked in Western Buffer
A (Table 4) for one hour at room temperature on a shaking platform
with two changes of buffer. Secreted PDGF.beta.-R analogs were
visualized on the filters described below.
[0243] B. Preparation of Protein Blot Filters
[0244] A nitrocellulose filter was soaked in Western Buffer A
(Table 4) without the gelatin and placed in a Minifold (Schleicher
& Schuell, Keene, N.H.). Five milliliters of culture
supernatant was added without dilution to the Minifold wells, and
the liquid was allowed to pass through the nitrocellulose filter by
gravity. The nitrocellulose filter was removed from the minifold
and was soaked in Western Buffer A (Table 3) for one hour on a
shaking platform at room temperature. The buffer was changed three
times during the hour incubation.
[0245] C. Preparation of Western Blot Filters
[0246] The transformants were analyzed by Western blot, essentially
as described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76:
4350-4354, 1979) and Gordon et al. (U.S. Pat. No. 4,452,901).
Culture supernatants from appropriately grown transformants were
diluted with three volumes of 95% ethanol. The ethanol mixtures
were incubated overnight at -70.degree. C. The precipitates were
spun out of solution by centrifugation in an SS-24 rotor at 18,000
rpm for 20 minutes. The supernatants were discarded and the
precipitate pellets were resuspended in 200 .mu.l of dH.sub.2O. Two
hundred microliters of 2.times.loading buffer (Table 4) was added
to each sample, and the samples were incubated in a boiling water
bath for 5 minutes.
[0247] The samples were electrophoresed in a 15% sodium
dodecylsulfate polyacrylamide gel under non-reducing conditions.
The proteins were electrophoretically transferred to nitrocellulose
paper using conditions described by Towbin et al. (ibid.). The
nitrocellulose filters were then incubated in Western Buffer A
(Table 4) for 75 minutes at room temperature on a platform
rocker.
[0248] D. Processing the Filters for Visualization with
Antibody
[0249] Filters prepared as described above were screened for
proteins recognized by the binding of a PDGF .beta.-receptor
specific monoclonal antibody, designated PR7212. The filters were
removed from the Western Buffer A (Table 4) and placed in sealed
plastic bags containing a 10 ml solution comprising 10 .mu.g/ml
PR7212 monoclonal antibody diluted in Western Buffer A. The filters
were incubated on a rocking platform overnight at 420 C. or for one
hour at room temperature. Excess antibody was removed with three
15-minute washes with Western Buffer A on a shaking platform at
room temperature.
[0250] Ten microliters biotin-conjugated horse anti-mouse antibody
(Vector Laboratories, Burlingame, Calif.) in 20 ml Western Buffer A
was added to the filters. The filters were re-incubated for one
hour at room temperature on a platform shaker, and unbound
conjugated antibody was removed with three fifteen-minute washes
with Western Buffer A.
[0251] The filters were pre-incubated for one hour at room
temperature with a solution comprising 50 .mu.l Vectastain Reagent
A (Vector Laboratories) in 10 ml of Western Buffer A that had been
allowed to incubate at room temperature for 30 minutes before use.
The filters were washed with one quick wash with distilled water
followed by three 15-minute washes with Western Buffer B (Table 4)
at room temperature. The Western Buffer B washes were followed by
one wash with distilled water.
[0252] During the preceding wash step, the substrate reagent was
prepared. Sixty mg of horseradish peroxidase reagent (Bio-Rad,
Richmond, Calif.) was dissolved in 20 ml HPLC grade methanol.
Ninety milliliters of distilled water was added to the dissolved
peroxidase followed by 2.5 ml 2 M Tris, pH 7.4 and 3.8 ml 4 M NaCl.
One hundred microliters of 30% H.sub.2O.sub.2 was added just before
use. The washed filters were incubated with 75 ml of substrate and
incubated at room temperature for 10 minutes with vigorous shaking.
After the 10 minute incubation, the buffer was changed, and the
filters were incubated for an additional 10 minutes. The filters
were then washed in distilled water for one hour at room
temperature. Positives were scored as those samples which exhibited
coloration.
[0253] E. Processing the Filters For Visualization with an
Anti-Substance P Antibody
[0254] Filters prepared as described above were probed with an
anti-substance P antibody. The filters were removed from the
Western Buffer A and rinsed with Western transfer buffer, followed
by a 5-minute wash in phosphate buffered saline (PBS, Sigma, St.
Louis, Mo). The filters were incubated with a 10 ml solution
containing 0.5 M 1-ethyl-3-3-dimethylamino propyl carbodiimide
(Sigma) in 1.0 M NH.sub.4Cl for 40 minutes at room temperature.
After incubation, the filters were washed three times, for 5
minutes per wash, in PBS. The filters were blocked with 2% powdered
milk diluted in PBS.
[0255] The filters were then incubated with a rat anti-substance P
monoclonal antibody (Accurate Chemical & Scientific Corp.,
Westbury, N.Y.). Ten microliters of the antibody was diluted in 10
ml of antibody solution (PBS containing 20% fetal calf serum and
0.5% Tween-20). The filters were incubated at room temperature for
1 hour. Unbound antibody was removed with four 5-minute washes with
PBS.
[0256] The filters were then incubated with a biotin-conjugated
rabbit anti-rat peroxidase antibody (Cappel Laboratories, Melvern,
Pa.). The conjugated antibody was diluted 1:1000 in 10 ml of
antibody solution for 2 hours at room temperature. Excess
conjugated antibody was removed with four 5-minute washes with
PBS.
[0257] The filters were pre-incubated for 30 minutes at room
temperature with a solution containing 50 .mu.l Vectastain Reagent
A (Vector Laboratories) and 50 .mu.l Vectastain Reagent B (Vector
Laboratories) in 10 ml of antibody solution that had been allowed
to incubate for 30 minutes before use. Excess Vectastain reagents
were removed by four 5-minute washes with PBS.
[0258] During the preceding wash step, the substrate reagent was
prepared. Sixty milligrams of horseradish peroxidase reagent
(Bio-Rad Laboratories, Richmond, Calif.) was dissolved in 25 ml of
HPLC grade methanol. Approximately 100 ml of PBS and 200 .mu.l
H.sub.2O.sub.2 were added just before use. The filters were
incubated with the substrate reagent for 10 to 20 minutes. The
substrate was removed by a vigorous washing distilled water.
[0259] F. Iodination of PDGF-BB
[0260] A PDGF-BB mutant molecule having a tyrosine replacing the
phenylalanine at position 23 (PDGF-BB.sub.Tyr) was iodinated and
subsequently purified, using a purification method which produces
125I-labeled PDGF-BB with a higher specific activity than
primary-labeled material and which was found to substantially
decrease the nonspecific binding component. The PDGF-BB.sub.Tyr was
labeled using the Iodobead method (Pierce Chemical). The labeled
protein was gel filtered over a C-25 desalting column (Pharmacia
LKB Technologies) equilibrated with 10 mM acetic acid, 0.25%
gelatin and 100 mM NaCl. The peak fractions were pooled and pH
adjusted to 7.2 by the addition of Tris-base. The labeled mixture
was chromatographed over an affinity column composed of PDGF
.beta.-receptor analog protein coupled to CnBr-activated Sepharose
(Pharmacia LKB Technologies, Inc.). The column was washed with
phosphate buffered saline and eluted with 0.1 M citrate, pH 2.5
containing 0.25% gelatin. The peak eluate fractions were pooled and
assayed by ELISA to determine the PDGF-BA concentration.
[0261] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be evident that certain changes and
modifications may be practiced within the scope of the appended
claims.
Sequence CWU 1
1
* * * * *