U.S. patent application number 11/548639 was filed with the patent office on 2007-06-14 for novel type-1 cytokine receptor glm-r.
Invention is credited to Frederic J. De Sauvage, Nico P. Ghilardi, Audrey Goddard, Paul J. Godowski, J. Christopher Grimaldi, Austin Gurney, William I. Wood.
Application Number | 20070136829 11/548639 |
Document ID | / |
Family ID | 27766136 |
Filed Date | 2007-06-14 |
United States Patent
Application |
20070136829 |
Kind Code |
A1 |
Ghilardi; Nico P. ; et
al. |
June 14, 2007 |
NOVEL TYPE-1 CYTOKINE RECEPTOR GLM-R
Abstract
The present invention is directed to novel polypeptides and
variants thereof of GLM-R polypeptides and to nucleic acid
molecules encoding those polypeptides. Also provided herein are
vectors and host cells comprising those nucleic acid sequences,
chimeric polypeptide molecules comprising the polypeptides of the
present invention fused to heterologous polypeptide sequences,
antibodies which bind to the polypeptides of the present invention
and to methods for producing the polypeptides of the present
invention. Also provided are methods for detecting agents that
modulate the activity of GLM-R. Also provided are methods for
diagnosing and for treating disorders characterized by the over or
under abundance of monocytes or macrophages.
Inventors: |
Ghilardi; Nico P.;
(Millbrae, CA) ; De Sauvage; Frederic J.; (Foster
City, CA) ; Goddard; Audrey; (San Francisco, CA)
; Godowski; Paul J.; (Hillsborough, CA) ;
Grimaldi; J. Christopher; (San Francisco, CA) ;
Gurney; Austin; (San Francisco, CA) ; Wood; William
I.; (Cupertino, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Family ID: |
27766136 |
Appl. No.: |
11/548639 |
Filed: |
October 11, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10373512 |
Feb 24, 2003 |
|
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11548639 |
Oct 11, 2006 |
|
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60359806 |
Feb 25, 2002 |
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Current U.S.
Class: |
800/14 ;
435/320.1; 435/325; 435/353; 435/6.16; 435/69.1; 435/7.1; 435/7.2;
530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 37/00 20180101; C07K 14/7158 20130101 |
Class at
Publication: |
800/014 ;
435/006; 435/007.2; 435/069.1; 435/320.1; 435/325; 530/350;
530/388.22; 536/023.5; 435/007.1; 435/353 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12Q 1/68 20060101 C12Q001/68; G01N 33/53 20060101
G01N033/53; G01N 33/567 20060101 G01N033/567; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/705 20060101
C07K014/705 |
Claims
1. An isolated nucleic acid molecule which comprises DNA having at
least about 80% sequence identity to (a) a DNA molecule encoding a
GLM-R polypeptide comprising the sequence of amino acid residues
from about 1 or about 20 to about 732 of FIG. 2 (SEQ ID NO:2), or
(b) the complement of the DNA molecule of (a).
2. The isolated nucleic acid molecule of claim 1 comprising the
sequence of nucleotide positions from about 63 or about 120 to
about 2258 of FIG. 1 (SEQ ID NO:1).
3. The isolated nucleic acid molecule of claim 1 comprising the
nucleotide sequence of FIG. 1 (SEQ ID NO:1).
4. The isolated nucleic acid molecule of claim 1 comprising a
nucleotide sequence that encodes the sequence of amino acid
residues from about 1 or about 20 to about 732 of FIG. 2 (SEQ ID
NO:2).
5. An isolated nucleic acid molecule comprising DNA which comprises
at least about 80% sequence identity to (a) a DNA molecule encoding
the same mature polypeptide encoded by the human protein cDNA
deposited with the ATCC on May 16, 2000 under ATCC Deposit No.
1874-PTA (DNA173920-2924), or (b) the complement of the DNA
molecule of (a).
6. The isolated nucleic acid molecule of claim 5 comprising DNA
encoding the same mature polypeptide encoded by the human protein
cDNA deposited with the ATCC on May 16, 2000 under ATCC Deposit No.
1874-PTA (DNA173920-2924).
7. An isolated nucleic acid molecule comprising DNA which comprises
at least about 80% sequence identity to (a) the full-length
polypeptide coding sequence of the human protein cDNA deposited
with the ATCC on May 16, 2000 under ATCC Deposit No. 1874-PTA
(DNA173920-2924), or (b) the complement of the coding sequence of
(a).
8. The isolated nucleic acid molecule of claim 7 comprising the
full-length polypeptide coding sequence of the human protein cDNA
deposited with the ATCC on May 16, 2000 under ATCC Deposit No.
1874-PTA (DNA173920-2924).
9. An isolated nucleic acid molecule encoding a GLM-R polypeptide
comprising DNA that hybridizes to the complement of the nucleic
acid sequence that encodes amino acids 1 or about 20 to about 732
of FIG. 2 (SEQ ID NO:2).
10. The isolated nucleic acid molecule of claim 9, wherein the
nucleic acid that encodes amino acids 1 or about 20 to about 732 of
FIG. 2 (SEQ ID NO:2) comprises nucleotides 63 or about 120 to about
2258 of FIG. 1 (SEQ ID NO:1).
11. The isolated nucleic acid molecule of claim 9, wherein the
hybridization occurs under stringent hybridization and wash
conditions.
12. An isolated nucleic acid molecule comprising at least about 702
nucleotides and which is produced by hybridizing a test DNA
molecule under stringent hybridization conditions with (a) a DNA
molecule which encodes a GLM-R polypeptide comprising a sequence of
amino acid residues from 1 or about 20 to about 732 of FIG. 2 (SEQ
ID NO:2), or (b) the complement of the DNA molecule of (a), and
isolating the test DNA molecule.
13. The isolated nucleic acid molecule of claim 12, which has at
least about 80% sequence identity to (a) or (b).
14. A vector comprising the nucleic acid molecule of any one of
claims 1 to 13.
15. The vector of claim 14, wherein said nucleic acid molecule is
operably linked to control sequences recognized by a host cell
transformed with the vector.
16. A nucleic acid molecule deposited with the ATCC under accession
number 1874-PTA (DNA173920-2924).
17. A host cell comprising the vector of claim 16.
18. The host cell of claim 17, wherein said cell is a CHO cell.
19. The host cell of claim 17, wherein said cell is an E. coli.
20. The host cell of claim 17, wherein said cell is a yeast
cell.
21. A process for producing a GLM-R polypeptide comprising
culturing the host cell of claim 17 under conditions suitable for
expression of said GLM-R polypeptide and recovering said GLM-R
polypeptide from the cell culture.
22. An isolated GLM-R polypeptide comprising an amino acid sequence
comprising at least about 80% sequence identity to the sequence of
amino acid residues from about 1 or about 20 to about 732 of FIG. 2
(SEQ ID NO:2).
23. The isolated GLM-R polypeptide of claim 22 comprising amino
acid residues 1 or about 20 to about 732 of FIG. 2 (SEQ ID
NO:2).
24. An isolated GLM-R polypeptide having at least about 80%
sequence identity to the polypeptide encoded by the cDNA insert of
the vector deposited with the ATCC on May 16, 2000 as ATCC Deposit
No. 1874-PTA (DNA173920-2924).
25. The isolated GLM-R polypeptide of claim 24 which is encoded by
the cDNA insert of the vector deposited with the ATCC on May 16,
2000 as ATCC Deposit No. 1874-PTA (DNA173920-2924).
26. An isolated GLM-R polypeptide comprising the sequence of amino
acid residues from 1 or about 20 to about 732 of FIG. 2 (SEQ ID
NO:2), or a fragment thereof sufficient to provide a binding site
for an anti-GLM-R antibody.
27. An isolated polypeptide produced by (i) hybridizing a test DNA
molecule under stringent conditions with (a) a DNA molecule
encoding a GLM-R polypeptide comprising the sequence of amino acid
residues from 1 or about 20 to about 732 of FIG. 2 (SEQ ID NO:2),
or (b) the complement of the DNA molecule of (a), (ii) culturing a
host cell comprising said test DNA molecule under conditions
suitable for the expression of said polypeptide, and (iii)
recovering said polypeptide from the cell culture.
28. The isolated polypeptide of claim 27, wherein said test DNA has
at least about 80% sequence identity to (a) or (b).
29. A chimeric molecule comprising a GLM-R polypeptide fused to a
heterologous amino acid sequence.
30. The chimeric molecule of claim 29, wherein said heterologous
amino acid sequence is an epitope tag sequence.
31. The chimeric molecule of claim 29, wherein said heterologous
amino acid sequence is a Fc region of an immunoglobulin.
32. An antibody which specifically binds to a GLM-R
polypeptide.
33. The antibody of claim 32, wherein said antibody is a monoclonal
antibody.
34. The antibody of claim 32, wherein said antibody is a humanized
antibody.
35. The antibody of claim 32, wherein said antibody is an antibody
fragment.
36. An agonist to a GLM-R polypeptide.
37. An antagonist to a GLM-R polypeptide.
38. A composition of matter comprising (a) a GLM-R polypeptide, (b)
an agonist to a GLM-R polypeptide, (c) an antagonist to a GLM-R
polypeptide, or (d) an anti-GLM-R antibody in admixture with a
pharmaceutically acceptable carrier.
39. An isolated nucleic acid molecule which comprises a nucleotide
sequence having at least about 80% sequence identity to (a) a DNA
molecule encoding amino acids 1 to X of FIG. 2 (SEQ ID NO:2), where
X is any amino acid from 510 to 519 of FIG. 2 (SEQ ID NO:2), or (b)
the complement of the DNA molecule of (a).
40. The isolated nucleic acid of claim 39 which comprises (a) a
nucleotide sequence encoding amino acids 1 to X of FIG. 2 (SEQ ID
NO:2), where X is any amino acid from 510 to 519 of FIG. 2 (SEQ ID
NO:2), or (b) the complement of the nucleotide sequence of (a).
41. An isolated soluble GLM-R polypeptide comprising an amino acid
sequence having at least about 80% sequence identity to amino acids
1 to X of FIG. 2 (SEQ ID NO:2), where X is any amino acid from 510
to 519 of FIG. 2 (SEQ ID NO:2).
42. The isolated soluble GLM-R polypeptide of claim 41 which
comprises amino acids 1 to X of FIG. 2 (SEQ ID NO:2), where X is
any amino acid from 510 to 519 of FIG. 2 (SEQ ID NO:2).
43. A method for screening for a bioactive agent capable of binding
to GLM-R comprising: a) adding a candidate bioactive agent to a
sample of GLM-R; and b) determining the binding of said candidate
agent to said GLM-R, wherein binding indicates a bioactive agent
capable of binding to GLM-R.
44. A method for screening for a bioactive agent capable of
modulating the activity of GLM-R, said method comprising the steps
of: a) adding a candidate bioactive agent to a sample of GLM-R; and
b) determining an alteration in the biological activity of GLM-R,
wherein an alteration indicates a bioactive agent capable of
modulating the activity of GLM-R.
45. A method of identifying a receptor for GLM-R, said method
comprising combining GLM-R with a composition comprising cell
membrane material wherein said GLM-R complexes with a receptor on
said cell membrane material, and identifying said receptor as a
GLM-R receptor.
46. The method of claim 44 wherein GLM-R binds to said receptor,
and said method further includes a step of crosslinking said GLM-R
and receptor.
47. The method of claim 44, wherein said composition is a cell.
48. The method of claim 44, wherein said composition is a cell
membrane extract preparation.
49. A rodent comprising a genome comprising a transgene encoding
GLM-R.
Description
[0001] This application is a continuation application of U.S. Ser.
No. 10/373,512, filed Feb. 24, 2003, now pending, which claims the
benefit under 35 U.S.C. .sctn. 119 of U.S. Ser. No. 60/359,806,
filed Feb. 26, 2002; the contents of which are incorporated herein
by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to GLM-R genes, including the
human GLM-R gene, which are novel genes involved in the development
and function of monocytes and macrophages. The scope of the
invention includes the identification and isolation of novel DNA
encoding and to the recombinant production of novel polypeptides
designated herein as GLM-R polypeptides, and to methods,
compositions and assays utilizing such polypeptides in the
diagnosis and treatment of disorder characterized by the over or
under abundance of monocytes or macrophages. The invention
encompasses nucleotide sequences of the GLM-R nucleic acid, host
cell expression systems and hosts which have been transformed by
these expression systems, including transgenic animals. Further
included are GLM-R proteins, polypeptides and peptides containing
GLM-R amino acid sequences, fusion proteins of GLM-R proteins,
polypeptides and peptides, and antibodies specifically binding
thereto.
BACKGROUND OF THE INVENTION
[0003] Helical cytokines control multiple biological processes,
ranging from host defense to development and body homeostasis. This
family of ligands, consisting of Interleukin (IL-x) 2, 3, 4, 5, 6,
7, 9, 11, 12, 13, 15, 21, 23, thymic stromal lymphopoietin (TSLP),
granulocyte factor (GM-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin
(TPO), prolactin (PRL), growth hormone (GN), leukemia inhibitory
factor (LIF), oncostatin-M (OSM), cardiotrophin-1 (CT-1),
cardiotrophin-like cytokine (CLC), ciliary neurotrophic factor
(CNTF), and leptin (OB), has a rich source of molecules with highly
specific biological effects and important therapeutic
potential.
[0004] The helical cytokine family is defined by a common
three-dimensional structure consisting of an anti-parallel four
helix bundle with a characteristic "up-up-down-down" topology.
Bazan, J. F., Immunol. Today 11(10): 350-4(1990), Rozwarski, D. A.
et al, Structure 2(3): 159-73 (1994). Unfortunately, the lack of
significant sequence homology has hampered the identification of
novel members of this family by homology screens, and more
recently, data mining. The cognate receptors, however, form a
family of so-called type I cytokine receptors and share several
structural motifs, including a cytokine receptor homology (CRH)
domain with 2 pairs of conserved cysteine residues and a WSXWS
sequence motif in the extracellular domain [Bazan, J. F., Proc.
Natl. Acad. Sci. USA 87(18): 6934-8 (1990)], a single transmembrane
domain and an intracellular domain without intrinsic enzymatic
activity. These features allow for homology-based identification of
novel receptors, which in turn can be used as tools to subsequently
identify their ligands by a variety of different screening
techniques. De Sauvage et al., Nature 369(6481): 533-8 (1994);
Parrish-Novak J., et al, Nature 408(6808): 57-63 (2000); Lok, S. et
al., Nature 369 (6481): 565-8 (1994).
[0005] Ligand binding induces homo- or heteromerization of at least
two receptor subunits. In the former case, two identical receptor
subunits form a homodimeric receptor that is sufficient for ligand
binding and signaling [e.g., GH-R, de Vos, A. M. et al., Science
255(5042): 306-12 (1992)]. Heteromerization is induced when a
ligand-specific .alpha.-chain forms a high affinity receptor in
combination with a signal transducing .beta.-chain. This
.beta.-chain is shared amongst several other .alpha.-chains, e.g.,
IL-3, IL-5, GM-CSF. Itoh, N. et al, Science 247 (4940): 324-7
(1990). In either instance, ligand binding to the receptor leads to
activation of cytoplasmic tyrosine kinases of the Janus kinase
(Jak) family, which associate with the receptor subunits through
conserved box-1 and box-2 motifs within the membrane proximal part
of the intracellular domain. Ihle, J. N., Nature 377(6550): 591-4
(1995). Jak activation leads to phosphorylation of cytoplasmic
target proteins, in particular the intracellular domains of the
receptors and members of the STAT protein family, which are
recruited to phosphotyrosines on the receptor by means of their
src-homology type 2 (SH2) domains. Ihle, J. H. Cell 84(3): 331-4
(1996); Ihle, J. H. et al., Stem Cells 15 (Suppl. 1): 105-11,
discussion 112 (1997). Phosphorylation of STATs induces
dimerization and translocation to the nucleus and results in
specific activiation of gene trancription. Darnell, J. E., Jr.,
Science 277 (5332): 1630-5 (1997). Seven STAT proteins are known to
date (STATs 1, 2, 3, 4, 5a, 5b and 6). Analysis of animals
deficient for STAT isoforms indicates that STATs mediate many of
the specific effects of cytokines, Ihle J. N. Curr. Opin. Cell
Biol. 13(2): 211-7 (2001), highlighting their key importance in
cytokine receptor signaling. In addition to specific target gene
regulation, and in combination with other signaling pathways
activated by cytokine receptors, such as mitogen-activated protein
kinase and phosphatidylinositol-3 kinase, STATs can contribute to
anti-apoptotic and mitogenic signals upon activation Ihle, J. N.
Nature 377(6550): 591-4 (1995).
SUMMARY OF THE INVENTION
[0006] The present invention relates to the identification of
nucleic acid that encode novel GLM-R polypeptides that are involved
in the development and function of monocytes and macrophage, and
physiological conditions associated therewith. The nucleic acid
molecules represent nucleotide sequences corresponding to the
mammalian GLM-R polynucleotides, including human GLM-R
polynucleotides. Particular examples of the nucleic acids molecules
of the present invention are designated herein as
DNA173920-2924.
[0007] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a GLM-R polypeptide. An example GLM-R polypeptide is PRO21073.
[0008] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% nucleic acid
sequence identity, alternatively at least about 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% nucleic acid sequence identity to (a) a DNA molecule
encoding a polypeptide having the sequence of amino acid residues
from about: 1 or about 20 to about 732, inclusive, of FIG. 2 (SEQ
ID NO:2), or (b) the complement of the DNA molecule of (a).
[0009] In another aspect, the isolated nucleic acid molecule
comprises (a) a nucleotide sequence encoding a GLM-R polypeptide
having the sequence of amino acid residues from about: (i) 1 or
about 20 to about 732, inclusive, of FIG. 2 (SEQ ID NO:2), or (b)
the complement of the DNA molecule of (a).
[0010] In yet another aspect, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 8%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% nucleic acid sequence identity to (a) a DNA
molecule having the sequence of nucleotides from about 63 or about
120 to about 2258, inclusive, of FIG. 1 (SEQ ID NO:1), or (b) the
complement of the DNA molecule of (a).
[0011] In yet another aspect, the isolated nucleic acid molecule
comprises (a) the nucleotide sequence from about 63 or about 120 to
about 2258, inclusive, of FIG. 1 (SEQ ID NO:1), or (b) the
complement of the DNA molecule of (a).
[0012] In yet another aspect, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% nucleic acid sequence identity to: (a) a DNA
molecule that encodes the same mature polypeptide encoded by the
human protein cDNA deposited with the ATCC on May 16, 2000 under
ATCC Dep. No. 1874-PTA (DNA173920-2924) or (b) the complement of
the nucleotide sequence of (a).
[0013] In yet another aspect, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% nucleic acid sequence identity to: (a) the
full-length polypeptide coding sequence of the DNA deposited with
the ATCC on May 16, 2000 under ATCC Dep. No. 1874-PTA (DNA
173920-2924) or (b) the complement of the nucleotide sequence of
(a). In a specific aspect, the isolated nucleic acid molecule
comprises: (a) the full-length polypeptide coding sequence of the
DNA deposited with the ATCC on May 16, 2000 under ATCC Deposit No.
1874-PTA (DNA173920-2924), or (b) the complement of the nucleotide
sequence of (a).
[0014] In yet another aspect, the isolated nucleic acid molecule is
a nucleotide sequence which encodes an active GLM-R polypeptide as
defined below comprising a nucleotide sequence that hybridizes to
the complement of (a) a nucleic acid sequence that encodes amino
acid residues from about 1 or about 20 to about 732, inclusive, of
FIG. 2 (SEQ ID NO:2). Preferably, hybridization occurs under
stringent hybridization and wash conditions.
[0015] In yet another aspect, the isolated nucleic acid molecule is
a nucleotide sequence which encodes an active GLM-R polypeptide as
defined below comprising a nucleotide sequence that hybridizes to
the complement of (a) the nucleic acid sequence between about
nucleotides 63 or about 120 and about 2258, inclusive, of FIG. 1
(SEQ ID NO:1). Preferably, hybridization occurs under stringent
hybridization and wash conditions.
[0016] In yet another aspect, the isolated nucleic acid is a
nucleotide sequence having at least about 702 nucleotide residues
and which is produced by hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a GLM-R
polypeptide having the sequence of amino acid residues from about 1
or about 20 to about 732, inclusive, of FIG. 2A (SEQ ID NO:2), or
(b) the complement of the DNA molecule of (a), and, if the test DNA
molecule has at least about an 80% nucleic acid sequence identity,
alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
nucleic acid sequence identity to (a) or (b), and isolating the
test DNA molecule.
[0017] In yet another aspect, the isolated nucleic acid molecule
comprises DNA encoding a GLM-R polypeptide without the N-terminal
signal sequence and/or the initiating methionine, or is
complementary to such encoding nucleic acid molecule. The signal
peptide has been tentatively identified as extending from about
amino acid position 1 to about amino acid position 19, inclusive,
in the sequence of FIG. 2 (SEQ ID NO:2). It is noted, however, that
the C-terminal boundary of the signal peptide may vary, but most
likely by no more than about 5 amino acids on either side of the
signal peptide C-terminal boundary as initially identified herein,
wherein the C-terminal boundary of the signal peptide may be
identified pursuant to criteria routinely employed in the art for
identifying that type of amino acid sequence element (e.g., Nielsen
et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl.
Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognized
that, in some cases, cleavage of a signal sequence from a secreted
polypeptide is not entirely uniform, resulting in more than one
secreted species. These polypeptides, and the polynucleotides
encoding them, are contemplated by the present invention. As such,
for purposes of the present application, the signal peptide of the
GLM-R polypeptide shown in FIG. 2 (SEQ ID NO:2) extends from amino
acids 1 to X of FIG. 2 (SEQ ID NO:2), respectively, wherein X is
any amino acid from 15 to 24 of FIG. 2 (SEQ ID NO:2), respectively.
Therefore, mature forms of the GLM-R polypeptide which are
encompassed by the present invention include those comprising amino
acid residues X to 732 of FIG. 2 (SEQ ID NO:2); wherein X is any
amino acid from 15 to 24 of FIG. 2 (SEQ ID NO:2) and variants
thereof as described below. Isolated nucleic acid molecules
encoding these polypeptides are also contemplated.
[0018] In yet another embodiment, the invention provides an
isolated nucleic acid molecule comprising a nucleotides sequence
encoding a GLM-R polypeptide which is either transmembrane
domain-deleted or transmembrane domain-inactivated, or is
complementary to such encoding nucleotide sequence, wherein the
transmembrane domain has been tentatively identified as extending
from about amino acid position 515 to about amino acid position 539
in the sequence of FIG. 2 (SEQ ID NO:2). Therefore, soluble
extracellular domains of the herein described GLM-R polypeptides
are contemplated.
[0019] In yet another embodiment, the invention provided an
isolated nucleic acid molecule comprising a nucleotide sequence
having at least about 80% nucleic acid sequence identity,
alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleic
acid sequence identity to (a) a DNA molecule encoding amino acids 1
to X of FIG. 2 (SEQ ID NO:2), where X is any amino acid from 510 to
519 of FIG. 2 (SEQ ID NO:2), or (b) the complement of the DNA
molecule of (a). In a specific aspect, the isolated nucleic acid
molecule comprises a nucleotide sequence which encodes amino acids
1 to X of FIG. 2 (SEQ ID NO:2), where X is any amino acid from 510
to 519 of FIG. 2 (SEQ ID NO:2), or (b) is the complement of the DNA
molecule of (a).
[0020] In yet another embodiment, the invention provides fragments
of a GLM-R polypeptide sequence which includes the coding sequence
that may find use as, for example, hybridization probes or for
encoding fragments of a GLM-R polypeptide that may optionally
encode a polypeptide comprising a binding site for an anti-GLM-R
antibody. Such nucleic acid fragments are usually at least about
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900, 1000 nucleotides in length, wherein in this context the term
"about" means the referenced nucleotide sequence length plus or
minus up to 10% of that referenced length. In a preferred
embodiment, the nucleotide sequence fragment is derived from any
coding region of the nucleotide sequence shown in FIG. 1 (SEQ ID
NO:1). It is noted that novel fragments of a GLM-R
polypeptide-encoding nucleotide sequence may be determined in a
routine manner by aligning the GLM-R polypeptide-encoding
nucleotide sequence with other known nucleotide sequences using any
of a number of well known sequence alignment programs and
determining which GLM-R polypeptide-encoding nucleotide sequence
fragment(s) are novel. All of such GLM-R polypeptide-encoding
nucleotide sequences are contemplated herein and can be determined
without undue experimentation. Also contemplated are the GLM-R
polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those GLM-R polypeptide fragments that
comprise a binding site for an anti-GLM-R antibody.
[0021] In yet another embodiment, the invention provides a vector
(e.g., expression vectors) comprising a nucleotide sequence
encoding GLM-R or its variants. The vector may comprise any of the
isolated nucleic acid molecules hereinabove identified. A host cell
comprising such a vector is also provided. By way of example, the
host cells may be CHO cells, E. coli, baculovirus infected insect
cells, or yeast. In one aspect, the invention comprises host
organisms that have been transformed with GLM-R-encoding nucleotide
sequence, including, for example, transgenic animals.
[0022] In another aspect, the transgenic animals of the invention
express a GLM-R variant, in particular a variant that is associated
with a weight disorder such as obesity, cachexia or anorexia. In
particular, such transgenic animals comprise those that express a
GLM-R transgene at higher or lower levels than normal. In another
particular aspect, the transgenic animals include those which
express GLM-R in all or some ("mosaic") of their cells. In yet a
further particular aspect, such transgenic animals further includes
those in which GLM-R nucleic acid is introduced into and expressed
in only specific cell types. In yet another particular aspect, the
invention includes "knock-out" animals, or animals which have been
modified to no longer express, or express in a lower quantity,
GLM-R polynucleotides.
[0023] In yet another embodiment, the invention provides isolated
GLM-R polypeptide encoded by any of the isolated nucleic acid
sequences hereinabove identified. In one aspect, the invention
provides isolated native sequence GLM-R polypeptide, which in
certain embodiments, includes an amino acid sequence comprising
residues from about 1 or about 20 to about 732, inclusive, of FIG.
2 (SEQ ID NO:2).
[0024] In yet another aspect, the invention provides an isolated
GLM-R polypeptide, comprising an amino acid sequence having at
least about 80% amino acid sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence
identity to the sequence of amino acid residues from about 1 or
about 20 to about 732, inclusive, of FIG. 2 (SEQ ID NO:2).
[0025] In yet another aspect, the invention provides an isolated
GLM-R polypeptide comprising an amino acid sequence having at least
about 80% amino acid sequence identity, alternatively at least
about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to
an amino acid sequence encoded by the human protein cDNA deposited
with the ATCC on May 16, 2000, under ATCC Deposit No. 1874-PTA
(DNA173920-2924). In a particular embodiment, the isolated GLM-R
polypeptide comprises an amino acid sequence encoding by the human
protein cDNA deposited with the ATCC on May 16, 2000 under ATCC
Deposit No. 1874-PTA (DNA 173920-2924).
[0026] In yet another aspect, the isolated GLM-R polypeptide
comprises a polypeptide without the N-terminal signal sequence
and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino acid sequence as hereinbefore
described. Processes for producing the same are also herein
described, wherein those processes comprise culturing a host cell
comprising a vector which comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of
the GLM-R polypeptide and recovering the GLM-R polypeptide from the
cell culture.
[0027] In yet another aspect, the invention provides an isolated
GLM-R polypeptide that is either transmembrane domain-deleted or
transmembrane domain-inactivated. Processes for producing the same
are also herein described, wherein those processes comprise
culturing a host cell comprising a vector comprising the
appropriate encoding nucleic acid molecule under conditions
suitable for expression of the GLM-R polypeptide and recovering the
GLM-R polypeptide from the cell culture.
[0028] In a specific aspect, the invention provides an isolated
soluble GLM-R polypeptide comprising an amino acid sequence having
at least about 80% amino acid sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity
to amino acids 1 to X of FIG. 2 (SEQ ID NO:2), where X is any amino
acid from 510 to 519 of FIG. 2 (SEQ ID NO:2). In a specific aspect,
the isolated soluble GLM-R polypeptide comprises amino acids 1 to X
of FIG. 2 (SEQ ID NO:2), where X is any amino acid from 510 to 519
of FIG. 2 (SEQ ID NO:2).
[0029] In yet another aspect, the isolated GLM-R polypeptide is a
polypeptide comprising the sequence of amino acid residues from
about 1 or about 20 to about 732, inclusive, of FIG. 2 (SEQ ID
NO:2), inclusive, of FIG. 2, or a fragment thereof which is
biologically active or sufficient to provide a binding site for an
anti-GLM-R antibody, wherein the identification of GLM-R
polypeptide fragments that possess biological activity or provide a
binding site for an anti-GLM-R antibody may be accomplished in a
routine manner using techniques which are well known in the art.
Preferably, the GLM-R fragment retains a qualitative biological
activity of a native GLM-R polypeptide, including the affect the
development or function of monocytes or macrophages.
[0030] In yet another aspect, the isolated GLM-R polypeptide is a
polypeptide produced by (1) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a GLM-R
polypeptide having the sequence of amino acid residues from about 1
or about 20 to about 732, inclusive, of FIG. 2 (SEQ ID NO:2), (b)
the complement of the DNA molecule of (a), and if the test DNA
molecule has at least about an 80% sequence identity, alternatively
at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleic acid sequence
identity to (a) or (b), (2) culturing a host cell comprising the
test DNA molecule under conditions suitable for expression of the
polypeptide, and (3) recovering the polypeptide from the cell
culture.
[0031] In yet another embodiment, the invention provides chimeric
molecules comprising an GLM-R polypeptide fused to a heterologous
polypeptide or amino acid sequence, wherein the GLM-R polypeptide
may comprise any GLM-R polypeptide, variant or fragment thereof as
hereinbefore described. An example of such a chimeric molecule
comprises a GLM-R polypeptide fused to an epitope tag sequence or
an Fc region of an immunoglobulin.
[0032] In another embodiment, the invention provides an antibody as
defined below which specifically binds to a GLM-R polypeptide as
hereinbefore described. Optionally, the antibody is a monoclonal
antibody, an antibody fragment or a single chain antibody.
[0033] In yet another embodiment, the invention provides a method
of identifying agonists or antagonists to a GLM-R polypeptide which
comprises contacting the GLM-R polypeptide with a candidate
molecule and monitoring a biological activity mediated by said
GLM-R polypeptide. In a particular aspect, the GLM-R polypeptide is
a native sequence GLM-R polypeptide.
[0034] In yet another embodiment, the invention provides a
composition of matter comprising a GLM-R polypeptide, or an agonist
or antagonist of a GLM-R polypeptide as herein described, or an
anti-GLM-R antibody, in combination with a carrier. Optionally, the
carrier is a pharmaceutically acceptable carrier.
[0035] In yet another embodiment, the invention provides a use of a
GLM-R polypeptide, or an agonist or antagonist thereof as herein
described, or an anti-GLM-R antibody, for the preparation of a
medicament useful in the treatment of a condition which is
responsive to the GLM-R polypeptide, an agonist or antagonist
thereof or an anti-GLM-R antibody.
[0036] In yet another embodiment, the invention provides a method
of screening for a bioactive agent capable of binding to GLM-R. In
one aspect, the method comprises adding a candidate bioactive agent
to a sample of GLM-R and determining the binding of said candidate
agent to said GLM-R, wherein binding indicates a bioactive agent
capable of binding to GLM-R.
[0037] In yet another embodiment, the invention provides a method
of screening for a bioactive agent capable of modulating the
activity of GLM-R. In one aspect, the method comprises the steps of
adding a candidate bioactive agent to a sample of GLM-R and
determining an alteration in the biological activity of GLM-R,
wherein an alteration indicates a bioactive agent capable of
modulating the activity of GLM-R. In a particular aspect, GLM-R
activity is the activation of STAT-3 or STAT-5 on peripheral blood
mononuclear cells (PBMC).
[0038] In yet another embodiment, the invention provides a method
of identifying a receptor for GLM-R. In one aspect, the method
comprises combining GLM-R with a composition comprising cell
membrane material wherein said GLM-R complexes with a receptor on
said cell membrane material, and identifying said receptor as a
GLM-R receptor. In one aspect, the method includes a step of
crosslinking said GLM-R and receptor. The cell membrane can be from
an intact cell or a cell membrane extract preparation.
[0039] In yet another embodiment, a method is provided for the
activation of STAT-3 or STAT-5 in cells. In one aspect, the method
comprises administering GLM-R to cells in at least an amount
effective to induce activation of STAT-3 or STAT-5.
[0040] In yet another embodiment, a method is provided for the
regulation of the development and function of monocytes or
macrophages. In one aspect, the method comprises administering
GLM-R to cells in at least an amount effective to affect the
development and differentiation of monocytes or macrophages.
[0041] In yet another embodiment, the invention provides cellular
and non-cellular assays to identify compounds that interact with
GLM-R polynucleotide and/or GLM-R polypeptide. In a particular
aspect, the cell-based assays of the invention utilize cells, cell
lines, or engineered cells or cell lines that express the GLM-R
polypeptide.
[0042] In yet another embodiment, the invention provides a method
for identifying a compound which modulates the expression of the
mammalian GLM-R polynucleotide and/or its level of biological
activity. In one aspect, the method comprises: [0043] (a)
contacting a compound to a cell that expresses a GLM-R
polynucleotide; [0044] (b) measuring the level of GLM-R DNA
expression in the cell; and [0045] (c) comparing the level obtained
in (b) to GLM-R expression level obtained in the absence of the
compound; such that if the level obtained in (b) differs from that
obtained in the absence of the compound, a compound that modulates
GLM-R activity is identified.
[0046] In yet another embodiment, the invention provides a method
for identifying compounds which modulates the biological activity
of a GLM-R polypeptide, comprising: [0047] (a) contacting a
compound to a cell that contains a GLM-R polypeptide; [0048] (b)
measuring the level of GLM-R polypeptide or activity in the cell;
and [0049] (c) comparing the level obtained in (b) to the level of
GLM-R polypeptide or activity obtained in the absence of the
compound; such that if the level obtained in (b) differs from that
obtained in the absence of the compound, a compound that modulates
a GLM-R activity is identified.
[0050] In yet another embodiment, the invention provides a method
for identifying compounds which modulate the biological activity of
a GLM-R polypeptide, comprising: [0051] (a) administering a
compound to a host (e.g., transgenic animal that expresses a GLM-R
transgene); [0052] (b) measuring the level of GLM-R gene
transcription, GLM-R expression or activity of GLM-R activity; and
[0053] (c) comparing the level obtained in (b) to the level present
in the absence of the compound; such that if the level in (b)
differs from that obtained in the absence of the compound, a
compound that modulates a GLM-R activity is identified.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) of a cDNA
encoding a nucleotide sequence (nucleotides 63-2258) encoding
native sequence GLM-R, wherein the nucleotide sequence (SEQ ID
NO:1) is a clone designated herein as "DNA173920-2924". Also
presented in bold font and underline are the positions of the
respective start and stop codons.
[0055] FIG. 2 shows the amino acid sequence (SEQ ID NO:2) of a
native sequence GLM-R (PRO21073) polypeptide as derived from the
coding sequence of SEQ ID NO:1. Also shown are the approximate
locations of various other important polypeptide domains.
[0056] FIGS. 3A-3C. FIG. 3A is a schematic to-scale representation
of the domain structure of GLM-R. A block box labeled "S"
represents the signal peptide. The cytokine receptor homology
domain is depicted as a pair of oval shapes. The positions of four
conserved cysteine residues and the WSDWS signature motif are
indicated. Three repeats of a Fibronectin type III (FNIII) repeat
complete the extracellular domain, and a block box labeled "tm"
represents the transmembrane domain. FIG. 3B is a graphical
alignment of human (SEQ ID NO:2) and murine (SEQ ID NO:16) GLM-R
protein sequences. Identical amino acid residues are shaded.
Predicted disulfide bridges are indicated by lines, and the WSXWS
motif, transmembrane domain, and box 1 motif are boxed. The open
arrowheads show the positions of introns, which were found to be
conserved in both species by analysis of genomic sequences (Celara
genomic databases and contig NT.sub.--016864.7, NCBI Annotation
Project, National Center for Biotechnology Information, NIH,
Bethesda, Md. 20894, USA). Cytoplasmic tyrosine residues are
printed in boldface. FIG. 3C shows the homology and chromosomal
localizations of GLM-R and related cytokine receptors. The
percentage of amino acid identity was calculated by the align
program.
[0057] FIG. 4 is an expression pattern of GLM-R by Taqman.TM. and
FACS. In panels A, B, D and E, GLM-R mRNA expression levels are
given as arbitrary units calculated from the expression of GLM-R
mRNA and expression of a housekeeping gene mRNA, rpl-19. FIG. 4A
shows the tissue distribution of GLM-R transcripts in human organs.
FIG. 4B shows expression of GLM-R in sorted human blood cells. FIG.
4C shows the detection of GLM-R expression by FACS on human blood
cells. Freshly isolated PBMC were double stained with biotinylated
antibodies and streptavidin-conjugated phycoerythrin (PE) in
combination with marker antibodies coupled to FITC or CyChrome.
Histograms are gated on cells positive for the indicated makers.
Grey histograms, staining with biotinylated isotype antibody; white
histograms, staining with biotinylated anti-GLM-R. FIG. 4D shows
the expression of GLM-R in human cell lines. FIG. 4E shows the
upregulation of GLM-R transcripts in monocytes from three healthy
volunteers upon activation with LPS/IFNg for 4 hours. (Note ND=not
detectable).
[0058] FIG. 5 depicts the results of introducing a chimeric
hGH-R/GLM-R chimeric receptor into 32D cells and proliferation
assay. FIG. 5A is a sequence proximal to the junction of the hGH
transmembrane domain and the GLM-R intracellular domain. The amino
acids predicted to be within the transmembrane region are boxed.
FIG. 5B is a FACS analysis of 32D cells overexpressing the chimeric
receptor. Cells were stained with a monoclonal anti-hGH-R antibody
or an isotype control antibody (black), followed by an FITC-coupled
goat anti-mouse antibody. FIGS. 5C and 5D depict thymidine
incorporation in response to growth hormone (5C) or WEHI-3B
conditioned medium (5D). A representative experiment is shown.
Round symbols, parental 32D cells, squares, hGH-R/GLM-R transfected
cells.
[0059] FIG. 6 shows STAT activation by an hGH-R/GLM-R chimera.
FIGS. 6A and 6B show an electrophoretic mobility shift assay using
the m67(A) and .beta.CAS (B) probes. Cells were stimulated with 10
ng/ml IL-3 or 100 ng/ml hGH, and complexes were supershifted with
polyclonal antibodies as indicated. FIG. 6C shows tyrosine
phosphorylation of STAT-3 and STAT-5. Phosphorylated proteins were
immunoprecipitated from stimulated cell lysates with
anti-phosphotyrosine antibodies and detected by western blot, using
polyclonal antibodies specific for STAT-3 and STAT-5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0060] The terms "GLM-R polypeptide", "GLM-R protein" and "GLM-R"
when used herein encompass native sequence GLM-R and GLM-R
polypeptide variants (which are further defined herein). The GLM-R
polypeptide may be isolated from a variety of sources, such as from
human tissue types or from another source, or prepared by
recombinant and/or synthetic methods. The term "GLM-R
polynucleotide" includes nucleic acids which encode the
polypeptides described in this paragraph.
[0061] A "native sequence GLM-R" comprises a polypeptide having the
same amino acid sequence as a GLM-R derived from nature. Such
native sequence GLM-R can be isolated from nature or can be
produced by recombinant and/or synthetic means. The term "native
sequence GLM-R" specifically encompasses naturally-occurring
truncated or secreted forms (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the
GLM-R. In one embodiment of the invention, the native sequence
GLM-R is a mature or full-length native sequence GLM-R comprising
amino acids 1 or about 20 to about 732, inclusive, of FIG. 2 (SEQ
ID NO:2). Also, while the GLM-R polypeptides disclosed in FIG. 2
(SEQ ID NO:2) is shown to begin with the methionine residue
designated herein as amino acid position 1, it is conceivable and
possible that another methionine residue located either upstream or
downstream from amino acid position 1 in FIG. 2 may be employed as
the starting amino acid residue for the respective GLM-R
polypeptide.
[0062] A GLM-R polypeptide "extracellular domain" or "ECD" refers
to a form of the GLM-R polypeptide which is essentially free of the
transmembrane and cytoplasmic domains. Ordinarily, a GLM-R
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of
such domains. It will be understood that any transmembrane domains
identified for the GLM-R polypeptides of the present invention are
identified pursuant to criteria routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain may vary but most likely by no more than
about 5 amino acids at either end of the domain as initially
identified herein. As such, in a specific aspect, the extracellular
domain of a GLM-R polypeptide comprises amino acids 1 or about 20
to X, wherein X is any amino acid from amino acid 510 to 519 or
FIG. 2 (SEQ ID NO:2).
[0063] The approximate location of the "signal peptides" of the
various GLM-R polypeptides disclosed herein may be shown in the
present specification and/or the accompanying figures. For example,
for the proteins encoded by DNA173920-2924 (SEQ ID NO:1), the
signal sequences are identified in FIG. 1, respectively. It is
noted, however, that the C-terminal boundary of a signal peptide
may vary, but most likely by no more than about 5 amino acids on
either side of the signal peptide C-terminal boundary as initially
identified herein, wherein the C-terminal boundary of the signal
peptide may be identified pursuant to criteria routinely employed
in the art for identifying that type of amino acid sequence element
(e.g., Nielsen et al., Prot. Eng. 10: 1-6 (1997) and von Heinje et
al., Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from
a secreted polypeptide is not entirely uniform, resulting in more
than one secreted species. These mature polypeptides, where the
signal peptide is cleaved within no more than about 5 amino acids
on either side of the C-terminal boundary of the signal peptide as
identified herein, and the polynucleotides encoding them, are
contemplated by the present invention.
[0064] "GLM-R variant polypeptide" (including "GLM-R mutant" or
"GLM-R polymorphism") means an active GLM-R polypeptide as defined
below having at least about 80% amino acid sequence identity with
the amino acid sequence of (a) 1 or about 20 to about 732,
inclusive, of FIG. 2 (SEQ ID NO:2), (b) X to 732 of GLM-R
polypeptide shown in FIG. 2 (SEQ ID NO:2), wherein X is any amino
acid from 15 to 24 of FIG. 2 (SEQ ID NO:2), (c) 1 or about 20 to X
of FIG. 2 (SEQ ID NO:2), wherein X is any amino acid from amino
acid residues 510-519 of FIG. 2 (SEQ ID NO:2), or (d) another
specifically derived fragment of the amino acid sequence shown in
FIG. 2 (SEQ ID NO:2). Such GLM-R variant polypeptides include, for
instance, GLM-R polypeptides wherein one or more amino acid
residues are added, or deleted, at the N- and/or C-terminus, as
well as within one or more internal domains, of the sequence of
FIG. 2. Ordinarily, a GLM-R variant polypeptide will have at least
about 80% amino acid sequence identity, alternatively at least
about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 98% or 99% amino acid sequence
identity with (a) 1 or about 20 to about 732, inclusive, of the
GLM-R polypeptide shown in FIG. 2 (SEQ ID NO:2), (b) X to 732 of
FIG. 2 (SEQ ID NO:2), wherein X is any amino acid from 15 to 24 of
FIG. 2 (SEQ ID NO:2), (c) 1 or about 20 to X of FIG. 2 (SEQ ID
NO:2), wherein X is any amino acid from amino acid residues 510 to
519 of FIG. 2 (SEQ ID NO:2) or (d) another specifically derived
fragment of the amino acid sequence shown in FIG. 2 (SEQ ID NO:2).
GLM-R variant polypeptides explicitly do not encompass the native
GLM-R polypeptide sequence. Ordinarily, GLM-R variant polypeptides
are at least about 10 amino acids in length, alternatively at least
about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,
420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,
550, 560, 570, 580, 590, 600 amino acids in length, or more.
[0065] "Percent (%) amino acid sequence identity" with respect to
the GLM-R polypeptide sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in a GLM-R sequence, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign
(DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full-length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are obtained as described below
by using the sequence comparison computer program ALIGN-2, wherein
the complete source code for the ALIGN-2 program is provided in
Table 1 below. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Table 1
has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Table 1.
The ALIGN-2 program should be compiled for use on a UNIX operating
system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
[0066] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. As examples of % amino acid
sequence identity calculations, Tables 2 and 3 demonstrate how to
calculate the % amino acid sequence identity of the amino acid
sequence designated "Comparison Protein" to the amino acid sequence
designated "PRO".
[0067] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described
above using the ALIGN-2 sequence comparison computer program.
However, % amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 [Altschul et al.,
Nucleic Acids Res. 25: 3389-3402 (1997]. The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0068] In situations where NCBI-BLAST2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program NCBI-BLAST2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity B to A.
[0069] "GLM-R variant polynucleotide" or "GLM-R variant nucleic
acid sequence" means a nucleic acid molecule which encodes an
active GLM-R polypeptide as defined below and which has at least
about 80% nucleic acid sequence identity with the nucleic acid
sequence encoding amino acid residues: (a) 1 or about 20 to about
732, inclusive, of the GLM-R polypeptide of FIG. 2 (SEQ ID NO:2),
(b) X to 732 of the GLM-R polypeptide of FIG. 2 (SEQ ID NO:2),
wherein X is any amino acid residues from 15 to 24 of FIG. 2 (SEQ
ID NO:2), (c) 1 or about 20 to X, wherein X is any amino acid
residue from 510 to 519 of FIG. 2 (SEQ ID NO:2), (d) another
specifically derived fragment of the amino acid sequence shown in
FIG. 2 (SEQ ID NO:2). Ordinarily, a GLM-R variant polynucleotide
will have at least about 80% nucleic acid sequence identity,
alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%
nucleic acid sequence identity with: (a) a nucleic acid sequence
which encodes residues 1 or about 20 to about 732, inclusive, of
the GLM-R polypeptide shown in FIG. 2 (SEQ ID NO:2), (b) a nucleic
acid sequence that encodes amino acids X to 732 of the GLM-R
polypeptide shown in FIG. 2 (SEQ ID NO:2), wherein X is any amino
acid residue from 15 to 24 of FIG. 2 (SEQ ID NO:2), (c) a nucleic
acid sequence that encodes amino acids 1 or about 20 to X of FIG. 2
(SEQ ID NO:2), wherein X is any amino acid from residues 510 to 519
of FIG. 2 (SEQ ID NO:2) or (d) a nucleic acid sequence which
encodes another specifically derived fragment of the amino acid
sequence shown in FIG. 2 (SEQ ID NO:2). GLM-R polynucleotide
variants do not encompass the native GLM-R nucleotide sequence.
[0070] Ordinarily, GLM-R variant polynucleotides are at least about
5 nucleotides in length, alternatively at least about 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,
390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510,
520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,
650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,
780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900,
910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in
length, wherein in this context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that
referenced length.
[0071] "Percent (%) nucleic acid sequence identity" with respect to
the GLM-R polypeptide-encoding nucleic acid sequences identified
herein is defined as the percentage of nucleotides in a candidate
sequence that are identical with the nucleotides in a GLM-R
polypeptide-encoding nucleic acid sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of
determining percent nucleic acid sequence identity can be achieved
in various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the
art can determine appropriate parameters for measuring alignment,
including any algorithms needed to achieve maximal alignment over
the full-length of the sequences being compared. For purposes
herein, however, % nucleic acid sequence identity values are
obtained as described below by using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program is provided in Table 1 below. The ALIGN-2 sequence
comparison computer program was authored by Genentech, Inc. and the
source code shown in Table 1 has been filed with user documentation
in the U.S. Copyright Office, Washington D.C., 20559, where it is
registered under U.S. Copyright Registration No. TXU510087. The
ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco, Calif. or may be compiled from the source code
provided in Table 1. The ALIGN-2 program should be compiled for use
on a UNIX operating system, preferably digital UNIX V4.0D. All
sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
[0072] For purposes herein, the % nucleic acid sequence identity of
a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can alternatively be phrased as a
given nucleic acid sequence C that has or comprises a certain %
nucleic acid sequence identity to, with, or against a given nucleic
acid sequence D) is calculated as follows: 100 times the fraction
W/Z where W is the number of nucleotides scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of C and D, and where Z is the total number of
nucleotides in D. It will be appreciated that where the length of
nucleic acid sequence C is not equal to the length of nucleic acid
sequence D, the % nucleic acid sequence identity of C to D will not
equal the % nucleic acid sequence identity of D to C. As examples
of % nucleic acid sequence identity calculations, Tables 4 and 5
demonstrate how to calculate the % nucleic acid sequence identity
of the nucleic acid sequence designated "Comparison DNA" to the
nucleic acid sequence designated "PRO-DNA". Unless specifically
stated otherwise, all % nucleic acid sequence identity values used
herein are obtained as described above using the ALIGN-2 sequence
comparison computer program.
[0073] Unless specifically stated otherwise, all % nucleic acid
sequence identity values used herein are obtained as described
herein using the ALIGN-2 sequence comparison computer program.
However, % nucleic acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 [Altschul et al.,
Nucleic Acids Res. 25: 3389-3402 (1997)]. The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov or otherwise obtained from the National
Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0074] In situations where NCBI-BLAST2 is employed for sequence
comparisons, the % nucleic acid sequence identity of a given
nucleic acid sequence C to, with or against a given nucleic acid
comprises a certain % nucleic acid sequence identity to, with or
against a given nucleic acid sequence D) is calculated as follows:
100 times the fraction W/Z where W is the number of nucleotides
scored as identical matches by the sequence alignment program
NCBI-BLAST2 in that program's alignment of C and D, and where Z is
the total number of nucleotides in D. It will be appreciated that
where the length of nucleic acid sequence C is not equal to the
length of nucleic acid sequence D, the % nucleic acid sequence
identity of C to D will not equal the % nucleic acid sequence
identity of D to C.
[0075] In other embodiments, GLM-R variant polynucleotides are
nucleic acid molecules that encode an active GLM-R polypeptide and
which are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to nucleotide sequences encoding
the full-length GLM-R polypeptide shown in FIG. 2 (SEQ ID NO:2).
GLM-R variant polypeptides may be those that are encoded by a GLM-R
variant polynucleotide.
[0076] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Preferably, the isolated polypeptide is free of
association with all components with which it is naturally
associated. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the GLM-R
natural environment will not be present. Ordinarily, however,
isolated polypeptide will be prepared by at least one purification
step.
[0077] An "isolated" nucleic acid molecule encoding a GLM-R
polypeptide is a nucleic acid molecule that is identified and
separated from at least one contaninant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
GLM-R-encoding nucleic acid. Preferably, the isolated nucleic is
free of association with all components with which it is naturally
associated. An isolated GLM-R-encoding nucleic acid molecule is
other than in the form or setting in which it is found in nature.
Isolated nucleic acid molecules therefore are distinguished from
the GLM-R-encoding nucleic acid molecule as it exists in natural
cells. However, an isolated nucleic acid molecule encoding a GLM-R
polypeptide includes GLM-R-encoding nucleic acid molecules
contained in cells that ordinarily express GLM-R where, for
example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0078] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0079] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0080] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-GLM-R monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-GLM-R antibody compositions with polyepitopic
specificity, single chain anti-GLM-R antibodies, and fragments of
anti-GLM-R antibodies (see below). The term "monoclonal antibody"
as used herein refers to an antibody obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally-occurring mutations that may be present in minor
amounts.
[0081] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0082] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0083] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0084] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a GLM-R polypeptide fused to a "tag
polypeptide". The tag polypeptide has enough residues to provide an
epitope against which an antibody can be made, yet is short enough
such that it does not interfere with activity of the polypeptide to
which it is fused. The tag polypeptide preferably also is fairly
unique so that the antibody does not substantially cross-react with
other epitopes. Suitable tag polypeptides generally have at least
six amino acid residues and usually between about 8 and 50 amino
acid residues (preferably, between about 10 and 20 amino acid
residues).
[0085] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0086] "Active" or "activity" for the purposes herein refers to
form(s) of GLM-R which retain a biological and/or an immunological
activity of native or naturally-occurring GLM-R, wherein
"biological" activity refers to a biological function (either
inhibitory or stimulatory) caused by a native or
naturally-occurring GLM-R other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring GLM-R and an "immunological"
activity refers to the ability to induce the production of an
antibody against an antigenic epitope possessed by a native or
naturally-occurring GLM-R. A preferred biological activity includes
any one or more of the following activities: decreased body weight,
decreased adiposity (e.g., fat/body weight ratio), increased lean
muscle mass. Alternative definitions of biological activity include
the ability to activate STAT-3 or STAT-5.
[0087] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits or
neutralizes a biological activity of a native GLM-R polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native GLM-R polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native GLM-R polypeptides,
peptides, small organic molecules, etc. Methods for identifying
agonists or antagonists of a GLM-R polypeptide may comprise
contacting a GLM-R polypeptide with a candidate agonist or
antagonist molecule and measuring a detectable change in one or
more biological activities normally associated with the GLM-R
polypeptide.
[0088] It is understood that some of the activities of GLM-R are
directly induced by GLM-R and some are indirectly induced, however,
each are the result of the presence of GLM-R and would not
otherwise have the result in the absence of GLM-R.
[0089] "Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology of a
disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) the targeted
pathological condition or disorder. Individuals in need of
treatment include those already with the disorder as well as those
in which the disorder is to be prevented.
[0090] The term "effective amount" is at least the minimum
concentration of GLM-R which causes, induces or results in either a
detectable improvement in an in vitro cell-based model of a body
weight disorder. For example, decreased glucose uptake into
adipocytes, increased leptin release from adipocytes, etc.
Furthermore, a "therapeutically effective amount" is at least the
minimum concentration (amount) of GLM-R administered to a mammal
which would be effective in at least attenuating or improving a
pathological symptom associated with a body weight disorder. For
example, decreased body weight, decreased fat/body weight ratio,
increase lean muscle mass/body weight ratio, increased metabolic
rate, decreased serum triglycerides or fatty acids, etc.
[0091] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0092] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, goats, rabbits, ferrets, etc.
Preferably, the mammal is human.
[0093] "Individual" is any subject patient, preferably a mammal,
more preferably a human.
[0094] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0095] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native GLM-R polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native GLM-R polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native GLM-R polypeptides,
peptides, small organic molecules, etc. Methods for identifying
agonists or antagonists of a GLM-R polypeptide may comprise
contacting a GLM-R polypeptide with a candidate agonist or
antagonist molecule and measuring a detectable change in one or
more biological activities normally associated with the GLM-R
polypeptide.
[0096] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.RTM., polyethylene glycol (PEG), and PLURONICS.RTM..
[0097] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies
(Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0098] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0099] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the VH-VL
dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three CDRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0100] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0101] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa and lambda, based on the amino acid sequences
of their constant domains.
[0102] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0103] "Single-chain Fv" or "sFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Preferably, the Fv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the sFv to form the desired structure for antigen binding.
For a review of sFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
[0104] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO
93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993).
[0105] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0106] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope.
[0107] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0108] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0109] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a GLM-R polypeptide or antibody
thereto) to a mammal. The components of the liposome are commonly
arranged in a bilayer formation, similar to the lipid arrangement
of biological membranes.
[0110] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons. TABLE-US-00001 TABLE 2 GLM-R
XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison XXXXXYYYYYYY
(Length = 12 amino acids) Protein % amino acid sequence identity =
(the number of identically matching amino acid residues between the
two polypeptide sequences as determined by ALIGN-2) divided by (the
total number of amino acid residues of the GLM-R polypeptide) = 5
divided by 15 = 33.3%
[0111] TABLE-US-00002 TABLE 3 GLM-R XXXXXXXXXX (Length = 10 amino
acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein
% amino acid sequence identity = (the number of identically
matching amino acid residues between the two polypeptide sequences
as determined by ALIGN-2) divided by (the total number of amino
acid residues of the GLM-R polypeptide) = 5 divided by 10 = 50%
[0112] TABLE-US-00003 TABLE 4 GLM-R-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the GLM-R-DNA nucleic acid sequence) = 6 divided by 14 =
42.9%
[0113] TABLE-US-00004 TABLE 5 GLM-R-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the GLM-R-DNA nucleic acid sequence) = 4 divided by 12 =
33.3%
II. Compositions and Methods of the Invention
[0114] The present invention describes a novel molecule that
displays the typical architecture and structural features of type I
receptors. It shares significant homology to known members of this
receptor family, most notably gp130 and GCSF-R, and is found in
close physical proximity to gp 130 on human chromosome 5 and mouse
chromosome 13.
[0115] GLM-R was found to be expressed predominantly on activated
monocytes. In support of this finding, GLM-R was expressed in two
monocytic cell lines, TMP-1 and U937, but not in a number of other
cell lines of lyphoid and myeloid origin. Furthermore, strong
induction of GLM-R upon stimulation with LPS and IFN-.gamma. was
seen in monocytes and in the two cell lines (not shown). Together,
these expression data suggest that monocytes and possibly
macrophages are a likely site of physiologic activity of this
receptor, and prompt further analysis of its function in those
cells. Expression in monocytes is accounts for the elevated GLM-R
levels detected in thymus and bone marrow. On the other hand,
presence of GLM-R in testis and prostate suggests that it was
additional functions outside the immune system
[0116] The capacity of GLM-R to signal was examined by fusing its
intracellular domain to the ligand binding domain of hGH-R, a
receptor that is well known to homodimerize upon stimulation with
hGH. The resulting chimeric molecule was able to transduce a
proliferative signal into myeloid 32D cells, and caused activation
of the transcription factors STAT-3 and, to a lesser extent,
STAT-5. Thus, GLM-R is capable of signaling, but it remains to be
determined whether its extracellular domain binds a ligand by
itself, or whether additional receptor subunits are required to
form a functional receptor. Using proliferation of 32D cells
transfected with the full length human molecule as a readout, we
found that GLM-R is not a sufficient receptor for IL-2, -3, -4, -5,
-6, -7, -9, -11, -12, -13, -15, -23, GCSF, GM-CSF, EPO, TPO, PRL,
GH, OSM, CT-1 and OB (not shown). These cells can now be used to
screen a variety of sources for a GLM-R ligand, but such a strategy
will only be successful if GLM-R can act as a homodimer, or if
necessary accessory chains are endogenously expressed in 32D
cells.
[0117] GLM-R preferentially activated STAT-3, while STAT-5
activation was low but nonetheless detectable. These two proteins
were shown to have very different functions in myeloid cells.
Enforced expression of constitutively active STAT-5a or STAT-5b
resulted in factor independence and myeloid differentiation of BaF3
cells [Nosaka et al., Embo J. 18(17): 4754-65 (1999)], and
macrophage differentiation of Ml cells [Kawashima et al., J.
Immunol. 167(7):3652-60 (2001)], while macrophages deficient in
STAT-5a displayed a defect in GM-CSF induced proliferation and gene
expression. Feldman et al., Blood 90(5): 1768-76 (1997). Moreover,
repopulation of all blood cell lineages, including monocytes, was
severely compromised when STAT-5a.sup.-/-5b.sup.-/- bone marrow
cells instead of wild-type cells were sued as a graft to rescue
lethally irradiated animals. Bunting et al., Blood 99(2): 479-487
(2002). Together, these data suggest that STAT-5 plays an important
role in the development and proliferation of monocytes/macrophages.
On the other hand, STAT-3 appears to be involved in the negative
regulation of macrophage activation, a function mainly exerted by
IL-10. Riley et al., J. Biol. Chem. 274(23), 16513-16521 (1999),
O'Farrell et al., Embo. J. 17(4): 1006-18 (1998). In a mouse model
in which STAT-3 was deleted in a tissue specific fashion in
macrophages and neutrophils, macrophages were constitutively
activated, which led to chronic enterocolitis through activation of
Th1 cells in vivo [Takeda, K. et al., Immunity 10(1): 39-49
(1999)], a phenotype that is mimicked by IL-10 deficient mice. Kuhn
et al., Cell 75(2): 263-74 (1993).
[0118] Taken together, our data suggests that GLM-R is a receptor
for a yet unknown helical cytokine that likely acts on monocytes
and possibly also macrophages. Using the receptor as a tool, it
will hopefully be possible to identify this ligand, which is
critical in order to further understand the biological function of
GLM-R.
[0119] A. Full-length GLM-R Polypeptide
[0120] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as GLM-R (alternatively PRO21073 or UNQ6368).
In particular, cDNA encoding a GLM-R polypeptide has been
identified and isolated, as disclosed in further detail in the
Examples below. It is noted that proteins produced in separate
expression rounds may be given different PRO numbers but the UNQ
number is unique for any given DNA and the encoded protein, and
will not be changed. However, for sake of simplicity, in the
present specification the protein encoded by DNA173920-2924 as well
as all further native homologues and variants included in the
foregoing definition of GLM-R (also sometimes referred to as
PRO21073), will be referred to as "GLM-R", regardless of their
origin or mode of preparation.
[0121] As disclosed in the Examples below, cDNA clones designated
herein as DNA173920-2924 have been deposited with the ATCC. The
actual nucleotide sequence of the clones can readily be determined
by the skilled artisan by sequencing of the deposited clones using
routine methods in the art. The predicted amino acid sequence can
be determined from the nucleotide sequence using routine skill. For
the GLM-R polypeptides and encoding nucleic acid described herein,
Applicants have identified what is believed to be the reading frame
best identifiable with the sequence information available at the
time.
[0122] B. GLM-R Variants
[0123] In addition to the full-length native sequence GLM-R
polypeptides described herein, it is contemplated that GLM-R
variants can be prepared. GLM-R variants can be prepared by
introducing appropriate nucleotide changes into the GLM-R DNA,
and/or by synthesis of the desired GLM-R polypeptide. Those skilled
in the art will appreciate that amino acid changes may alter
post-translational processes of the GLM-R, such as changing the
number or position of glycosylation sites or altering the membrane
anchoring characteristics.
[0124] Variations in the native full-length sequence GLM-R or in
various domains of the GLM-R described herein, can be made, for
example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the GLM-R that results in a change in the amino acid sequence of
the GLM-R as compared with the native sequence GLM-R. Optionally
the variation is by substitution of at least one amino acid with
any other amino acid in one or more of the domains of the GLM-R.
Guidance in determining which amino acid residue may be inserted,
substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the GLM-R with
that of homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high
homology. Amino acid substitutions can be the result of replacing
one amino acid with another amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine
with a serine, i.e., conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1
to 5 amino acids. The variation allowed may be determined by
systematically making insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for
activity exhibited by the full-length or mature native
sequence.
[0125] GLM-R polypeptide fragments are provided herein. Such
fragments may be truncated at the N-terminus or C-terminus, or may
lack internal residues, for example, when compared with a full
length native protein. Certain fragments lack amino acid residues
that are not essential for a desired biological activity of the
GLM-R polypeptide.
[0126] GLM-R fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
GLM-R fragments by enzymatic digestion, e.g., by treating the
protein with an enzyme known to cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with
suitable restriction enzymes and isolating the desired fragment.
Yet another suitable technique involves isolating and amplifying a
DNA fragment encoding a desired polypeptide fragment, by polymerase
chain reaction (PCR). Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers
in the PCR. Preferably, GLM-R polypeptide fragments share at least
one biological and/or immunological activity with the native GLM-R
polypeptide shown in FIG. 2 (SEQ ID NO:2).
[0127] In particular embodiments, conservative substitutions of
interest are shown in Table 6 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are introduced and the products
screened. TABLE-US-00005 TABLE 6 Original Exemplary Preferred
Residue Substitutions Substitutions Ala (A) val; leu; ile val Arg
(R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu
glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro;
ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala;
phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile
Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu;
val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser
ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V)
ile; leu; met; phe; ala; norleucine leu
[0128] Substantial modifications in function or immunological
identity of the GLM-R polypeptide are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
[0129] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0130] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the GLM-R variant DNA.
[0131] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0132] C. Modifications of GLM-R
[0133] Covalent modifications of GLM-R are included within the
scope of this invention. One type of covalent modification includes
reacting targeted amino acid residues of GLM-R polypeptides with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the GLM-R.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking GLM-R to a water-insoluble support matrix or
surface for use in the method for purifying anti-GLM-R antibodies,
and vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0134] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0135] Another type of covalent modification of the GLM-R
polypeptide included within the scope of this invention comprises
altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence GLM-R (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence GLM-R. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0136] Addition of glycosylation sites to the GLM-R polypeptide may
be accomplished by altering the amino acid sequence. The alteration
may be made, for example, by the addition of, or substitution by,
one or more serine or threonine residues to the native sequence
GLM-R (for O-linked glycosylation sites). The GLM-R amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the GLM-R
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0137] Another means of increasing the number of carbohydrate
moieties on the GLM-R polypeptide is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0138] Removal of carbohydrate moieties present on the GLM-R
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys, 259:52 (1987) and by
Edge et al., Anal. Biochem 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0139] Another type of covalent modification of GLM-R comprises
linking the GLM-R polypeptide to one of a variety of
nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0140] The GLM-R of the present invention may also be modified in a
way to form a chimeric molecule comprising GLM-R fused to another,
heterologous polypeptide or amino acid sequence.
[0141] In one embodiment, such a chimeric molecule comprises a
fusion of the GLM-R with a tag polypeptide which provides an
epitope to which an anti-tag antibody can selectively bind. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the GLM-R. The presence of such epitope-tagged forms of the
GLM-R can be detected using an antibody against the tag
polypeptide. Also, provision of the epitope tag enables the GLM-R
to be readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al, Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0142] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the GLM-R with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule (also referred to as an "immunoadhesin"), such a
fusion could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a GLM-R polypeptide in place
of at least one variable region within an Ig molecule. In a
particularly preferred embodiment, the immunoglobulin fusion
includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3
regions of an IgG1 molecule. For the production of immunoglobulin
fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0143] D. Preparation of GLM-R
[0144] The description below relates primarily to production of
GLM-R by culturing cells transformed or transfected with a vector
containing GLM-R nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed to prepare GLM-R. For instance, the GLM-R sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
GLM-R may be chemically synthesized separately and combined using
chemical or enzymatic methods to produce the full-length GLM-R.
1. Isolation of DNA Encoding GLM-R
[0145] DNA encoding GLM-R may be obtained from a cDNA library
prepared from tissue believed to possess the GLM-R mRNA and to
express it at a detectable level. Accordingly, human GLM-R DNA can
be conveniently obtained from a cDNA library prepared from human
tissue, such as described in the Examples. The GLM-R-encoding gene
may also be obtained from a genomic library or by known synthetic
procedures (e.g., automated nucleic acid synthesis).
[0146] Libraries can be screened with probes (such as antibodies to
the GLM-R or oligonucleotides of at least about 20-80 bases)
designed to identify the gene of interest or the protein encoded by
it. Screening the cDNA or genomic library with the selected probe
may be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding GLM-R is to use PCR methodology [Sambrook
et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 1995)].
[0147] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0148] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0149] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
[0150] Host cells are transfected or transformed with expression or
cloning vectors described herein for GLM-R production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: A
Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook
et al., supra.
[0151] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al, J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al, Methods in
Enzymology 185:527-537 (1990) and Mansour et al, Nature,
336:348-352 (1988).
[0152] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0153] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for GLM-R-encoding vectors. Saccharomyces cerevisiae is a commonly
used lower eukaryotic host microorganism. Others include
Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 [1981];
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat.
No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such
as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al.,
J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424),
K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221
[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not
limited to, yeast capable of growth on methanol selected from the
genera consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry of Methylotrophs 269 (1982).
[0154] Suitable host cells for the expression of glycosylated GLM-R
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
3. Selection and Use of a Replicable Vector
[0155] The nucleic acid (e.g., cDNA or genomic DNA) encoding GLM-R
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. Various vectors are publicly
available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0156] The GLM-R may be produced recombinantly not only directly,
but also as a fusion polypeptide with a heterologous polypeptide,
which may be a signal sequence or other polypeptide having a
specific cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the GLM-R-encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published 4 Apr. 1990), or the signal described in WO 90/13646
published 15 Nov. 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0157] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0158] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0159] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the GLM-R-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line deficient in DHFR activity, prepared
and propagated as described by Urlaub et al., Proc. Natl. Acad.
Sci. USA, 77:4216 (1980). A suitable selection gene for use in
yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene
7:141 (1979); Tschemper et al., Gene 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1 [Jones, Genetics 85:12 (1977)].
[0160] Expression and cloning vectors usually contain a promoter
operably linked to the GLM-R-encoding nucleic acid sequence to
direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well known. Promoters suitable for use
with prokaryotic hosts include the .beta.-lactamase and lactose
promoter systems [Chang et al., Nature 275:615 (1978); Goeddel et
al., Nature 281:544 (1979)], alkaline phosphatase, a tryptophan
(trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid promoters such as the tac promoter [deBoer
et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for
use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding GLM-R.
[0161] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0162] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0163] GLM-R transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0164] Transcription of a DNA encoding the GLM-R by higher
eukaryotes may be increased by inserting an enhancer sequence into
the vector. Enhancers are cis-acting elements of DNA, usually about
from 10 to 300 bp, that act on a promoter to increase its
transcription. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, .alpha.-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples include the SV40 enhancer on the
late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus enhancers.
The enhancer may be spliced into the vector at a position 5' or 3'
to the GLM-R coding sequence, but is preferably located at a site
5' from the promoter.
[0165] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
GLM-R.
[0166] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of GLM-R in recombinant vertebrate cell
culture are described in Gething et al., Nature 293:620-625 (1981);
Mantei et al., Nature 281:40-46 (1979); EP 117,060; and EP
117,058.
4. Detecting Gene Amplification/Expression
[0167] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0168] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence GLM-R polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to GLM-R DNA and encoding a specific antibody
epitope.
5. Purification of Polypeptide
[0169] Forms of GLM-R may be recovered from culture medium or from
host cell lysates. If membrane-bound, it can be released from the
membrane using a suitable detergent solution (e.g. Triton-X.RTM.
100) or by enzymatic cleavage. Cells employed in expression of
GLM-R can be disrupted by various physical or chemical means, such
as freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents.
[0170] It may be desired to purify GLM-R from recombinant cell
proteins or polypeptides. The following procedures are exemplary of
suitable purification procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms of the GLM-R.
Various methods of protein purification may be employed and such
methods are known in the art and described for example in
Deutscher, Methods in Enzymology 182 (1990); Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular
GLM-R produced.
[0171] E. Uses for GLM-R
[0172] Nucleotide sequences (or their complement) encoding GLM-R
have various applications in the art of molecular biology,
including uses as hybridization probes, in chromosome and gene
mapping and in the generation of anti-sense RNA and DNA. GLM-R
nucleic acid will also be useful for the preparation of GLM-R
polypeptides by the recombinant techniques described herein.
[0173] The full-length native sequence GLM-R DNA (SEQ ID NO:1), or
portions thereof, may be used as hybridization probes for a cDNA
library to isolate the full-length GLM-R cDNA or to isolate still
other cDNAs (for instance, those encoding naturally-occurring
variants of GLM-R or GLM-R from other species) which have a desired
sequence identity to the GLM-R sequence disclosed in FIG. 1 (SEQ ID
NO:1). Optionally, the length of the probes will be about 20 to
about 50 bases. The hybridization probes may be derived from at
least partially novel regions of the nucleotide sequence of SEQ ID
NO:1 wherein such regions may be determined without undue
experimentation or from genomic sequences including promoters,
enhancer elements and introns of native sequence GLM-R. By way of
example, a screening method may comprise isolating the coding
region of the GLM-R gene using the known DNA sequence to synthesize
a selected probe of about 40 bases. Hybridization probes may be
labeled by a variety of labels, including radionucleotides such as
.sup.32P or .sup.35S, or enzymatic labels such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling
systems. Labeled probes having a sequence complementary to that of
the GLM-R gene of the present invention can be used to screen
libraries of human cDNA, genomic DNA or mRNA to determine which
members of such libraries the probe hybridizes to. Hybridization
techniques are described in further detail in the Examples
below.
[0174] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0175] Other useful fragments of the GLM-R nucleic acids include
antisense or sense oligonucleotides comprising a singe-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target GLM-R mRNA (sense) or GLM-R DNA (antisense) sequences.
Antisense or sense oligonucleotides, according to the present
invention, comprise a fragment of the coding region of GLM-R DNA.
Such a fragment generally comprises at least about 14 nucleotides,
preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense oligonucleotide, based upon a cDNA sequence
encoding a given protein is described in, for example, Stein and
Cohen, Cancer Res. 48:2659 (1988) and van der Krol et al.,
BioTechniques 6:958 (1988).
[0176] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. The antisense oligonucleotides thus may be used to block
expression of GLM-R proteins. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0177] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10048, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0178] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0179] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0180] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0181] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
GLM-R coding sequences.
[0182] Nucleotide sequences encoding a GLM-R can also be used to
construct hybridization probes for mapping the gene which encodes
that GLM-R and for the genetic analysis of individuals with genetic
disorders. The nucleotide sequences provided herein may be mapped
to a chromosome and specific regions of a chromosome using known
techniques, such as in situ hybridization, linkage analysis against
known chromosomal markers, and hybridization screening with
libraries.
[0183] When the coding sequences for GLM-R encode a protein which
binds to another protein (for example, where the GLM-R is a
receptor or co-ligand), the GLM-R can be used in assays to identify
the other proteins or molecules involved in the binding
interaction. By such methods, inhibitors of the receptor/ligand
binding interaction can be identified. Proteins involved in such
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding interaction.
Also, the receptor GLM-R can be used to isolate correlative
ligand(s). Screening assays can be designed to find lead compounds
that mimic the biological activity of a native GLM-R or a receptor
for GLM-R. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them
particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are
well characterized in the art.
[0184] Nucleic acids which encode GLM-R or its modified forms can
also be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. A transgenic animal (e.g., a
mouse or rat) is an animal having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of
the animal at a prenatal, e.g., an embryonic stage. A transgene is
a DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, cDNA encoding GLM-R
can be used to clone genomic DNA encoding GLM-R in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
GLM-R. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for GLM-R
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding GLM-R
introduced into the germ line of the animal at an embryonic stage
can be used to examine the effect of increased expression of DNA
encoding GLM-R. Such animals can be used as tester animals for
reagents thought to confer protection from, for example,
pathological conditions associated with its overexpression. In
accordance with this facet of the invention, an animal is treated
with the reagent and a reduced incidence of the pathological
condition, compared to untreated animals bearing the transgene,
would indicate a potential therapeutic intervention for the
pathological condition.
[0185] Alternatively, non-human homologues of GLM-R can be used to
construct a GLM-R "knock out" animal which has a defective or
altered DNA encoding GLM-R as a result of homologous recombination
between the endogenous DNA encoding GLM-R and altered genomic DNA
encoding GLM-R introduced into an embryonic stem cell of the
animal. For example, cDNA encoding GLM-R can be used to clone
genomic DNA encoding GLM-R in accordance with established
techniques. A portion of the genomic DNA encoding GLM-R can be
deleted or replaced with another gene, such as a gene encoding a
selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking DNA (both at the
5' and 3' ends) are included in the vector [see e.g., Thomas and
Capecchi, Cell 51:503 (1987) for a description of homologous
recombination vectors]. The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogenous DNA
are selected [see e.g., Li et al, Cell 69:915 (1992)]. The selected
cells are then injected into a blastocyst of an animal (e.g., a
mouse or rat) to form aggregation chimeras [see e.g., Bradley, in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term to create a "knock
out" animal. Progeny harboring the homologously recombined DNA in
their germ cells can be identified by standard techniques and used
to breed animals in which all cells of the animal contain the
homologously recombined DNA. Knockout animals can be characterized
for instance, for their ability to defend against certain
pathological conditions and for their development of pathological
conditions due to absence of the GLM-R polypeptide.
[0186] Nucleic acid encoding the GLM-R polypeptides may also be
used in gene therapy. In gene therapy applications, genes are
introduced into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al, Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0187] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al, Trends in Biotechnology 11 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262 4429-4432
(1987); and Wagner et al, Proc. Natl. Acad. Sci. USA 87: 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al, Science 256: 808-813 (1992).
[0188] The GLM-R polypeptides described herein may also be employed
as molecular weight markers for protein electrophoresis
purposes.
[0189] The nucleic acid molecules encoding the GLM-R polypeptides
or fragments thereof described herein are useful for chromosome
identification. In this regard, there exists an ongoing need to
identify new chromosome markers, since relatively few chromosome
marking reagents, based upon actual sequence data are presently
available. Each GLM-R nucleic acid molecule of the present
invention can be used as a chromosome marker.
[0190] The GLM-R polypeptides and nucleic acid molecules of the
present invention may also be used for tissue typing, wherein the
GLM-R polypeptides of the present invention may be differentially
expressed in one tissue as compared to another. GLM-R nucleic acid
molecules will find use for generating probes for PCR, Northern
analysis, Southern analysis and Western analysis.
[0191] The GLM-R polypeptides and modulators thereof described
herein may also be employed as therapeutic agents. The GLM-R
polypeptides and modulators thereof of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the GLM-R product hereof is combined
in admixture with a pharmaceutically acceptable carrier vehicle.
Therapeutic formulations are prepared for storage by mixing the
active ingredient having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients or stabilizers are
nontoxic to recipients at the dosages and concentrations employed,
and include buffers such as phosphate, citrate and other organic
acids; antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides and
other carbohydrates including glucose, mannose, or dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or
sorbitol; salt-forming counterions such as sodium; and/or nonionic
surfactants such as TWEEN.RTM., PLURONICS.RTM. or PEG.
[0192] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to or following lyophilization
and reconstitution.
[0193] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0194] The route of administration is in accord with known methods,
e.g. injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by sustained
release systems.
[0195] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0196] When in vivo administration of a GLM-R polypeptide or
agonist or antagonist thereof is employed, normal dosage amounts
may vary from about 10 ng/kg to up to 100 mg/kg of mammal body
weight or more per day, preferably about 1 .mu.g/kg/day to 10
mg/kg/day, depending upon the route of administration. Guidance as
to particular dosages and methods of delivery is provided in the
literature; see, for example, U.S. Pat. Nos. 4,657,760; 5,206,344;
or 5,225,212. It is anticipated that different formulations will be
effective for different treatment compounds and different
disorders, that administration targeting one organ or tissue, for
example, may necessitate delivery in a manner different from that
to another organ or tissue.
[0197] Where sustained-release administration of a GLM-R
polypeptide or modulator is desired in a formulation with release
characteristics suitable for the treatment of any disease or
disorder requiring administration of the GLM-R polypeptide or
modulator, microencapsulation is contemplated. Microencapsulation
of recombinant proteins for sustained release has been successfully
performed with human growth hormone (rhGH), interferon-(rhIFN-),
interleukin-2, and MN rgp120. Johnson et al., Nat. Med. 2:795-799
(1996); Yasuda, Biomed. Ther. 27:1221-1223 (1993); Hora et al.,
Bio/Technology 8:755-758 (1990); Cleland, "Design and Production of
Single Immunization Vaccines Using Polylactide Polyglycolide
Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant
Approach, Powell and Newman, eds, (Plenum Press: New York, 1995),
pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.
No. 5,654,010.
[0198] The sustained-release formulations of these proteins can be
developed using poly-lactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. Lewis, "Controlled release of
bioactive agents from lactide/glycolide polymer," in: M. Chasin and
R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems
(Marcel Dekker: New York, 1990), pp. 1-41.
[0199] GLM-R and compositions comprising GLM-R are preferably used
in viva. However, as discussed below, administration can be in
vitro such as in the methods described below for screening for
modulators of GLM-R. Although, it is understood that modulators of
GLM-R can also be identified by the use of animal models and
samples from patients.
[0200] This invention encompasses methods of screening compounds to
identify those that mimic or enhance the GLM-R polypeptide
(agonists) or prevent or inhibit the effect of the GLM-R
polypeptide (antagonists). Agonists and antagonists are referred to
as modulators herein. Screening assays for antagonist drug
candidates are designed to identify compounds that bind or complex
with the GLM-R polypeptides encoded by the genes identified herein,
or otherwise interfere with the interaction of the encoded
polypeptides with other cellular proteins. Such screening assays
will include assays amenable to high-throughput screening of
chemical libraries, making them particularly suitable for
identifying small molecule drug candidates.
[0201] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0202] All assays for antagonists are common in that they call for
contacting the drug candidate with a GLM-R polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0203] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the GLM-R polypeptide encoded by the
gene identified herein or the drug candidate is immobilized on a
solid phase, e.g., on a microtiter plate, by covalent or
non-covalent attachments. Non-covalent attachment generally is
accomplished by coating the solid surface with a solution of the
GLM-R polypeptide and drying. Alternatively, an immobilized
antibody, e.g., a monoclonal antibody, specific for the GLM-R
polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed by adding the non-immobilized
component, which may be labeled by a detectable label, to the
immobilized component, e.g., the coated surface containing the
anchored component. When the reaction is complete, the non-reacted
components are removed, e.g., by washing, and complexes anchored on
the solid surface are detected. When the originally non-immobilized
component carries a detectable label, the detection of label
immobilized on the surface indicates that complexing occurred.
Where the originally non-immobilized component does not carry a
label, complexing can be detected, for example, by using a labeled
antibody specifically binding the immobilized complex.
[0204] If the candidate compound interacts with but does not bind
to a particular GLM-R polypeptide encoded by a gene identified
herein, its interaction with that polypeptide can be assayed by
methods well known for detecting protein-protein interactions. Such
assays include traditional approaches, such as, e.g.,
cross-linking, co-immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers (Fields and Song, Nature
(London) 340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci.
USA 88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc.
Natl. Acad. Sci. USA 89: 5789-5793 (1991). Many transcriptional
activators, such as yeast GAL4, consist of two physically discrete
modular domains, one acting as the DNA-binding domain, the other
one functioning as the transcription-activation domain. The yeast
expression system described in the foregoing publications
(generally referred to as the "two-hybrid system") takes advantage
of this property, and employs two hybrid proteins, one in which the
target protein is fused to the DNA-binding domain of GAL4, and
another, in which candidate activating proteins are fused to the
activation domain. The expression of a GAL1-lacZ reporter gene
under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0205] Compounds that interfere with the interaction of a DNA
encoding a GLM-R polypeptide identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the GLM-R polypeptide and
the intra- or extracellular component under conditions and for a
time allowing for the interaction and binding of the two products.
To test the ability of a candidate compound to inhibit binding, the
reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex
formation) between the test compound and the intra- or
extracellular component present in the mixture is monitored as
described hereinabove. The formation of a complex in the control
reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the
interaction of the test compound and its reaction partner.
[0206] To assay for antagonists, the GLM-R polypeptide may be added
to a cell along with the compound to be screened for a particular
activity and the ability of the compound to inhibit the activity of
interest in the presence of the GLM-R polypeptide indicates that
the compound is an antagonist to the GLM-R polypeptide.
Alternatively, antagonists may be detected by combining the GLM-R
polypeptide and a potential antagonist with membrane-bound GLM-R
polypeptide receptors or recombinant receptors under appropriate
conditions for a competitive inhibition assay. The GLM-R
polypeptide can be labeled, such as by radioactivity, such that the
number of GLM-R polypeptide molecules bound to the receptor can be
used to determine the effectiveness of the potential antagonist.
The gene encoding the receptor can be identified by numerous
methods known to those of skill in the art, for example, ligand
panning and FACS sorting. Coligan et al, Current Protocols in
Immun. 1(2): Chapter 5 (1991). Preferably, expression cloning is
employed wherein polyadenylated RNA is prepared from a cell
responsive to the GLM-R polypeptide and a cDNA library created from
this RNA is divided into pools and used to transfect COS cells or
other cells that are not responsive to the GLM-R polypeptide.
Transfected cells that are grown on glass slides are exposed to
labeled GLM-R polypeptide. The GLM-R polypeptide can be labeled by
a variety of means including iodination or inclusion of a
recognition site for a site-specific protein kinase. Following
fixation and incubation, the slides are subjected to
autoradiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an interactive
sub-pooling and re-screening process, eventually yielding a single
clone that encodes the putative receptor.
[0207] As an alternative approach for receptor identification,
labeled GLM-R polypeptide can be photoaffinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0208] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled GLM-R polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0209] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
GLM-R polypeptide, and, in particular, antibodies including,
without limitation, poly- and monoclonal antibodies and antibody
fragments, single-chain antibodies, anti-idiotypic antibodies, and
chimeric or humanized versions of such antibodies or fragments, as
well as human antibodies and antibody fragments. Alternatively, a
potential antagonist may be a closely related protein, for example,
a mutated form of the GLM-R polypeptide that recognizes the
receptor but imparts no effect, thereby competitively inhibiting
the action of the GLM-R polypeptide.
[0210] In one embodiment herein where competitive binding assays
are performed, GLM-R receptor or an antibody to GLM-R may be used
as a competitor.
[0211] Another potential GLM-R polypeptide antagonist is an
antisense RNA or DNA construct prepared using antisense technology,
where, e.g., an antisense RNA or DNA molecule acts to block
directly the translation of mRNA by hybridizing to targeted mRNA
and preventing protein translation. Antisense technology can be
used to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to DNA or RNA. For example, the 5' coding portion
of the polynucleotide sequence, which encodes the mature GLM-R
polypeptides herein, is used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241: 456 (1988);
Dervan et al., Science 251:1360 (1991)), thereby preventing
transcription and the production of the GLM-R polypeptide. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the GLM-R polypeptide
(antisense--Okano, Neurochem. 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 1988). The oligonucleotides described above can also be
delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the GLM-R polypeptide.
When antisense DNA is used, oligodeoxyribonucleotides derived from
the translation-initiation site, e.g., between about -10 and +10
positions of the target gene nucleotide sequence, are
preferred.
[0212] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the GLM-R polypeptide, thereby
blocking the normal biological activity of the GLM-R polypeptide.
Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides,
and synthetic non-peptidyl organic or inorganic compounds.
[0213] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology 4:469-471 (1994),
and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
[0214] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0215] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0216] It is appreciated that all the assays provided herein can be
used to screen a wide variety of candidate bioactive agents. The
term "candidate bioactive agent", "candidate agent" or "drug
candidate" or grammatical equivalents as used herein describes any
molecule, e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, purine analog, etc., to be tested
for bioactive agents that are capable of directly or indirectly
altering either the cellular activity phenotype or the expression
of a GLM-R sequence, including both nucleic acid sequences and
protein sequences.
[0217] Candidate agents can encompass numerous chemical classes,
though typically they are organic molecules, preferably small
organic compounds having a molecular weight of more than 100 and
less than about 2,500 daltons (d). Small molecules are further
defined herein as having a molecular weight of between 50 d and
2000 d. In another embodiment, small molecules have a molecular
weight of less than 1500, or less than 1200, or less than 1000, or
less than 750, or less than 500 d. In one embodiment, a small
molecule as used herein has a molecular weight of about 100 to 200
d. Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0218] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0219] In a preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes amino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration. In the preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents may be used, for example to
prevent or retard in vivo degradations.
[0220] In a preferred embodiment, the candidate bioactive agents
are naturally occurring proteins or fragments of naturally
occurring proteins. Thus, for example, cellular extracts containing
proteins, or random or directed digests of proteinaceous cellular
extracts, may be used. In this way libraries of procaryotic and
eucaryotic proteins may be made for screening in the methods of the
invention. Particularly preferred in this embodiment are libraries
of bacterial, fungal, viral, and mammalian proteins, with the
latter being preferred, and human proteins being especially
preferred.
[0221] In a preferred embodiment, the candidate bioactive agents
are peptides of from about 5 to about 30 amino acids, with from
about 5 to about 20 amino acids being preferred, and from about 7
to about 15 being particularly preferred. The peptides may be
digests of naturally occurring proteins as is outlined above,
random peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents herein is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0222] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of nucleic acid binding domains, the
creation of cysteines, for cross-linking, prolines for SH-3
domains, serines, threonines, tyrosines or histidines for
phosphorylation sites, etc., or to purines, etc.
[0223] In a preferred embodiment, the candidate bioactive agents
are nucleic acids. By "nucleic acid" or "oligonucleotide" or
grammatical equivalents herein means at least two nucleotides
covalently linked together. A nucleic acid of the present invention
will generally contain phosphodiester bonds, although in some
cases, as outlined below, nucleic acid analogs are included that
may have alternate backbones, comprising, for example,
phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein; Letsinger, J. Org. Chem. 35:3800 (1970);
Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al.,
Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805
(1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 (1986)), phosphorothioate
(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature 365:566 (1993);
Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy et al, Proc. Natl. Acad. Sci.
USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,
5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al.,
Angew. Chem. Intl. (Ed. English) 30:423 (1991); Letsinger et al, J.
Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside
&Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &
Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular
NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose
backbones, including those described in U.S. Pat. Nos. 5,235,033
and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,
"Carbohydrate Modifications in Antisense Research", Ed. Y. S.
Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp
169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xathanine
hypoxathanine, isocytosine, isoguanine, etc.
[0224] As described above generally for proteins, nucleic acid
candidate bioactive agents may be naturally occurring nucleic
acids, random nucleic acids, or "biased" random nucleic acids. For
example, digests of prokaryotic or eukaryotic genomes may be used
as is outlined above for proteins.
[0225] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0226] In a preferred embodiment, as outlined above, screens may be
done on individual genes and gene products (proteins). In a
preferred embodiment, the gene or protein has been identified as
described below in the Examples as a differentially expressed gene
associated with particular tissues and thus conditions related to
those tissues. Thus, in one embodiment, screens are designed to
first find candidate agents that can bind to GLM-R, and then these
agents may be used in assays that evaluate the ability of the
candidate agent to modulate GLM-R activity. Thus, as will be
appreciated by those in the art, there are a number of different
assays which may be run.
[0227] Screening for agents that modulate the activity of GLM-R may
also be done. In a preferred embodiment, methods for screening for
a bioactive agent capable of modulating the activity of GLM-R
comprise the steps of adding a candidate bioactive agent to a
sample of GLM-R and determining an alteration in the biological
activity of GLM-R. "Modulating the activity of GLM-R" includes an
increase in activity, a decrease in activity, or a change in the
type or kind of activity present. Thus, in this embodiment, the
candidate agent should both bind to GLM-R (although this may not be
necessary), and alter its biological or biochemical activity as
defined herein. The methods include both in vitro screening
methods, as are generally outlined above, and in vivo screening of
cells for alterations in the presence, expression, distribution,
activity or amount of GLM-R.
[0228] Thus, in this embodiment, the methods comprise combining a
sample and a candidate bioactive agent, and evaluating the effect
on GLM-R activity. By "GLM-R protein activity" or grammatical
equivalents herein is meant at least one of the GLM-R protein's
biological activities as described above.
[0229] In a preferred embodiment, the activity of the GLM-R protein
is increased; in another preferred embodiment, the activity of the
GLM-R protein is decreased. Thus, bioactive agents that are
antagonists are preferred in some embodiments, and bioactive agents
that are agonists may be preferred in other embodiments.
[0230] In one aspect of the invention, cells containing GLM-R
sequences are used in drug screening assays by evaluating the
effect of drug candidates on GLM-R. Cell types include normal
cells, tumor cells, and adipocytes.
[0231] Methods of assessing GLM-R activity such as changes in
glucose uptake, leptin release, metabolism, triglyceride and free
fatty acid levels, body weight and body fat, are known in the art
and are exemplified below in the examples.
[0232] In a preferred embodiment, the methods comprise adding a
candidate bioactive agent, as defined above, to a cell comprising
GLM-R. Preferred cell types include almost any cell. The cells
contain a nucleic acid, preferably recombinant, that encodes a
GLM-R protein. In a preferred embodiment, a library of candidate
agents are tested on a plurality of cells.
[0233] In one aspect, the assays are evaluated in the presence or
absence or previous or subsequent exposure to physiological
signals, for example hormones, antibodies, peptides, antigens,
cytokines, growth factors, action potentials, pharmacological
agents including chemotherapeutics, radiation, carcinogenics, or
other cells (i.e., cell-cell contacts). In another example, the
determinations are determined at different stages of the cell cycle
process.
[0234] The GLM-R sequences provided herein can also be used in
methods of diagnosis. Overexpression of GLM-R may indicate an
abnormally high metabolic rate and underexpression may indicate a
propensity for obesity and related disorders. Moreover, a sample
from a patient may be analyzed for mutated or disfunctional GLM-R.
Generally, such methods include comparing a sample from a patient
and comparing GLM-R expression to that of a control.
[0235] A potential use of a GLM-R would be in the regulation of the
development or function of monocytes or macrophage and the
treatment of disorders related thereto.
[0236] F. Anti-GLM-R Antibodies
[0237] The present invention further provides anti-GLM-R
antibodies. Exemplary antibodies include polyclonal, monoclonal,
humanized, bispecific, and heteroconjugate antibodies.
[0238] 1. Polyclonal Antibodies
[0239] The anti-GLM-R antibodies may comprise polyclonal
antibodies. Methods of preparing polyclonal antibodies are known to
the skilled artisan. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant. Typically, the immunizing agent
and/or adjuvant will be injected in the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent
may include the GLM-R polypeptide or a fusion protein thereof. It
may be useful to conjugate the immunizing agent to a protein known
to be immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0240] 2. Monoclonal Antibodies
[0241] The anti-GLM-R antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0242] The immunizing agent will typically include the GLM-R
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0243] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0244] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against GLM-R. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0245] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0246] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0247] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention can be
readily isolated and sequenced using conventional procedures (e.g.,
by using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the invention serve as a
preferred source of such DNA. Once isolated, the DNA may be placed
into expression vectors, which are then transfected into host cells
such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al, supra] or by covalently joining
to the immunoglobulin coding sequence all or part of the coding
sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0248] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0249] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0250] 3. Human and Humanized Antibodies
[0251] The anti-GLM-R antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0252] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0253] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J.
Immunol, 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0254] 4. Bispecific Antibodies
[0255] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the GLM-R, the other one is for any other
antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
[0256] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0257] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0258] According to another approach described in WO 96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. The preferred interface comprises at
least a part of the CH3 region of an antibody constant domain. In
this method, one or more small amino acid side chains from the
interface of the first antibody molecule are replaced with larger
side chains (e.g. tyrosine or tryptophan). Compensatory "cavities"
of identical or similar size to the large side chain(s) are created
on the interface of the second antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as
homodimers.
[0259] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al, Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab').sub.2 fragments. These fragments are
reduced in the presence of the dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies produced can be used as agents
for the selective immobilization of enzymes.
[0260] Fab' fragments may be directly recovered from E. coli and
chemically coupled to form bispecific antibodies. Shalaby et al.,
J. Exp. Med. 175:217-225 (1992) describe the production of a fully
humanized bispecific antibody F(ab').sub.2 molecule. Each Fab'
fragment was separately secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific
antibody. The bispecific antibody thus formed was able to bind to
cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity of human cytotoxic
lymphocytes against human breast tumor targets.
[0261] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See, Gruber et al., J.
Immunol. 152:5368 (1994).
[0262] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991). Exemplary bispecific antibodies may bind
to two different epitopes on a given GLM-R polypeptide herein.
Alternatively, an anti-GLM-R polypeptide arm may be combined with
an arm which binds to a triggering molecule on a leukocyte such as
a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc
receptors for IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16) so as to focus
cellular defense mechanisms to the cell expressing the particular
GLM-R polypeptide. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express a particular GLM-R
polypeptide. These antibodies possess a GLM-R-binding arm and an
arm which binds a cytotoxic agent or a radionuclide chelator, such
as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of
interest binds the GLM-R polypeptide and further binds tissue
factor (TF).
[0263] 5. Heteroconjugate Antibodies
[0264] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0265] 6. Effector Function Engineering
[0266] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J.
Immunol., 148: 2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design. 3: 219-230 (1989).
[0267] 7. Immunoconjugates
[0268] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0269] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re.
[0270] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0271] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) that is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
[0272] 8. Immunoliposomes
[0273] The antibodies disclosed herein may also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0274] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst. 81(19): 1484 (1989).
[0275] 9. Pharmaceutical Compositions of Antibodies
[0276] Antibodies specifically binding a GLM-R polypeptide
identified herein, as well as other molecules identified by the
screening assays disclosed hereinbefore, can be administered for
the treatment of various disorders in the form of pharmaceutical
compositions.
[0277] If the GLM-R polypeptide is intracellular and whole
antibodies are used as inhibitors, internalizing antibodies are
preferred. However, lipofections or liposomes can also be used to
deliver the antibody, or an antibody fragment, into cells. Where
antibody fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,
90: 7889-7893 (1993). The formulation herein may also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Alternatively, or in addition,
the composition may comprise an agent that enhances its function,
such as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0278] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
supra.
[0279] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0280] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0281] G. Generalized Uses for Anti-GLM-R Antibodies
[0282] The anti-GLM-R antibodies of the invention have various
utilities. For example, anti-GLM-R antibodies may be used in
diagnostic assays for GLM-R, e.g., detecting its expression in
specific cells, tissues, or serum. Various diagnostic assay
techniques known in the art may be used, such as competitive
binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in either heterogeneous or
homogeneous phases [Zola, Monoclonal Antibodies: A Manual of
Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies
used in the diagnostic assays can be labeled with a detectable
moiety. The detectable moiety should be capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32P, .sup.35S, or .sup.125I, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for conjugating the antibody to the
detectable moiety may be employed, including those methods
described by Hunter et al, Nature, 144:945 (1962); David et al,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 4:219
(1981); and Nygren, J. Histochem. Cytochem., 30:407 (1982).
[0283] Anti-GLM-R antibodies also are useful for the affinity
purification of GLM-R from recombinant cell culture or natural
sources. In this process, the antibodies against GLM-R are
immobilized on a suitable support, such a Sephadex resin or filter
paper, using methods well known in the art. The immobilized
antibody then is contacted with a sample containing the GLM-R to be
purified, and thereafter the support is washed with a suitable
solvent that will remove substantially all the material in the
sample except the GLM-R, which is bound to the immobilized
antibody. Finally, the support is washed with another suitable
solvent that will release the GLM-R from the antibody.
[0284] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0285] H. Trangenic Animals
[0286] Nucleic acids which encode novel human GLM-R or analogous
GLM-R from other species, such as the murine, can be used to
generate either transgenic animals or "knock out" animals which, in
turn, are useful in the development and screening of
therapeutically useful reagents. A transgenic animal (e.g., a
mouse) is an animal having cells that contain a transgene, which
transgene was introduced into the animal or an ancestor of the
animal at a prenatal, e.g., an embryonic stage. A transgene is a
DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, murine cDNA encoding
or an appropriate sequence thereof can be used to clone genomic DNA
encoding in accordance with established techniques and the genomic
sequences used to generate transgenic animals that contain cells
which express DNA encoding GLM-R. Methods for generating transgenic
animals, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells
would be targeted for transgene incorporation with tissue-specific
enhancers, which could result in production of GLM-R. Transgenic
animals that include a copy of a transgene encoding introduced into
the germ line of the animal at an embryonic stage can be used to
examine the effect of increased expression of DNA encoding. Such
animals can be used as tester animals for reagents thought to
confer protection from weight related disorders, such as, obesity,
cachexia or anorexia. In accordance with this facet of the
invention, an animal is treated with the reagent and a reduced
incidence of the disease, compared to untreated animals bearing the
transgene, would indicate a potential therapeutic intervention for
the disease.
[0287] Alternatively, the non-human homologues of GLM-R can be used
to construct a "knock out" animal which has a defective or altered
gene encoding GLM-R as a result of homologous recombination between
the endogenous gene encoding and altered genomic DNA encoding
introduced into an embryonic cell of the animal. For example,
murine cDNA encoding GLM-R can be used to clone genomic DNA
encoding in accordance with established techniques. A portion of
the genomic DNA encoding (e.g., such as an exon) GLM-R can be
deleted or replaced with another gene, such as a gene encoding a
selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking DNA (both at the
5' and 3' ends) are included in the vector (see e.g., Thomas and
Capecchi, Cell, 51:503 (1987) for a description of homologous
recombination vectors). The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogenous DNA
are selected (see e.g., Li et al., Cell, 69: 915 [1992]). The
selected cells are then injected into a blastocyst of an animal
(e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. [IRL, Oxford, 1987], pp. 113-152). A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term to create a "knock
out" animal. Progeny harboring the homologously recombined DNA in
their germ cells can be identified by standard techniques and used
to breed animals in which all cells of the animal contain the
homologously recombined DNA. Knockout animals can be characterized
for their ability to accept grafts, reject tumors and defend
against infectious diseases and can be used in the study of basic
immunobiology.
[0288] One particular technique used for the creation of transgenic
animals involves the use of yeast artificial chromosomes (YAC).
Yeast artificial chromosomes are cloning vectors constructed from
elements of yeast chromosomes, and allow the vector to be
replicated and maintained in yeast cells in vivo. Yeast elements
include a centromere, an autonomous replication sequence, a pair of
telomeres, yeast selectable markers, and usually a bacterial origin
of relication and selectable marker for replication and selection
of the YAC vector arms in bacteria.
[0289] YACs may be used in combination with gene targetting of
endogenous loci for insertion into the host animal's genome. An
advantage of using YACs is that hundreds of kilobases of DNA may be
inserted into a host cell. Therefore, the use of YAC cloning
vehicles permits inclusion of a substantial portion of the
transgene region. A further advantage is that sequences can be
deleted or inserted onto the YAC by utilizing high frequency
homologous recombination in yeast. This provides for facile
engineering of the YAC transgenes.
[0290] Another strategy of incorporating large segments of human
nucleic acid into mammals, such as occurs for the creation of human
antibodies is known as the "minilocus approach". The "minilocus
approach" is directed to facsimile reproduction of the locus for
the gene of interest (such as an immunoglobulin) through inclusion
of individual genes which comprise the locus. For example, when the
locus is an immunoglobulin, the component genes may be one or more
VH genes, one or more DH genes, one or more JH genes, a mu constant
region, a second constant region can be formed into a single
construct for insertion into the animal. Examples of this approach
are described in U.S. Pat. No. 5,545,807 to Surani et al., U.S.
Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016,
5,770,429, 5,789,650 and 5,814,318 each to Lonberg and Kay, U.S.
Pat. No. 5,591,669 to Krimpenfort and Berns, U.S. Pat. Nos.
5,612,205, 5,721,367, 5,789,215 to Berns et al. and U.S. Pat. No.
5,643,763 to Choi and Dunn, European Patent No. 0 546 073 B1 and
International Patent Application Nos. WO 92/03918, WO 92/22645, WO
92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO
96/14436, WO 97/13852 and 98/24884. Additional examples appear in
Taylor et al., Nucleic Acids Res. 20: 6287-6295 (1992), Chen et
al., Inter. Immunol. 5: 647-656 (1993), Tuaillon et al., J.
Immunol. 154: 6453-6465 (1995), Choi et al., Nature Genetics 4:
117-123 (1993), Lonberg et al., Nature 368: 856-859 (1994), Taylor
et al., Internat. Immunol. 6: 579-591 (1994), Tuaillon et al., J.
Immunol. 154: 6453-6465 (1995) and Fishwild et al., Nature Biotech.
14: 845-851 (1996).
[0291] Yet another strategy for introducing human nucleic acid into
mammalian cells is termed "microcell fusion". In microcell fusion,
portions or whole human chromosomes can be introduced into mice as
described in European Patent No. EP 0 843 961 A1. Because this
approach results in a transgene comprising a substantial amount of
genetic material, when a particular locus comprises many genes,
there is typically not a great degree of control over the profile
of expression of each particular gene. Another difficulty with
microcell fusion is that the transchromosomes are mitotically and
meiotically unstable. For example, when the transgene was an
immunoglobulin locus, the transchromosomes encoding human IgH, IgK
or both were lost with a frequency approaching 80%.
[0292] I. Uses of GLM-R Polynucleotide and Polypeptides Encoded
Thereby for Disorders, Related to the Over or Under Abundance of
Monocytes or Macrophages.
[0293] The invention is also directed to the use of GLM-R DNA,
polypeptides encoded therefrom, including peptide fragments thereof
and antibodies directed thereagainst and peptide fragments for
various particular uses related to the diagnosis and treatment
disorders related to the over or under abundance of monocytes or
macrophages.
[0294] Such uses include, for example: (1) prognostic and
diagnostic evaluation disorders related to the over or under
abundance of monocytes or macrophages and the identification of
individuals at risk for developing such disorders; (2) methods for
the treatment of disorders related to the over or under abundance
of monocytes or macrophages; (3) identification of compounds which
modulate the expression of the GLM-R DNA or activity of GLM-R
polypeptide (4). More specifically, such uses on an individual
include, the detection of the presence of a GLM-R mutant, or the
detection of either over- or under-expression of GLM-R polypeptide
relative to wild type expression levels, non-diseased organisms
having genetic profile which correlates with a diseased state, or
the susceptibility toward disorders related to the over or under
abundance of monocytes or macrophages.
[0295] The methods described herein may be performed, for example,
by utilizing pre-packaged diagonistic kits comprising at least one
specific GLM-R nucleic acid or anti-GLM-R polypeptide described
herein, which may be used to screen and diagnose individuals
exhibiting body weight disorder abnormalities, and then screen such
individuals having a predisposition to developing a disorder
related to the over or under abundance of monocytes or
macrophages.
[0296] In the detection of GLM-R mutants, any nucleated cell from
the individual in question can be used as a source for genomic
nucleic acid. In the detection of GLM-R expression, any cell type
or tissue in which the GLM-R DNA is expressed may be utilized, such
as, for example, tissues or cells shown herein to express the GLM-R
DNA. Examples of both nucleic acid-based as well as peptide-based
detection techniques are described below.
[0297] (1) Detection of GLM-R Nucleic Acid
[0298] Mutations or polymorphisms within the GLM-R DNA can be
detected through a number of techniques. Nucleic acid from any
nucleated cell can be used as the starting point for such assay
techniques, and may be isolated according to standard nucleic acid
preparation procedures which are well known to those of skill in
the art.
[0299] Genomic DNA may be used in hybridization or amplification
assays of biological samples to detect abnormalities involving
GLM-R gene structure, including point mutations, insertions,
deletions and chromosomal rearrangements. Such assays may include,
but are not limited to, Southern analyses, single stranded
conformation polymorphism analyses (SSCP), and PCR analyses.
[0300] Diagnostic methods for the detection of GLM-R gene-specific
mutations can involve for example, contacting and incubating
nucleic acids obtained from a sample, e.g., derived from a patient
sample or other appropriate cellular source with one or more
labeled nucleic acid reagents including recombinant DNA molecules,
cloned genes or degenerate variants thereof, under conditions
favorable for the specific annealing of these reagents to their
complementary sequences within or flanking the GLM-R gene.
Preferably, the lengths of such nucleic acid reagents can be at
least 15 to 30 nucleotides.
[0301] After incubation, all non-annealed nucleic acids are removed
from the nucleic acid:GLM-R molecule hybrid. The presence of
nucleic acids that have hybridized, if any such molecules exist, is
then detected. Using such a detection scheme, the nucleic acid from
the cell type or tissue of interest can be immobilized, for
example, to a solid support such as a membrane, or a plastic
surface such as that on a microtiter plate or polystyrene beads. In
this case, after incubation, non-annealed, labeled nucleic acid
reagents are easily removed. Detection of the remaining, annealed,
labeled GLM-R nucleic acid reagents is accomplished using standard
techniques well-known to those in the art. The GLM-R DNA sequences
to which the nucleic acid reagents have annealed can be compared to
the annealing pattern expected from a normal GLM-R DNA sequence in
order to determine whether a GLM-R DNA mutation is present.
[0302] In a preferred embodiment, GLM-R gene mutations or
polymorphisms can be detected by using a microassay of GLM-R
nucleic acid sequences immobilized to a substrate or "gene chip"
(see, e.g. Cronin, et al., Human Mutation 7:244-255)(1996).
[0303] Alternative diagnostic methods for the detection of GLM-R
gene specific nucleic acid molecules, in patient samples or other
appropriate cell sources, may involve their amplification, e.g., by
PCR (the experimental embodiment set forth in U.S. Pat. No.
4,683,202), followed by the analysis of the amplified molecules
using techniques well known to those of skill in the art, such as,
for example, those listed above. The resulting amplified sequences
can be compared to those that would be expected if the nucleic acid
being amplified contained only normal copies of the GLM-R gene in
order to determine whether a GLM-R gene mutation exists.
[0304] Among those GLM-R nucleic acid sequences which are preferred
for such amplification-related diagnostic screening analyses are
cligonucleotide primers which amplify GLM-R exon sequences. The
sequences of such oligonucleotide primers are, therefore,
preferably derived from GLM-R intron sequences so that the entire
exon, or coding region, can be analyzed as discussed below. Primer
pairs useful for amplification of GLM-R exons are preferably
derived from adjacent introns. Appropriate primer pairs can be
chosen such that each of the 25 GLM-R exons are amplified.
[0305] Primers for the amplification of GLM-R exons can be
routinely designed by one of ordinary skill in the art by utilizing
the coding and unstranslated sequences of GLM-R shown in FIG. 1.
Additional GLM-R nucleic acid sequences which are preferred for
such amplification-related analyses are those which will detect the
presence of a GLM-R polymorphism. Such polymorphisms include ones
which represent mutations associated with disorders related to the
over or under abundance of monocytes or macrophages.
[0306] Further, well-known genotyping techniques can be performed
to type polymorphisms that are in close proximity to mutations in
the GLM-R gene itself, including mutations associated with
disorders related to the over or under abundance of monocytes or
macrophages. Such polymorphisms can be used to identify individuals
in families likely to carry mutations in the GLM-R gene. If a
polymorphism exhibits linkage disequilibrium with mutations in the
GLM-R gene, the polymorphism can also be used to identify
individuals in the general population who are likely to carry such
mutations.
[0307] Polymorphisms that can be used in this way include
restriction fragment length polymorphisms (RFLPs), which involve
sequence variations in restriction enzyme target sequences,
single-base polymorphisms, and simple sequence length polymorphisms
(SSLPs). For example, U.S. Pat. No. 5,075,217 describes a DNA
marker based on length polymorphisms in blocks of
(dC-dA).sub.n-(dG-dT).sub.n short tandem repeats. The average
separation of (dC-dA).sub.n-(dG-dT).sub.n blocks is estimated to be
30,000-60,000 bp. Markers that are so closely spaced exhibit a high
frequency co-inheritance, and are extremely useful in the
identification of genetic mutations, such as, for example,
mutations within the GLM-R gene, and the diagnosis of diseases and
disorders related to mutations in the GLM-R gene.
[0308] Also, U.S. Pat. No. 5,364,759 describes a DNA profiling
assay for detecting short tri and tetra nucleotide repeat
sequences. The process includes extracting the DNA of interest,
such as the GLM-R gene, amplifying the extracted DNA, and labelling
the repeat sequences to form a genotypic map of the individual's
DNA.
[0309] A GLM-R probe could additionally be used to directly
identify RFLPs. Further, a GLM-R probe or primers derived from the
GLM-R sequence could be used to isolate genomic clones such as
YACs, BACs, PACs, cosmids, phage, or plasmids.
[0310] The DNA contained in these clones can be screened for
single-base polymorphisms or SSLPs using standard hybridization or
sequencing procedures. The level of GLM-R gene expression can also
be assayed. For example, RNA from a cell type or tissue known, or
suspected, to express the GLM-R gene, such as muscle, brain,
kidney, testes, heart, liver, lung, skin, hypothalamus, spleen, and
adipose tissue may be isolated and tested utilizing hybridization
or PCR techniques such as are described, above. The isolated cells
can be derived from cell culture or from a patient. The analysis of
cells taken from culture may be a necessary step in the assessment
of cells to be used as part of a cell-based gene therapy technique
or, alternatively, to test the effect of compounds on the
expression of the GLM-R gene. Such analyses may reveal both
quantitative and qualitative aspects of the expression pattern of
the GLM-R gene, including activation or inactivation of GLM-R gene
expression.
[0311] In one embodiment of such a detection scheme, a cDNA
molecule is synthesized from an RNA molecule of interest (e.g., by
reverse transcription of the RNA molecule into cDNA). All or part
of the resulting cDNA is then used as the template for a nucleic
acid amplification reaction, such as a PCR amplification reaction,
or the like. The nucleic acid reagents used as synthesis initiation
reagents (e.g., primers) in the reverse transcription and nucleic
acid amplification steps of this method are chosen from among the
GLM-R gene nucleic acid reagents described herein.
[0312] The preferred lengths of such nucleic acid reagents are at
least 9-30 nucleotides. For detection of the amplified product, the
nucleic acid amplification may be performed using radioactively or
non-radioactively labeled nucleotides. Alternatively, enough
amplified product may be made such that the product may be
visualized by standard ethidium bromide staining or by utilizing
any other suitable nucleic acid staining method.
[0313] As an alternative to amplification techniques, standard
Northern analyses can be performed to determine the level of mRNA
expression of the GLM-R gene, if a sufficient quantity of the
appropriate cells can be obtained.
[0314] Additionally, it is possible to perform such GLM-R gene
expression assays "in situ", i.e., directly upon tissue sections
(fixed and/or frozen) of patient tissue obtained from biopsies or
resections, such that no nucleic acid purification is necessary.
Nucleic acid reagents such as those described herein may be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo, G. J., 1992, "PCR In Situ Hybridization: Protocols
and Applications", Raven Press, NY).
[0315] (2) Detection of GLM-R Gene Products
[0316] GLM-R gene products, including both native sequence,
variants, and polypeptide fragments thereof, may be detected using
antibodies which are directed against such GLM-R gene products.
Such anti-GLM-R antibodies may be used as diagnostics and
prognostics for disorders related to the over or under abundance of
monocytes or macrophages. Such methods may be used to detect
abnormalities in the level of GLM-R gene expression or of GLM-R
gene product synthesis, or abnormalities in the structure, temporal
expression, and/or physical location of GLM-R gene product. The
antibodies and immunoassay methods described herein have, for
example, important in vitro applications in assessing the efficacy
of treatments for disorders related to the over or under abundance
of monocytes or macrophages. Antibodies, or fragments of
antibodies, such as those described below, may be used to screen
potentially therapeutic compounds in vitro to determine their
effects on GLM-R gene expression and GLM-R gene product production.
The compounds that have beneficial effects on disorders related to
the over or under abundance of monocytes or macrophages, can
thereby be identified, and a therapeutically effective dose
determined.
[0317] In vitro immunoassays may also be used, for example, to
assess the efficacy of cell-based gene therapy for disorders
related to the over or under abundance of monocytes or macrophages.
Antibodies directed against GLM-R gene products may be used in
vitro to determine, for example, the level of GLM-R gene expression
achieved in cells genetically engineered to produce GLM-R gene
product. In the case of intracellular GLM-R gene products, such an
assessment is done, preferably, using cell lysates or extracts.
Such analysis will allow for a determination of the number of
transformed cells necessary to achieve therapeutic efficacy in
vivo, as well as optimization of the gene replacement protocol.
[0318] The tissue or cell type to be analyzed will generally
include those that are known, or suspected, to express a GLM-R
gene. The protein isolation methods employed herein may, for
example, be such as those described in Harlow and Lane (1988,
"Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.). The isolated cells can be derived
from cell culture or from a patient. The analysis of cells taken
from culture may be a necessary step in the assessment of cells to
be used as part of a cell-based gene therapy technique or,
alternatively, to test the effect of compounds on the expression of
the GLM-R gene.
[0319] Preferred diagnostic methods for the detection of GLM-R gene
products, conserved variants or peptide fragments thereof, may
involve, for example, immunoassays wherein the GLM-R gene products
or conserved variants or peptide fragments are detected by their
interaction with an anti-GLM-R gene product-specific antibody.
[0320] For example, antibodies, or fragments of antibodies, such as
those described, above, may be used to quantitatively or
qualitatively detect the presence of GLM-R gene products or
conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody coupled with light
microscopic, flow cytometric, or fluorimetric detection. Such
techniques are especially preferred for GLM-R gene products that
are expressed on the cell surface.
[0321] The antibodies (or fragments thereof) useful in the present
invention may, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of GLM-R gene products, conserved variants or peptide
fragments thereof. In situ detection may be accomplished by
removing a histological specimen from a patient, and applying
thereto a labeled antibody that binds to a GLM-R polypeptide. The
antibody (or fragment) is preferably applied by overlaying the
labeled antibody (or fragment) onto a biological sample. Through
the use of such a procedure, it is possible to determine not only
the presence of the GLM-R gene product, conserved variants or
peptide fragments, but also its distribution in the examined
tissue. Using the present invention, those of ordinary skill will
readily recognize that any of a wide variety of histological
methods (such as staining procedures) can be modified in order to
achieve in situ detection of a GLM-R gene product.
[0322] Immunoassays for GLM-R gene products, conserved variants, or
peptide fragments thereof will typically comprise: (1) incubating a
sample, such as a biological fluid, a tissue extract, freshly
harvested cells, or lysates of cells in the presence of a
detectably labeled antibody capable of identifying GLM-R gene
products, conserved variants or peptide fragments thereof; and (2)
detecting the bound antibody by any of a number of techniques
well-known in the art.
[0323] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier, such as
nitrocellulose, that is capable of immobilizing cells, cell
particles or soluble proteins. The support may then be washed with
suitable buffers followed by treatment with the detectably labeled
GLM-R gene product specific antibody.
[0324] The solid phase support may then be washed with the buffer a
second time to remove unbound antibody. The amount of bound label
on the solid support may then be detected by conventional
means.
[0325] "Solid phase support or carrier" means any support capable
of binding an antigen or an antibody. Well-known supports or
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier
can be either soluble to some extent or insoluble for the purposes
of the present invention. The support material may have virtually
any possible structural configuration so long as the coupled
molecule is capable of binding to an antigen or antibody. Thus, the
support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0326] One method for detectably labeling an GLM-R gene
product-specific antibody is through linkage to a readily
detectable enzyme, such as an enzyme immunoassay (EIA) (Voller, A.,
"The Enzyme Linked Immunosorbent Assay (ELISA)II, Diagnostic
Horizons 2: 1-7, Microbiological Associates Quarterly Publication,
Walkersville, Md.) (1978); Voller, A. et al., J. Clin. Pathol. 31,
507-520 (1978); Butler, J. E., Meth. Enzymol. 73: 482-523 (1981);
Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla.
(1980); Ishikawa, E., et al. (eds.), Enzyme Immunoassay, Kgaku
Shoin, Tokyo (1981). The enzyme which is bound to the antibody will
react with an appropriate substrate, preferably a chromogenic
substrate, in such a manner as to produce a chemical moiety that
can be detected, for example, by spectrophotometric, fluorimetric
or by visual means. Enzymes that can be used to detectably label
the antibody include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, .beta.-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. The detection can be accomplished by
colorimetric methods that employ a chromogenic substrate for the
enzyme. Detection may also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0327] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect GLM-R
gene products through the use of a radioimmunoassay (RIA) (see, for
example, Weintraub, B., Principles of Radioimmunoassays, Seventh
Training Course on Radioligand Assay Techniques, The Endocrine
Society, March, 1986). The radioactive isotope can be detected by
such means as the use of a gamma counter or a scintillation counter
or by autoradiography.
[0328] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0329] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0330] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0331] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0332] (3) Screening Assays for Compounds that Interact with GLM-R
Nucleic Acid or Gene Product
[0333] The following assays are designed to identify compounds that
bind to an GLM-R gene product, compounds that bind to proteins, or
portions of proteins that interact with am GLM-R gene product,
compounds that interfere with the interaction of an GLM-R gene
product with proteins and compounds that modulate the activity of
the GLM-R gene (i.e., modulate the level of GLM-R gene expression
and/or modulate the level of GLM-R gene product activity). Assays
may additionally be utilized that identify compounds that bind to
GLM-R gene regulatory sequences (e.g., promoter sequences; see
e.g., Platt, J. Biol. Chem. 269: 28558-28562 (1994), which is
incorporated herein by reference in its entirety, and that can
modulate the level of GLM-R gene expression. Such compounds may
include, but are not limited to, small organic molecules, such as
ones that are able to cross the blood-brain barrier, gain to and/or
entry into an appropriate cell and affect expression of the GLM-R
gene or some other gene involved in the body weight regulatory
pathway, or intracellular proteins.
[0334] Methods for the identification of such proteins are
described, below. Such proteins may be involved in disorders
related to the over or under abundance of monocytes or macrophages.
Furthermore, among these compounds are compounds that affect the
level of GLM-R gene expression and/or GLM-R gene is product
activity and that can be used in the therapeutic treatment of
disorders related to the over or under abundance of monocytes or
macrophages.
[0335] Compounds may include, but are not limited to, peptides such
as, for example, soluble peptides, including but not limited to,
Ig-tailed fusion peptides, and members of random peptide libraries;
(see, e.g., Lam et al., Nature 354: 82-84 (1991); Houghten et al,
Nature 354: 84-86 (1991), and combinatorial chemistry-derived
molecular library made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to members of random or
partially degenerate, directed phosphopeptide libraries; see, e.g.,
Songyang et al., Cell 72: 767-778 (1993), antibodies (including,
but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab'), and FAb expression library fragments, and epitope-binding
fragments thereof), and small organic or inorganic molecules.
[0336] Compounds identified via assays such as those described
herein may be useful, for example, in elaborating the biological
function of the GLM-R gene product and for ameliorating disorders
related to the over or under abundance of monocytes or
macrophages.
[0337] (a) In vitro Screening Assays for Compounds that Bind to
GLM-R Gene Product
[0338] In vitro systems may be designed to identify compounds
capable of binding the GLM-R gene products of the invention.
Compounds identified may be useful, for example, in modulating the
activity of unimpaired and/or mutant GLM-R gene products, in
elaborating the biological function of the GLM-R gene product, in
screens for identifying compounds that disrupt normal GLM-R gene
product interactions, or may in themselves disrupt such
interactions.
[0339] The principle of the assays used to identify compounds that
bind to the GLM-R gene product involves preparing a reaction
mixture of the GLM-R gene product and the test compound under
conditions and for a time sufficient to allow the two components to
interact and bind, thus forming a complex that can be removed
and/or detected in the reaction mixture. These assays can be
conducted in a variety of ways. For example, one method to conduct
such an assay involves anchoring a GLM-R gene product or a test
substance onto a solid support and detecting GLM-R gene
product/test compound complexes formed on the solid support at the
end of the reaction. In one embodiment of such a method, the GLM-R
gene product may be anchored onto a solid support, and the test
compound, which is not anchored, may be labeled, either directly or
indirectly.
[0340] In practice, microtiter plates are conveniently utilized as
the solid support. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0341] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously non-immobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0342] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g., using an immobilized antibody
specific for GLM-R gene product or the test compound to anchor any
complexes formed in solution, and a labeled antibody specific for
the other component of the possible complex to detect anchored
complexes.
[0343] (b) Assays for Proteins that Interact with the GLM-R Gene
Product
[0344] Any method suitable for detecting protein-protein
interactions may be employed for identifying GLM-R gene
product-protein interactions. Among the traditional methods that
may be employed are co-immunoprecipitation, cross-linking and
co-purification through gradients or chromatographic columns.
Utilizing procedures such as these allows for the identification of
proteins that interact with GLM-R gene products. Such proteins can
include, but are not limited, the GLM-R gene product. Once
isolated, such a protein can be identified and can be used in
conjunction with standard techniques, to identify proteins it
interacts with. For example, at least a portion of the amino acid
sequence of a protein that interacts with the GLM-R gene product
can be ascertained using techniques well known to those of skill in
the art, such as via the Edman degradation technique (see, e.g.,
Creighton, "Proteins: Structures and Molecular Principles," W.H.
Freeman & Co., N.Y., pp. 34-49 (1983). The amino acid sequence
obtained may be used as a guide for the generation of
oligonucleotide mixtures that can be used to screen for gene
sequences encoding such proteins. Screening may be accomplished,
for example, by standard hybridization or PCR techniques.
Techniques for the generation of oligonucleotide mixtures and the
screening are well-known. (See, e.g., Ausubel, supra, and 1990,
"PCR Protocols: A Guide to Methods and Applications," Innis et al,
eds. Academic Press, Inc., New York).
[0345] Additionally, methods may be employed that result in the
simultaneous identification of genes that encode a protein which
interacts with a GLM-R gene product. These methods include, for
example, probing expression libraries with labeled GLM-R gene
product, using GLM-R gene product in a manner similar to the well
known technique of antibody probing of Xgt11 libraries. One method
that detects protein interactions in vivo, the two-hybrid system,
is described in detail for illustration only and not by way of
limitation. One version of this system has been described (Chien,
et al., Proc. Natl. Acad. Sci. USA 88: 9578-9582 (1991) and is
commercially available from Clontech (Palo Alto, Calif.).
[0346] Briefly, utilizing such a system, plasmids are constructed
that encode two hybrid proteins: one consists of the DNA-binding
domain of a transcription activator protein fused to the GLM-R gene
product and the other consists of the transcription activator
protein's activation domain fused to an unknown protein that is
encoded by a cDNA that has been recombined into this plasmid as
part of a cDNA library. The DNA-binding domain fusion plasmid and
the cDNA library are transformed into a strain of the yeast
Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS
or lacZ) whose regulatory region contains the transcription
activator's binding site. Either hybrid protein alone cannot
activate transcription of the reporter gene: the DNA-binding domain
hybrid cannot because it does not provide activation function and
the activation domain hybrid cannot because it cannot localize to
the activator's binding sites. Interaction of the two hybrid
proteins reconstitutes the functional activator protein and results
in expression of the reporter gene, which is detected by an assay
for the reporter gene product.
[0347] The two-hybrid system or related methodologies may be used
to screen activation domain libraries for proteins that interact
with the "bait" gene product. By way of example, and not by way of
limitation, GLM-R gene products may be used as the bait gene
product. Total genomic or cDNA sequences are fused to the DNA
encoding an activation domain.
[0348] This library and a plasmid encoding a hybrid of a bait GLM-R
gene product fused to the DNA-binding domain are co-transformed
into a yeast reporter strain, and the resulting transformants are
screened for those that express the reporter gene. For example, a
bait GLM-R gene sequence, such as the open reading frame of the
GLM-R gene, can be cloned into a vector such that it is
translationally fused to the DNA encoding the DNA-binding domain of
the GAL4 protein. These colonies are purified and the library
plasmids responsible for reporter gene expression are isolated. DNA
sequencing is then used to identify the proteins encoded by the
library plasmids.
[0349] A cDNA library of the cell line from which proteins that
interact with bait GLM-R gene product are to be detected can be
made using methods routinely practiced in the art. According to the
particular system described herein, for example, the cDNA fragments
can be inserted into a vector such that they are translationally
fused to the transcriptional activation domain of GAL4. Such a
library can be co-transformed along with the bait GLM-R gene-GAL4
fusion plasmid into a yeast strain that contains a lacZ gene driven
by a promoter that contains GAL4 activation sequence.
[0350] A cDNA encoded protein, fused to a GAL4 transcriptional
activation domain that interacts with bait GLM-R gene product will
reconstitute an active GAL4 protein and thereby drive expression of
the HIS3 gene. Colonies that express HIS3 can be detected by their
growth on petri dishes containing semi-solid agar based media
lacking histidine.
[0351] The cDNA can then be purified from these strains, and used
to produce and isolate the bait GLM-R gene product-interacting
protein using techniques routinely practiced in the art.
[0352] (c) Assays for Compounds that Interfere with GLM-R Gene
Product Macromolecule Interaction
[0353] The GLM-R gene products may, in vivo, interact with one or
more macromolecules, such as proteins. For example, the GLM-R
gene-products may, in vivo, interact with the GLM-R gene products.
Other macromolecules which interact with the GLM-R gene products
may include, but are not limited to, nucleic acid molecules and
those proteins identified via methods such as those described
herein. For purposes of this discussion, the macromolecules are
referred to herein as "binding partners". Compounds that disrupt
GLM-R gene product binding to a binding partner may be useful in
regulating the activity of the GLM-R gene product, especially
mutant GLM-R gene products. Such compounds may include, but are not
limited to molecules such as peptides, and the like.
[0354] The basic principle of an assay system used to identify
compounds that interfere with the interaction between the GLM-R
gene product and a binding partner or partners involves preparing a
reaction mixture containing the GLM-R gene product and the binding
partner under conditions and for a time sufficient to allow the two
to interact and bind, thus forming a complex. In order to test a
compound for inhibitory activity, the reaction mixture is prepared
in the presence and absence of the test compound. The test compound
may be initially included in the reaction mixture, or may be added
at a time subsequent to the addition of GLM-R gene product and its
binding partner. Control reaction mixtures are incubated without
the test compound or with a compound which is known not to block
complex formation. The formation of any complexes between the GLM-R
gene product and the binding partner is then detected. The
formation of a complex in the control reaction, but not in the
reaction mixture containing the test compound, indicates that the
compound interferes with the interaction of the GLM-R gene product
and the binding partner. Additionally, complex formation within
reaction mixtures containing the test compound and normal GLM-R
gene product may also be compared to complex formation within
reaction mixtures containing the test compound and a mutant GLM-R
gene product. This comparison may be important in those cases
wherein it is desirable to identify compounds that disrupt
interactions of mutant but not normal GLM-R gene product.
[0355] The assay for compounds that interfere with the interaction
of the GLM-R gene products and binding partners can be conducted in
a heterogeneous or homogeneous format. Heterogeneous assays involve
anchoring either the GLM-R gene product or the binding partner onto
a solid support and detecting complexes formed on the solid support
at the end of the reaction. In homogeneous assays, the entire
reaction is carried out in a liquid phase. In either approach, the
order of addition of reactants can be varied to obtain different
information about the compounds being tested. For example, test
compounds that interfere with the interaction between the GLM-R
gene products and the binding partners, e.g., by competition, can
be identified by conducting the reaction in the presence of the
test substance; i.e., by adding the test substance to the reaction
mixture prior to or simultaneously with the GLM-R gene product and
interactive intracellular binding partner. Alternatively, test
compounds that disrupt preformed complexes, e.g., compounds with
higher binding constants that displace one of the components from
the complex, can be tested by adding the test compound to the
reaction mixture after complexes have been formed. The various
formats are described briefly below.
[0356] In a heterogeneous assay system, either the GLM-R gene
product or the interactive binding partner, is anchored onto a
solid surface, while the non-anchored species is labeled, either
directly or indirectly. In practice, microtiter plates are
conveniently utilized. The anchored species may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished simply by coating the solid surface with a solution
of the GLM-R gene product or binding partner and drying.
Alternatively, an immobilized antibody specific for the species to
be anchored may be used to anchor the species to the solid surface.
The surfaces may be prepared in advance and stored.
[0357] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface; e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, may be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds that inhibit complex formation
or that disrupt preformed complexes can be detected.
[0358] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds that inhibit complex
formation or that disrupt preformed complexes can be
identified.
[0359] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of the
GLM-R gene product and the interactive binding partner is prepared
in which either the GLM-R gene product or its binding partners is
labeled, but the signal generated by the label is quenched due to
complex formation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein
which utilizes this approach for immunoassays). The addition of a
test substance that competes with and displaces one of the species
from the preformed complex will result in the generation of a
signal above background. In this way, test substances that disrupt
GLM-R gene product/binding partner interaction can be
identified.
[0360] In another embodiment of the invention, these same
techniques can be employed using peptide fragments that correspond
to the binding domains of the GLM-R gene product and/or the binding
partner (in cases where the binding partner is a protein), in place
of one or both of the full length proteins. Any number of methods
routinely practiced in the art can be used to identify and isolate
the binding sites. These methods include, but are not limited to,
mutagenesis of the gene encoding one of the proteins and screening
for disruption of binding in a co-immunoprecipitation assay.
Compensating mutations in the gene encoding the second species in
the complex can then be selected. Sequence analysis of the genes
encoding the respective proteins will reveal the mutations that
correspond to the region of the protein involved in interactive
binding. Alternatively, one protein can be anchored to a solid
surface using methods described in this Section above, and allowed
to interact with and bind to its labeled binding partner, which has
been treated with a proteolytic enzyme, such as trypsin. After
washing, a short, labeled peptide comprising the binding domain may
remain associated with the solid material, which can be isolated
and identified by amino acid sequencing. Also, once the gene coding
for the segments is engineered to express peptide fragments of the
protein, it can then be tested for binding activity and purified or
synthesized.
[0361] For example, and not by way of limitation, a GLM-R gene
product can be anchored to a solid material as described, above, in
this Section by making a GST-1 fusion is protein and allowing it to
bind to glutathione agarose beads. The binding partner can be
labeled with a radioactive isotope, such as .sup.35S, and cleaved
with a proteolytic enzyme such as trypsin. Cleavage products can
then be added to the anchored GST-1 fusion protein and allowed to
bind. After washing away unbound peptides, labeled bound material,
representing the binding partner binding domain, can be eluted,
purified, and analyzed for amino acid sequence by well-known
methods. Peptides so identified can be produced synthetically or
produced using recombinant DNA technology.
[0362] (d) Assays for the Identification of Compounds Useful in the
Treatment of Body Weight Disorders
[0363] Compounds, including but not limited to binding compounds
identified via assay techniques such as those described previously
can be tested for the ability to treat symptoms of body weight
disorders. It should be noted that the assays described herein can
identify compounds that affect GLM-R activity by either affecting
GLM-R gene expression or by affecting the level of GLM-R gene
product activity. For example, compounds may be identified that are
involved in another step in the pathway in which the GLM-R gene
and/or GLM-R gene product is involved, such as, for example, a step
which is either "upfield" or "downfield" of the step in the pathway
mediated by the GLM-R gene. Such compounds may, by affecting this
same pathway, modulate the effect of GLM-R on the development of
body weight disorders. Such compounds can be used as part of a
therapeutic method for the treatment of the disorder.
[0364] Described below are cell-based and animal model-based assays
for the identification of compounds exhibiting such an ability to
ameliorate symptoms of body weight disorders. First, cell-based
systems can be used to identify compounds that may act to
ameliorate symptoms of body weight disorders. Such cell systems can
include, for example, recombinant or non-recombinant cell, such as
cell lines, that express the GLM-R gene.
[0365] In utilizing such cell systems, cells that express GLM-R may
be exposed to a compound suspected of exhibiting an ability to
ameliorate body weight disorder symptoms, at a sufficient
concentration and for a sufficient time to elicit such an
amelioration of such symptoms in the exposed cells. After exposure,
the cells can be assayed to measure alterations in the expression
of the GLM-R gene, e.g., by assaying cell lysates for GLM-R mRNA
transcripts (e.g., by Northern analysis) or for GLM-R gene products
expressed by the cell; compounds that modulate expression of the
GLM-R gene are good candidates as therapeutics.
[0366] In addition, animal-based systems or models for a mammalian
body weight disorder, for example, transgenic mice containing a
human or altered form of GLM-R gene, may be used to identify
compounds capable of ameliorating symptoms of the disorder. Such
animal models may be used as test substrates for the identification
of drugs, pharmaceuticals, therapies and interventions. For
example, animal models may be exposed to a compound suspected of
exhibiting an ability to ameliorate symptoms, at a sufficient
concentration and for a sufficient time to elicit such an
amelioration of body weight disorder symptoms. The response of the
animals to the exposure may be monitored by assessing the reversal
of the symptoms of the disorder.
[0367] With regard to intervention, any treatments that reverse any
aspect of body weight disorder-like symptoms should be considered
as candidates for human therapeutic intervention in such a
disorder.
[0368] (4) Compounds and Methods for the Treatment of Disorders
Related to the Over or Under Abundance of Monocytes or
Macrophages
[0369] Described below are methods and compositions whereby
disorders related to the over or under abundance of monocytes or
macrophages, may be treated. Such methods can comprise, for example
administering compounds which modulate the expression of a
mammalian GLM-R gene and/or the synthesis or activity of a
mammalian GLM-R gene product, so that symptoms of the disorders
related to the over or under abundance of monocytes or macrophages
are ameliorated. Alternatively, in those instances whereby the
disorders related to the over or under abundance of monocytes or
macrophages results from GLM-R gene mutations, such methods can
comprise supplying the mammal with a nucleic acid molecule encoding
an unimpaired GLM-R gene product such that an unimpaired GLM-R gene
product is expressed and symptoms of the disorder are ameliorated.
In another embodiment of methods for the treatment of mammalian
body weight disorders resulting from GLM-R gene mutations, such
methods can comprise supplying the mammal with a cell comprising a
nucleic acid molecule that encodes an unimpaired GLM-R gene product
such that the cell expresses the unimpaired GLM-R gene product, and
symptoms of the disorder are ameliorated.
[0370] Alternatively, symptoms of disorders related to the over or
under abundance of monocytes or macrophages, may be ameliorated by
increasing the level of GLM-R gene expression and/or GLM-R gene
product activity.
[0371] (a) Inhibitory Antisense, Ribozyme and Triple Helix
Approaches
[0372] In another embodiment, symptoms of disorders related to the
over or under abundance of monocytes or macrophages may be
ameliorated by decreasing the level of GLM-R gene expression and/or
GLM-R gene product activity by using GLM-R gene sequences in
conjunction with well-known antisense, gene "knock-out," ribozyme
and/or triple helix methods to decrease the level of GLM-R gene
expression. Among the compounds that may exhibit the ability to
modulate the activity, expression or synthesis of the GLM-R gene,
including the ability to ameliorate the symptoms of a mammalian
body weight disorder, are antisense, ribozyme, and triple helix
molecules. Such molecules may be designed to reduce or inhibit
either unimpaired, or if appropriate, mutant target gene activity.
Techniques for the production and use of such molecules are well
known to those of skill in the art.
[0373] Antisense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense approaches involve the design of
oligonucleotides that are complementary to a target gene mRNA. The
antisense oligonucleotides will bind to the complementary target
gene mRNA transcripts and prevent translation. Absolute
complementarily, although preferred, is not required.
[0374] A sequence "complementary" to a portion of an RNA, as
referred to herein, means a sequence having sufficient
complementarily to be able to hybridize with the RNA, forming a
stable duplex; in the case of double-stranded antisense nucleic
acids, a single strand of the duplex DNA may thus be tested, or
triplex formation may be assayed. The ability to hybridize will
depend on both the degree of complementarily and the length of the
antisense nucleic acid. Generally, the longer the hybridizing
nucleic acid, the more base mismatches with an RNA it may contain
and still form a stable duplex (or triplex, as the case may be).
One skilled in the art can ascertain a tolerable degree of mismatch
by use of standard procedures to determine the melting point of the
hybridized complex.
[0375] In one embodiment, oligonucleotides complementary to
non-coding regions of the GLM-R gene could be used in an antisense
approach to inhibit translation of endogenous GLM-R mRNA. Antisense
nucleic acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects the oligonucleotide is at least
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at
least 50 nucleotides.
[0376] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit gene expression. It is
preferred that these studies utilize controls that distinguish
between antisense gene inhibition and nonspecific biological
effects of oligonucleotides. It is also preferred that these
studies compare levels of the target RNA or protein with that of an
internal control RNA or protein. Additionally, it is envisioned
that results obtained using the antisense oligonucleotide are
compared with those obtained using a control oligonucleotide. It is
preferred that the control oligonucleotide is of approximately the
same length as the test oligonucleotide and that the nucleotide
sequence of the oligonucleotide differs from the antisense sequence
no more than is necessary to prevent specific hybridization to the
target sequence.
[0377] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86: 6553-6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci. U.S.A. 84, 648-652
(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or
the blood-brain barrier (see e.g., PCT Publication No. WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents
(see, e.g., Krol et al., BioTechniques 6: 958-976 (1988) or
intercalating agents (see e.g., Zon, Pharm. Res. 5: 539-549 (1988).
To this end, the oligonucleotide may be conjugated to another
molecule, e.g., a peptide, hybridization triggered cross-linking
agent, transport agent, hybridization-triggered cleavage agent,
etc.
[0378] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethyl-aminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, NG-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethyl guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid-methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0379] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
Alternatively, the antisense oligonucleotide comprises at least one
modified phosphate backbone selected from the group consisting of a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof. Alternatively
still, the antisense oligonucleotide is an .alpha.-anomeric
oligonucieotide. An .alpha.-anomeric oligonucleotide forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .about.-units, the strands run parallel to each other
(Gautier et al., Nucl. Acids Res. 15: 6625-6641 (1987). The
oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., Nucl.
Acids Res. 15: 6131-6148 (1987), or a chimeric RNA-DNA analogue
(Inoue et al., FEBS Lett. 215: 327-330 (1987).
[0380] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.,
Nucl. Acids Res. 16: 3209 (1988), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:
7448-7451 (1988), etc.
[0381] While antisense nucleotides complementary to the target gene
coding region sequence could be used, those complementary to the
transcribed, untranslated region are most preferred.
[0382] Antisense molecules should be delivered to cells that
express the target gene in vivo. A number of methods have been
developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systemically.
[0383] However, it is often difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
of endogenous mRNAs. Therefore a preferred approach utilizes a
recombinant DNA construct in which the antisense oligonucleotide is
placed under the control of a strong pol III or pol II promoter.
The use of such a construct to transfect target cells in the
patient will result in the transcription of sufficient amounts of
single stranded RNAs that will form complementary base pairs with
the endogenous target gene transcripts and thereby prevent
translation of the target gene mRNA. For example, a vector can be
introduced e.g., such that it is taken up by a cell and directs the
transcription of an antisense RNA. Such a vector can remain
episomal or become chromosomally integrated, as long as it can be
transcribed to produce the desired antisense RNA. Such vectors can
be constructed by recombinant DNA technology methods standard in
the art. Vectors can be plasmid, viral, or others known in the art,
used for replication and expression in mammalian cells. Expression
of the sequence encoding the antisense RNA can be by any promoter
known in the art to act in mammalian, preferably human cells. Such
promoters can be inducible or constitutive. Such promoters include
but are not limited to: the SV40 early promoter region (Bernoist
and Chambon, Nature 290: 304-310 (1981), the promoter contained in
the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al,
Cell 22: 787-797 (1980), the herpes thymidine kinase promoter
(Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445 (1961),
the regulatory sequences of the metallothionein gene (Brinster et
al., Nature 296: 39-42 (1982), etc. Any type of plasmid, cosmid,
YAC or viral vector can be used to prepare the recombinant DNA
construct which can be introduced directly into the tissue site.
Alternatively, viral vectors can be used that selectively infect
the desired tissue, in which case administration may be
accomplished by another route (e.g., systemically). Ribozyme
molecules designed to catalytically cleave target gene mRNA
transcripts can also be used to prevent translation of target gene
mRNA and, therefore, expression of target gene product. (See e.g.,
PCT International Publication WO90/11364, published Oct. 4, 1990;
Sarver et al, Science 247: 1222-1225 (1990).
[0384] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. (For a review, see Rossi Current
Biology 4: 469-471 (1994). The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by an endonucleolytic
cleavage event. The composition of ribozyme molecules must include
one or more sequences complementary to the target gene mRNA, and
must include the well known catalytic sequence responsible for mRNA
cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246,
which is incorporated herein by reference in its entirety.
[0385] While ribozymes that cleave mRNA at site specific
recognition sequences can be used to destroy target gene mRNAs, the
use of hammerhead ribozymes is preferred. Hammerhead ribozymes
cleave mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Myers,
Molecular Biology and Biotechnology: A Comprehensive Desk
Reference, VCH Publishers, New York, (see especially FIG. 4, page
833 (1995) and in Haseloff and Gerlach, Nature 334: 585-591 (1988),
which is incorporated herein by reference in its entirety.
[0386] Preferably the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the target gene
mRNA, i.e., to increase efficiency and minimize the
intracellular-accumulation of non-functional mRNA transcripts.
[0387] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one that occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-19 IVS RNA) and that has been extensively described by
Thomas Cech and collaborators (Zaug et al., Science 224: 574-578
(1984); Zaug and Cech, Science 231: 470-475 (1986); Zaug et al.,
Nature 324: 429-433 (1986); published International patent
application No. WO 88/04300 by University Patents Inc.; Been and
Cech, Cell 47: 207-216 (1986). The Cech-type ribozymes have an
eight base pair active site which hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes which target eight
base-pair active site sequences that are present in the target
gene.
[0388] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells that express the
target gene in vivo. A preferred method of delivery involves using
a DNA construct "encoding" the ribozyme under the control of a
strong constitutive pol III or pol II promoter, so that transfected
cells will produce sufficient quantities of the ribozyme to destroy
endogenous target gene messages and inhibit translation. Because
ribozymes unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0389] Endogenous target gene expression can also be reduced by
inactivating or "knocking out" the target gene or its promoter
using targeted homologous recombination (e.g., see Smithies et al.,
Nature 317: 230-234 (1985); Thomas and Capecchi, Cell 51: 503-512
(1987); Thompson et al., Cell 5: 313-321 (1989); each of which is
incorporated by reference herein in its entirety). For example, a
mutant, non-functional target gene (or a completely unrelated DNA
sequence) flanked by DNA homologous to the endogenous target gene
(either the coding regions or regulatory regions of the target
gene) can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells that express the
target gene in vivo. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the target
gene. Such approaches are particularly suited in the agricultural
field where modifications to ES (embryonic stem) cells can be used
to generate animal offspring with an inactive target gene (e.g.,
see Thomas and Capecchi, 1987 and Thompson, 1989, supra). However
this approach can be adapted for use in humans provided the
recombinant DNA constructs are directly administered or targeted to
the required site in vivo using appropriate viral vectors.
[0390] Alternatively, endogenous target gene expression can be
reduced by targeting deoxyribonucleotide sequences complementary to
the regulatory region of the target gene (i.e., the target gene
promoter and/or enhancers) to form triple helical structures that
prevent transcription of the target gene in target cells in the
body. (See generally, Helene, Anticancer Drug Des., 6 (6): 569-584
(1991); Helene et al., Ann. N.Y. Acad. Sci., 660: 27-36 (1992); and
Maher, Bioassays 14(12): 807-815 (1992).
[0391] Nucleic acid molecules to be used in triplex helix formation
for the inhibition of transcription should be single stranded and
composed of deoxynucleotides. The base composition of these
oligonucleotides must be designed to promote triple helix formation
via Hoogsteen base pairing rules, which generally require sizeable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based,
which will result in TAT and CGC.sup.+ triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarily to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen that are purine-rich, for example, contain a stretch of G
residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in GGC triplets across the three strands in the
triplex.
[0392] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizeable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0393] In instances wherein the antisense, ribozyme, and/or triple
helix molecules described herein are utilized to inhibit mutant
gene expression, it is possible that the technique may so
efficiently reduce or inhibit the transcription (triple helix)
and/or translation (antisense, ribozyme) of mRNA produced by normal
target gene alleles that the possibility may arise wherein the
concentration of normal target gene product present may be lower
than is necessary for a normal phenotype. In such cases, to ensure
that substantially normal levels of target gene activity are
maintained, therefore, nucleic acid molecules that encode and
express target gene polypeptides exhibiting normal target gene
activity may, be introduced into cells via gene therapy methods
such as those described, below, that do not contain sequences
susceptible to whatever antisense, ribozyme, or triple helix
treatments are being utilized.
[0394] Alternatively, in instances whereby the target gene encodes
an extracellular protein, it may be preferable to co-administer
normal target gene protein in order to maintain the requisite level
of target gene activity.
[0395] Anti-sense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules, as discussed above. These
include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in
the art such as for example solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the
antisense RNA molecule. Such DNA sequences may be incorporated into
a wide variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
[0396] (b) Gene Replacement Therapy
[0397] The GLM-R gene nucleic acid sequences described herein can
be utilized for the treatment of a disorders related to the over or
under abundance of monocytes or macrophages. Such treatment can be
in the form of gene replacement therapy. Specifically, one or more
copies of a normal GLM-R gene or a portion of the GLM-R gene that
directs the production of a GLM-R gene product exhibiting normal
GLM-R gene function, may be inserted into the appropriate cells
within a patient, using vectors that include, but are not limited
to adenovirus, adeno-associated virus, and retrovirus vectors, in
addition to other particles that introduce DNA into cells, such as
liposomes.
[0398] Because the GLM-R gene is expressed in the brain, such gene
replacement therapy techniques should be capable delivering GLM-R
gene sequences to these cell types within patients. Thus, in one
embodiment, techniques that are well known to those of skill in the
art (see, e.g., PCT Publication No. WO89/10134, published Apr. 25,
1988) can be used to enable GLM-R gene sequences to cross the
blood-brain barrier readily and to deliver the sequences to cells
in the brain. With respect to delivery that is capable of crossing
the blood-brain barrier, viral vectors such as, for example, those
described above, are preferable.
[0399] In another embodiment, techniques for delivery involve
direct administration of such GLM-R gene sequences to the site of
the cells in which the GLM-R gene sequences are to be
expressed.
[0400] Additional methods that may be utilized to increase the
overall level of GLM-R gene expression and/or GLM-R gene product
activity include using target homologous recombination methods, to
modify the expression characteristic of an endogenous GLM-R gene in
a cell or microorganism by inserting a heterologous DNA regulatory
element such that the inserted regulatory element is operatively
linked with the endogenous GLM-R gene in question. Targeted
homologous recombination can be thus used to activate transcription
of an endogenous GLM-R gene that is "transcriptionally silent",
i.e., is not normally expressed, or to enhance the expression of an
endogenous GLM-R gene that is normally expressed.
[0401] Further, the overall level of GLM-R gene expression and/or
GLM-R gene product activity may be increased by the introduction of
appropriate GLM-R-expressing cells, preferably autologous cells,
into a patient at positions and in numbers that are sufficient to
ameliorate body weight disorder symptoms. Such cells may be either
recombinant or non-recombinant.
[0402] Among the cells that can be administered to increase the
overall level of GLM-R gene expression in a patient are normal
cells, preferably brain cells, that express the GLM-R gene.
Alternatively, cells, preferably autologous cells, can be
engineered to express GLM-R gene sequences, and may then be
introduced into a patient in positions appropriate for the
amelioration of a symptom of disorders related to the over or under
abundance of monocytes or macrophages. Alternatively, cells that
express an unimpaired GLM-R gene and that are from a MHC matched
individual can be utilized, and may include, for example, brain
cells. The expression of the GLM-R gene sequences is controlled by
the appropriate gene regulatory sequences to allow such expression
in the necessary cell types. Such gene regulatory sequences are
well known to the skilled artisan. Such cell-based gene therapy
techniques are well known to those skilled in the art, see e.g.,
U.S. Pat. No. 5,399,349. When the cells to be administered are
non-autologous cells, they can be administered using well known
techniques that prevent a host immune response against the
introduced cells from developing. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[0403] Additionally, compounds, such as those identified via
techniques such as those described herein, that are capable of
modulating GLM-R gene product activity can be administered using
standard techniques that are well known to those of skill in the
art. In instances in which the compounds to be administered are to
involve an interaction with brain cells, the administration
techniques should include well known ones that allow for a crossing
of the blood-brain barrier.
[0404] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0405] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Isolation of GLM-R cDNAs
[0406] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
sequence databases. The databases included public databases (e.g.,
GenBank). In this instance, genomic DNA sequence from GenBank
(i.e., AC008857) was analyzed using the gene prediction program
GENSCAN, licensed from Stanford University. GENSCAN analysis
predicts gene coding regions, creating sequences which can be
subjected to the ECD search. The search was performed using the
computer program BLAST or BLAST2 [Altschul et al., Methods in
Enzymology, 266:460-480 (1996)] as a comparison of the ECD protein
sequences to a 6 frame translation of the sequences. Those
comparisons resulting in a BLAST score of 70 (or in some cases, 90)
or greater that did not encode known proteins were clustered and
assembled into consensus DNA sequences with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.) if
necessary.
[0407] From the consensus sequence, oligonucleotides were
synthesized: 1) to identify by PCR a cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for PRO21073. Forward and
reverse PCR primers generally range from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 bp in
length. The probe sequences are typically 40-55 bp in length. In
some cases, additional oligonucleotides are synthesized when the
consensus sequence is greater than about 1-1.5 kbp. In order to
screen several libraries for a full-length clone, DNA from the
libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular Biology, supra, with the PCR primer
pair. A positive library was then used to isolate clones encoding
the gene of interest using the probe oligonucleotide and one of the
primer pairs.
[0408] The PCR primers (forward and reverse) were: TABLE-US-00006
forward PCR primer 5'-GTCAAGGAGTCAAAGTTCTGGAGTGACTGG-3' (SEQ ID
NO:3) reverse PCR primer 5'-CGCACATCGCAGAGCTATGACATATTC-3' (SEQ ID
NO:4)
[0409] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA172257 sequence which
had the following nucleotide sequence: TABLE-US-00007 hybridization
probe 5'-CGTACAACCTCACGGGGCTGCAGCCTTTTACAG- (SEQ ID NO:5) 3'
[0410] A pool of 50 different human cDNA libraries from various
tissues was used in cloning. The cDNA libraries used to isolate the
cDNA clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with oligo dT containing a NotI site, linked
with blunt to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[0411] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length GLM-R nucleic
acid (designated herein as DNA173920-2924, FIG. 1, SEQ ID NO:1) and
the derived full length PRO21073 polypeptide.
[0412] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 63-65 and a stop signal at nucleotide
positions 2259-2261 (FIG. 1, SEQ ID NO:1). The predicted
polypeptide precursor is 732 amino acids long, has a calculated
molecular weight of approximately 82954 daltons and an estimated pI
of approximately 7.15. Analysis of the full-length PRO21073
sequence shown in FIG. 2 (SEQ ID NO:2) evidences the presence of a
variety of important polypeptide domains as shown in FIG. 2,
wherein the locations given for those important polypeptide domains
are approximate as described above. Clone DNA173920-2924 was
deposited with ATCC on May 16, 2000 and is assigned ATCC Deposit
No. 1874-PTA.
[0413] The GLM-R (PRO21073) sequence of FIG. 3A has features
characteristic of type I cytokine receptors. A predicted signal
peptide of 19 amino acid residues is followed by a cytokine
receptor homology domain (residue 20-227) with two pairs of
conserved cysteine residues and a WSDWS signature motif. Three
modules with homology to fibronectin type III domains (residues
228-324, 325-420, 421-516) complete the extracellular domain, and a
single transmembrane region (residues 517-539) connects to an
intracellular domain of 193 amino acids (residues 540-732). Within
the cytoplasmic tyrosine kinases of the Jak family, and four
tyrosine residues that may serve as docking sites for downstream
signaling molecules with SH2 domains are present.
Example 2
Use of GLM-R Polynucleotides as Hybridization Probes
[0414] The following method describes use of a nucleotide sequence
encoding SRT as a hybridization probe.
[0415] DNA comprising the coding sequence of full-length or mature
SRT is employed as a probe to screen for homologous DNAs (such as
those encoding naturally-occurring variants of SRT) in human tissue
cDNA libraries or human tissue genomic libraries.
[0416] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled SRT-derived probe to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times. Denhardt's solution, and 10% dextran sulfate at 42oC
for 20 hours. Washing of the filters is performed in an aqueous
solution of 0.1.times.SSC and 0.1% SDS at 42.degree. C.
[0417] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence SRT can then be identified
using standard techniques known in the art.
Example 3
Expression of GLM-R in E. coli
[0418] This example illustrates preparation of an unglycosylated
form of GLM-R by recombinant expression in E. coli.
[0419] The DNA sequence encoding GLM-R is initially amplified using
selected PCR primers. The primers should contain restriction enzyme
sites which correspond to the restriction enzyme sites on the
selected expression vector. A variety of expression vectors may be
employed. An example of a suitable vector is pBR322 (derived from
E. coli; see Bolivar et al, Gene 2:95 (1977)) which contains genes
for ampicillin and tetracycline resistance. The vector is digested
with restriction enzyme and dephosphorylated. The PCR amplified
sequences are then ligated into the vector. The vector will
preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the GLM-R coding region, lambda transcriptional terminator,
and an argU gene.
[0420] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0421] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0422] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized GLM-R protein can then be purified using a
metal chelating column under conditions that allow tight binding of
the protein.
[0423] GLM-R may be expressed in E. coli in a poly-His tagged form,
using the following procedure. The DNA encoding GLM-R is initially
amplified using selected PCR primers. The primers will contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector, and other useful sequences
providing for efficient and reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H.sub.2O, 1.07 g
KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500
mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7
mM MgSO.sub.4) and grown for approximately 20-30 hours at
30.degree. C. with shaking. Samples are removed to verify
expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell pellets are frozen until
purification and refolding.
[0424] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02 M, respectively, and the
solution is stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0425] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0426] Fractions containing the desired folded GLM-R polypeptide
are pooled and the acetonitrile removed using a gentle stream of
nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile
filtered.
Example 4
Expression of GLM-R in Mammalian Cells
[0427] This example illustrates preparation of a potentially
glycosylated form of GLM-R by recombinant expression in mammalian
cells.
[0428] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the GLM-R DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the GLM-R DNA using ligation methods such as described
in Sambrook et al., supra. The resulting vector is called
pRK5-GLM-R.
[0429] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-GLM-R DNA is mixed with about 1 .mu.g DNA encoding
the VA RNA gene [Thimmappaya et al., Cell 31:543 (1982)] and
dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0430] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12-hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of GLM-R polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0431] In an alternative technique, GLM-R may be introduced into
293 cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-GLM-R DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed GLM-R can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0432] In another embodiment, GLM-R can be expressed in CHO cells.
The pRK5-GLM-R can be transfected into CHO cells using known
reagents such as CaPO.sub.4 or DEAE-dextran. As described above,
the cell cultures can be incubated, and the medium replaced with
culture medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of GLM-R
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed GLM-R can then be concentrated and purified by any
selected method.
[0433] Epitope-tagged GLM-R may also be expressed in host CHO
cells. The GLM-R may be subcloned out of the pRK5 vector. The
subclone insert can undergo PCR to fuse in frame with a selected
epitope tag such as a poly-his tag into a Baculovirus expression
vector. The poly-his tagged GLM-R insert can then be subcloned into
a SV40 driven vector containing a selection marker such as DHFR for
selection of stable clones. Finally, the CHO cells can be
transfected (as described above) with the SV40 driven vector.
Labeling may be performed, as described above, to verify
expression. The culture medium containing the expressed poly-His
tagged GLM-R can then be concentrated and purified by any selected
method, such as by Ni.sup.2+-chelate affinity chromatography.
[0434] GLM-R may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[0435] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0436] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0437] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.-7 cells are frozen in an ampule for further growth
and production as described below.
[0438] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH is
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0439] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0440] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Example 5
Expression of GLM-R in Yeast
[0441] The following method describes recombinant expression of
GLM-R in yeast.
[0442] First, yeast expression vectors are constructed for
intracellular production or secretion of GLM-R from the ADH2/GAPDH
promoter. DNA encoding GLM-R and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of GLM-R. For secretion, DNA encoding
GLM-R can be cloned into the selected plasmid, together with DNA
encoding the ADH2/GAPDH promoter, a native GLM-R signal peptide or
other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory signal/leader sequence, and
linker sequences (if needed) for expression of GLM-R.
[0443] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0444] Recombinant GLM-R can subsequently be isolated and purified
by removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing GLM-R may further be
purified using selected column chromatography resins.
Example 6
Expression of GLM-R in Baculovirus-Infected Insect Cells
[0445] The following method describes recombinant expression of
GLM-R in Baculovirus-infected insect cells.
[0446] The sequence coding for GLM-R is fused upstream of an
epitope tag contained within a baculovirus expression vector. Such
epitope tags include poly-his tags and immunoglobulin tags (like Fc
regions of IgG). A variety of plasmids may be employed, including
plasmids derived from commercially available plasmids such as
pVL1393 (Novagen). Briefly, the sequence encoding GLM-R or the
desired portion of the coding sequence of GLM-R such as the
sequence encoding the extracellular domain of a transmembrane
protein or the sequence encoding the mature protein if the protein
is extracellular is amplified by PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the
expression vector.
[0447] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0448] Expressed poly-his tagged GLM-R can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-179 (1993). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL
Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 0.1%
NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged GLM-R are pooled and dialyzed against loading
buffer.
[0449] Alternatively, purification of the IgG tagged (or Fc tagged)
GLM-R can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
Example 7
Preparation of Antibodies that Bind GLM-R
[0450] This example illustrates preparation of monoclonal
antibodies which can specifically bind GLM-R.
[0451] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified GLM-R, fusion
proteins containing GLM-R, and cells expressing recombinant GLM-R
on the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0452] A construct encoding the extracellular domain of human GLM-R
fused to an octahistidine tag was derived by recombinant PCR and
cloned into a modified version of the pVL1393 baculovirus
expression vector (BD Pharmingen, San Diego, Calif.).
GLM-R-His.sub.8 was expressed in High-five insect cells
(Invitrogen, Carlsbad, Calif.) and purified by
Nickel-nitrilo-triacetic and affinity column. Monoclonal antibodies
against GLM-R-His.sub.8 were raised in balb/c mice in a manner
similar to that described below.
[0453] Mice, such as Balb/c, are immunized with the GLM-R immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-GLM-R antibodies.
[0454] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of GLM-R. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU. 1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0455] The hybridoma cells will be screened in an ELISA for
reactivity against GLM-R. Determination of "positive" hybridoma
cells secreting the desired monoclonal antibodies against GLM-R is
within the skill in the art.
[0456] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-GLM-R monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or
protein G can be employed.
Example 8
Purification of GLM-R Polypeptides Using Specific Antibodies
[0457] Native or recombinant GLM-R polypeptides may be purified by
a variety of standard techniques in the art of protein
purification. For example, pro-GLM-R polypeptide, mature GLM-R
polypeptide, or pre-GLM-R polypeptide is purified by immunoaffinity
chromatography using antibodies specific for the GLM-R polypeptide
of interest. In general, an immunoaffinity column is constructed by
covalently coupling the anti-GLM-R polypeptide antibody to an
activated chromatographic resin.
[0458] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0459] Such an immunoaffinity column is utilized in the
purification of GLM-R polypeptide by preparing a fraction from
cells containing GLM-R polypeptide in a soluble form. This
preparation is derived by solubilization of the whole cell or of a
subcellular fraction obtained via differential centrifugation by
the addition of detergent or by other methods well known in the
art. Alternatively, soluble GLM-R polypeptide containing a signal
sequence may be secreted in useful quantity into the medium in
which the cells are grown.
[0460] A soluble GLM-R polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of GLM-R
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/GLM-R polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and GLM-R polypeptide
is collected.
Example 9
Drug Screening
[0461] This invention is particularly useful for screening
compounds by using GLM-R polypeptides or binding fragment thereof
in any of a variety of drug screening techniques. The GLM-R
polypeptide or fragment employed in such a test may either be free
in solution, affixed to a solid support, borne on a cell surface,
or located intracellularly. One method of drug screening utilizes
eukaryotic or prokaryotic host cells which are stably transformed
with recombinant nucleic acids expressing the GLM-R polypeptide or
fragment. Drugs are screened against such transformed cells in
competitive binding assays. Such cells, either in viable or fixed
form, can be used for standard binding assays. One may measure, for
example, the formation of complexes between GLM-R polypeptide or a
fragment and the agent being tested. Alternatively, one can examine
the diminution in complex formation between the GLM-R polypeptide
and its target cell or target receptors caused by the agent being
tested.
[0462] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a GLM-R
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an GLM-R polypeptide or fragment
thereof and assaying (I) for the presence of a complex between the
agent and the GLM-R polypeptide or fragment, or (ii) for the
presence of a complex between the GLM-R polypeptide or fragment and
the cell, by methods well known in the art. In such competitive
binding assays, the GLM-R polypeptide or fragment is typically
labeled. After suitable incubation, free GLM-R polypeptide or
fragment is separated from that present in bound form, and the
amount of free or uncomplexed label is a measure of the ability of
the particular agent to bind to GLM-R polypeptide or to interfere
with the GLM-R polypeptide/cell complex.
[0463] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on 13 Sep. 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to a GLM-R polypeptide, the peptide test compounds are reacted with
GLM-R polypeptide and washed. Bound GLM-R polypeptide is detected
by methods well known in the art. Purified GLM-R polypeptide can
also be coated directly onto plates for use in the aforementioned
drug screening techniques. In addition, non-neutralizing antibodies
can be used to capture the peptide and immobilize it on the solid
support.
[0464] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding GLM-R polypeptide specifically compete with a test compound
for binding to GLM-R polypeptide or fragments thereof. In this
manner, the antibodies can be used to detect the presence of any
peptide which shares one or more antigenic determinants with GLM-R
polypeptide.
Example 10
Rational Drug Design
[0465] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., an
GLM-R polypeptide) or of small molecules with which they interact,
e.g., agonists, antagonists, or inhibitors. Any of these examples
can be used to fashion drugs which are more active or stable forms
of the GLM-R polypeptide or which enhance or interfere with the
function of the GLM-R polypeptide in vivo (Cf., Hodgson,
Bio/Technology, 9: 19-21 (1991)).
[0466] In one approach, the three-dimensional structure of the
GLM-R polypeptide, or of an GLM-R polypeptide-inhibitor complex, is
determined by x-ray crystallography, by computer modeling or, most
typically, by a combination of the two approaches. Both the shape
and charges of the GLM-R polypeptide must be ascertained to
elucidate the structure and to determine active site(s) of the
molecule. Less often, useful information regarding the structure of
the GLM-R polypeptide may be gained by modeling based on the
structure of homologous proteins. In both cases, relevant
structural information is used to design analogous GLM-R
polypeptide-like molecules or to identify efficient inhibitors.
Useful examples of rational drug design may include molecules which
have improved activity or stability as shown by Braxton and Wells,
Biochemistry, 31:7796-7801 (1992) or which act as inhibitors,
agonists, or antagonists of native peptides as shown by Athauda et
al., J. Biochem., 113:742-746 (1993).
[0467] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The anti-id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides would then
act as the pharmacore.
[0468] By virtue of the present invention, sufficient amounts of
the GLM-R polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the GLM-R polypeptide amino acid sequence provided herein will
provide guidance to those employing computer modeling techniques in
place of or in addition to x-ray crystallography.
Example 11
Isolation of GLM-R cDNAs and Cloning of hGH-R/GLM-R Chimeric
Receptor
[0469] As reported in Example 1, cDNA encoding full length human
GLM-R was subsequently cloned from a pooled tissue cDNA library.
Murine GLM-R was obtained by a combination of cross-species library
screening, and polymerase chain reaction (PCR) from a murine spleen
library.
[0470] Murine GLM-R, a slightly shorter homolog with 716 amino acid
residues, was cloned from a spleen library and shows 59.1% identity
and 67.5% identity to the human molecule (FIG. 3B). All hallmark
features except the second pair of cysteines and two cytoplasmic
tyrosine residues are conserved between the two sequences. The lack
of the cysteines in the murine protein does not appear to be due to
alternative mRNA splicing, since analysis of murine genomic
sequences did not reveal the presence of an alternative exom with
those features (not shown).
[0471] As shown in FIG. 3C, human GLM-R is most homologous to the
IL-6 signal transducer chain, gp103 [Hibi et al., Cell 63(6):
1149-57 (1990)], at 24.73% identity, followed by GCSF-R at 23.94%
identity [Larsen et al., J. Exp. Med. 172(6): 1559-70 (1990)], and
IL-12b2 at 20.09% identity [Presky, D. H. et al., Proc. Natl. Acad.
Sci. USA 93(24): 14002-7 (1996)]. Interestingly, analysis of
genomic sequences indicated that GLM-R and gp130 are separated by
only 24 kilobases (kb) on human chromosome 5, and by 19 kb in a
synthetic region on mouse chromosome 13. In light of the relatively
low level of sequence conservation, this chromosomal localization
pattern further confirms that mGLM-R and hGLM-R are true
homologues.
[0472] Primers for the PCR were designated based on sequences
obtained by data mining in the murine genomic database (Celera).
Two clones stemming from independent PCR reactions were sequenced
and confirmed to match with each other and the mRNA sequence
predicted from genomic DNA using Genscan software. Burge, C. and
Karlin, S. J., Mol. Biol. 268 (1): 78-94 (1997). A cDNA encoding a
chimerica molecule, consisting of the extracellular domain of human
GH-R and the intracellular domain of human GLM-R was obtained by
recombinant PCR, [Ho, S. N. et al., Gene 77(1): 51-9 (1989)] using
the partially complementary primers
5'-CTTTTCGAAACAGCAAAGGAAACCCAACAAATTGACTCA-3'(sense) (SEQ ID NO:6)
and 5'-GGGTTTCCTTTGCTGTTTCGAAAAGAGAAAAAC-3' (antisense) (SEQ ID
NO:7). This construct was cloned under the control of a CMV
promoter into the expression vector pRK5tkneo.
[0473] To gain insight into the potential function of this
receptor, we next sought to determine the expression pattern of
GLM-R. The abundance of this transcript was generally so low that
we were unable to reliably detect it by northern blot analysis in
any organ (not shown). Supporting low expression levels is an
absence of human expressed sequence tags (EST) corresponding to
GLM-R in the public databases. We therefore analyzed GLM-R
expression in a comprehensive panel of human total RNAs by real
time quantitative PCR (Taqman.TM.), using primers located in exon
11 (FIG. 4A). Highest levels of GLM-R transcript were detected in
testis, prostate, thymus, bone marrow and trachea. GLM-R
amplification product from testis RNA became detectable after 25
cycles of PCR (thymus, 26 cycles; prostate, 27 cycles), whereas
rpl-19 amplification product was detectable after 18 cycles
(thymus, 17 cycles; prostate, 18 cycles). Therefore, GLM-R
expression in testis was roughly 2.sup.7=128 fold lower than rpl-19
expression (thymus, prostate, 2.sup.9=512 fold lower). Using a
similar calculation, it can be determined that in most tissues,
GLM-R is expressed at 10.sup.3 to 10.sup.4 fold lower levels than
rpl-19.
[0474] Because type I cytokine receptors frequently play a role in
blood cell development and function, and because GLM-R expression
levels were comparably high in thymus and bone marrow, we were
interested in the expression of GLM-R on blood cell subsets. To
this end, peripheral blood mononuclear cells (PBMC) subsets were
isolated from healthy human volunteers by Ficoll density gradient
centrifugation followed by magnetic bead separation. Taqman.TM. PCR
was then performed on RNA isolated from those cell fractions, using
primers located in exon 11 of human GLM-R (FIG. 4B). Again, the
absolute levels of GLM-R expression were very low, but CD14
positive and, to a lesser extent, CD56 positive cells displayed
significantly higher expression than CD4, CD8, or CD19 positive
cells. This expression pattern was confirmed by FACS analysis with
monoclonal antibodies raised against the extracellular domain of
GLM-R. GLM-R protein was only detectable at low to moderate levels
on CD14 positive cells, and was barely detectable on CD56 positive
cells. No GLM-R was expressed on CD4, CD8, or CD19 positive cells.
Similar results were obtained from 4 independent blood donors, and
a representative set of histograms is shown in FIG. 4. Compatible
with the monocyte-specific expression of GLM-R, we found high
levels of GLM-R transcripts in two monocytic human cell lines,
THP-1 and U937 (FIG. 4D), whereas all other cell lines tested did
not express GLM-R. Finally, we found that GLM-R was induced between
56 and 91 fold in freshly isolated human monocytes after 4 hours of
stimulation with a combination of 1 .mu.g/ml lipopolysaccharide
(LPS) and 100 ng/ml interferon-.gamma. (IFN-.gamma.) (FIG. 4E).
Again, GLM-R induction was confirmed at the protein level by FACS
(not shown). Upregulation of GLM-R was not observed upon activation
of T- or B-cells with appropriate stimuli, suggesting that this is
a phenomenon restricted to monocytes (not shown).
[0475] To address whether GLM-R is capable of transmitting a signal
upon activation, we constructed a chimeric molecule consisting of
the extracellular and transmembrane domains of human GH (hGH)
receptor, joined to the cytoplasmic region of human GLM-R (FIG.
5A). This construct was stably transfected into IL-3 dependent
murine 32D cells [Greenberger, J. S. et al., Proc. Natl. Acad. Sci.
USA 80(10): 2931-5 (1983)], and three clones staining positive with
an anti-hGH-R antibody were used for further analysis (FIG. 5B).
All clones gave comparable results in subsequent assays.
[0476] First, we examined whether the hGH-R/GLM-R chimera could
signal for proliferation when stimulated with hGH (FIG. 5C). We
found that only hGH-R/GLM-R transfected cells were able to
proliferate in a dose dependent manner in response to hGH, while
both transfected and parental cells proliferated comparably in IL-3
(FIG. 5D).
[0477] The Jak/STAT pathway is critical to transmit the signal
generated by cytokine receptors, Ihle, J. N., Nature 377 (6550):
591-4 (1995), and STAT proteins were previously shown to transmit
many of the specific effects of cytokines. Ihle, J. N., Curr. Opin.
Cell Biol. 13(2): 211-7 (2001). To analyze which of the STAT
proteins are activated upon stimulation of the chimeric receptor
with hGH, an electrophoretic mobility shift analysis (EMSA) was
performed. A mutated form of the serum inducible element of the fos
promoter (m67) [Wagnerm B. J. et al., Embo. J. 9(13): 4477-84
(1990)] was used to test for STAT-1, STAT-3, and STAT-4 activation,
and the mammary gland factor response element of the .beta.-casein
gene (.beta.CAS) [Schmitt-Ney et al., Mol. Cell. Biol. 11(7):
3745-55 (1991)] was used to test for STAT-5 and STAT-6 activation.
Upon hGH stimulation, hGH-R/GLM-R transfected cells displayed
formation of a strong complex on the m67 probe, while parental
cells did not respond. This complex was completely supershifted
with an antibody against STAT-3 (FIG. 6A). A less intense yet
clearly identifiable complex was present when extracts were
incubated with the .beta.CAS probe, and this complex was
supershifted completely with an antibody against STAT-5 (FIG. 6B).
STAT-3 and STAT-5 were also activated upon stimulation with IL-3 in
both parental and transfected cells, as described previously [Mu,
S. et al., Blood 86(12): 4532-4543; Pallard, C. et al., J. Biol.
Chem. 270(27): 15942-5 (1995)] (23,24) (FIGS. 6A and 6B). To
exclude the presence of interferon stimulated gene factor 3, a
complex containing activated STAT-1 and STAT-2, extracts were also
tested on the interferon stimulated response element (ISRE) (25)
probe [Reich, N. et al., Proc. Natl. Sci. USA 84(18): 6394-8
(1987)], but did nor observe any hGH specific gelshifts (not
shown). Thus, no STAT molecules other than STAT-3 and STAT-5 are
activated by hGH-R/GLM-R under these conditions. Specific
activation of STAT-3 and STAT-5 upon activation of hGH-R/GLM-R was
confirmed by phosphotyrosine-immunoprecipitation followed by
western blot with antibodies specific for STAT-3 and STAT-5 (FIG.
6C).
Example 12
Quantitative PCR Analysis of GLM-R Expression
[0478] Total RNA from human organs was obtained from Clontech (Palo
Alto, Calif.), and total RNA from cell lines or sorted cells was
isolated using the Rneasy kit and DNAse I (Qiagen, Valencia,
Calif.). Taqman.TM. quantitative RT-PCR using a sequence detector
7700 instrument was carried out according to the instructions of
the manufacturer (Applied Biosystems, Foster City, Calif.). For
each sample, duplicate test reactions and a control reaction into
which no reverse transcriptase had been added were analyzed for
expression of GLM-R mRNA and a housekeeper mRNA, rpl-19. If a
signal was observed in the control reaction due to contamination
with genomic DNA, it was subtracted from the signal in the test
reaction. Arbitrary expression units were calculated by dividing
GLM-R expression by rpl-19 expression. Probes and primers were
designed using Primer Express software (Applied Biosystems, Foster
City, Calif.). The primer triplets were 5'-CCTGGAGTCCCTGAAACGAA-3'
(sense)(SEQ ID NO:8), 5'-GTTGGTTCCCCCAGCACTG-3' (antisense)(SEQ ID
NO:9), 5'-CTCTTACATTGTTCAGGTCATGGCCAGCA-3' (probe)(SEQ ID NO:10)
for human GLM-R, and 5'-GATGCCGGAAAAACACCTTG-3' (sense)(SEQ ID
NO:11), 5'-TGGCTGTACCCTTCCGCTT-3' (antisense)(SEQ ID NO:12),
5'-CCTATGCCCATGTGCCTGCCCTT-3' (probe)(SEQ ID NO:13) for human
rpl-19.
Example 13
Isolation of Blood Cell Subsets, FACS Analysis and Activation of
Monocytes
[0479] Heparinized blood was obtained with informed consent from
healthy volunteers. 35 ml of a 1:2 dilution of blood in phosphate
buffered saline (PBS) were layered over 15 ml Ficoll-Hypaque (ICN
Biomedicals, Costa Mesa, Calif.) and centrifuged for 30 minutes at
500.times.g. Interphase peripheral blood mononuclear cells (PBMC)
were recovered and washed once with PBS. For RNA isolation,
leukocyte subsets were separated using paramagnetic beads coupled
to various marker antibodies according to the instructions of the
manufacturer (Milteny, Auburn, Calif.). For FACS analysis, PBMC
were incubated for 30 minutes on ice in a buffer containing 10
.mu.g/ml total human IgG and 5 .mu.g/ml murine IgG1 (Sigma, St.
Louis, Mo.) to prevent Fc-receptor mediated binding of GLM-R
antibodies. Cells were then stained with 1 .mu.g per million cells
of biotinylated anti-GLM-R (IgG1) or biotinylated isotype control
antibody for 15 minutes, followed by two washes with the same
buffer. In a second round of staining, cells were simultaneously
incubated with streptavidin-coupled phycoerythrin (strep-PE) and
various marker antibodies directly coupled to either
fluorescin-isothiocyanate or Cychrome (BD Pharmingen, San Diego,
Calif.). Fluorescence was detected using an Epics-XL flow cytometry
system (Beckman Coulter Inc., Fullerton, Calif.). For stimulation
experiments, isolation of monocytes from PBMC was performed by a
depletion strategy employing paramagnetic beads coupled to
antibodies against CD3, CD7, CD19, CD45RA, CD56, and IgE (Milteny,
Auburn, Calif.). We chose this approach to avoid activation of
monocytes by ligation of the CD14 antigen, which would occur in a
positive selection approach. These monocytes were stimulated in
RPMI supplemented with 10% bovine calf serum,
penicillin-streptomycin, and L-glutamine (Invitrogen, Carlsbad,
Calif.) at 2.5.times.10.sup.6 cells/ml with 1 .mu.g/ml LPS (Sigma,
St. Louis, Mo.) and 100 ng/ml IFN.gamma.(R&D Systems,
Minneapolis, Minn.) for 4 hours.
Example 14
Culture and Transfection of 32D Cells
[0480] 32D cells were maintained in RPMI supplemented with 10%
bovine calf serum, L-Glutamine and Penicillin-Streptomycin
(Invitrogen, Carlsbad, Calif.). Conditioned medium from WEHI-3B
cells was used as a source of IL-3 and added to the culture at 5 to
10% final concentration. Cells were transfected by electroporation
and bulk selected in 0.4 mg/ml G418 (Invitrogen, Carlsbad, Calif.)
for 10 days. G418-resistant cells were then stained with a
monoclonal antibody against hGH-R (Genentech, South San Francisco,
Calif.), and single positive cells were sorted by FACS into
individual wells of 96-well plates. After one week of expansion,
clones were re-examined by FACS for hGH-R surface expression and by
proliferation assay for factor dependence. Three clones with
significant hGH-R expression and low background proliferation were
selected for further experiments.
Example 15
Proliferation Assay
[0481] Cells were starved for 20 hours in complete medium without
growth factors at a density of 5.times.10.sup.5 cells/ml.
Subsequently, 5.times.10.sup.4 cells per well were seeded into 96
well plates containing different concentrations of hGH or WEHI-3B
conditioned medium in triplicates. Cells were allowed to
proliferate for 22 hours with addition of 1 .mu.Ci
.sup.3H-thymidine per well during the last six hour of the
incubation period. Thymidine incorporation was determined using a
Top Count liquid scintillation counter according to the
manufacturers instructions (Packard Instruments, Meriden,
Conn.).
Example 16
Analysis of STAT Activation
[0482] 10.sup.7 cells per condition were washed free of 1-3 and
starved for 6 hours in RPMI supplemented with 10% bovine calf
serum. Purified recombinant hGH (Genentech Inc., South San
Francisco, Calif.) or murine IL-3 (R&D Systems, Minneapolis,
Minn.) were added to final concentrations of 100 ng/ml and 10
ng/ml, respectively. After 15 minutes at 37.degree. C., cells were
quick-chilled in icewater and washed once with ice-cold PBS. EMSA
was performed as described in Levy et al., Genes Dev. 3(9): 1362-71
(1989), and gelshifts were detected with the oligonucleotide probes
m67 5'-CATTTCCCGTAAATCAT-3' (SEQ ID NO:14) [Wagner, B. J. et al.,
Embo J. 9(13): 4477-84 (1990)], and .beta.CAS
5'-GATTTCTAGGAATTCAATCC-3' (SEQ ID NO:15) [Schmitt-Ney, M. et al.,
Mol. Cell. Biol. 11(7): 3745-55 (1991)]. For supershift
experiments, polyclonal anti-STAT-1 (sc-464.times.), anti-STAT-3
(sc-482.times.) and anti STAT-5 (sc-835.times.) (all Santa Cruz
Biotechnology, Santa Cruz, Calif.) were used. For western blot
analysis, cells were lysed in a buffer containing 50 mM Tris pH
7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 1% Triton X-100,
2 mM NaVO.sub.4, and complete.TM. protease inhibitors (Roche
Molecular Biochemicals, Indianapolis, Ind.). After 20 minutes on
ice, the lysates were centrifuged at 20000.times.g at 2.degree. C.,
and the supernatants were used for immunoprecipitation.
Precipitation of tyrosine phosphorylated proteins was carried out
using a 1:1 mixture of 4G10-agarose (Upstate Biotechnology Inc.,
Lake Placid, N.Y.) and PY20-agarose (BD transduction labs,
Lexington, Ky.). After washing 3.times. with lysis buffer, the
immunoprecipitated proteins were separated by SDS-PAGE and
transferred to nitrocellulose by western blot. STAT-3 was detected
by sc-482, and STAT-5 was detected by sc-835 (Santa Cruz
Biotechnology, Santa Cruz, Calif.) and enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech, Piscataway, N.J.).
Example 17
Tissue Expression Distribution
[0483] Oligonucleotide probes were constructed from the PRO21073
polypeptide-encoding nucleotide sequence shown in FIG. 1 for use in
quantitative PCR amplification reactions. The oligonucleotide
probes were chosen so as to give an approximately 200-600 base pair
amplified fragment from the 3' end of its associated template in a
standard PCR reaction. The oligonucleotide probes were employed in
standard quantitative PCR amplification reactions with cDNA
libraries isolated from different human adult and/or fetal tissue
sources and analyzed by agarose gel electrophoresis so as to obtain
a quantitative determination of the level of expression of the
PRO21073 polypeptide-encoding nucleic acid in the various tissues
tested. Knowledge of the expression pattern or the differential
expression of the PRO21073 polypeptide-encoding nucleic acid in
various different human tissue types provides a diagnostic marker
useful for tissue typing, with or without other tissue-specific
markers, for determining the primary tissue source of a metastatic
tumor, disease diagnosis, and the like. These assays provided the
following results. TABLE-US-00008 DNA Molecule Tissues
w/Significant Expression DNA173920-2924 Highly expressed in testis,
HUVEC, prostate and uterus. Expressed in cartilage, heart, bone
marrow and spleen. Tissues w/o Significant Expression
DNA173290-2924 Not expressed in colon tumor, placenta, adrenal
gland and aortic endothelial cells.
[0484] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC): TABLE-US-00009 Material ATCC Dep. No.
Deposit Date DNA173920-2924 1874-PTA May 16, 2000
[0485] This deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 8860G 638).
[0486] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0487] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
15 1 2481 DNA Homo sapien 1 catgtgtctg tgaatgtccg caaaacattc
tctctcccca gccttcatgt 50 gttaacctgg ggatgatgtg gacctgggca
ctgtggatgc tcccttcact 100 ctgcaaattc agcctggcag ctctgccagc
taagcctgag aacatttcct 150 gtgtctacta ctataggaaa aatttaacct
gcacttggag tccaggaaag 200 gaaaccagtt atacccagta cacagttaag
agaacttacg cttttggaga 250 aaaacatgat aattgtacaa ccaatagttc
tacaagtgaa aatcgtgctt 300 cgtgctcttt tttccttcca agaataacga
tcccagataa ttataccatt 350 gaggtggaag ctgaaaatgg agatggtgta
attaaatctc atatgacata 400 ctggagatta gagaacatag cgaaaactga
accacctaag attttccgtg 450 tgaaaccagt tttgggcatc aaacgaatga
ttcaaattga atggataaag 500 cctgagttgg cgcctgtttc atctgattta
aaatacacac ttcgattcag 550 gacagtcaac agtaccagct ggatggaagt
caacttcgct aagaaccgta 600 aggataaaaa ccaaacgtac aacctcacgg
ggctgcagcc ttttacagaa 650 tatgtcatag ctctgcgatg tgcggtcaag
gagtcaaagt tctggagtga 700 ctggagccaa gaaaaaatgg gaatgactga
ggaagaagct ccatgtggcc 750 tggaactgtg gagagtcctg aaaccagctg
aggcggatgg aagaaggcca 800 gtgcggttgt tatggaagaa ggcaagagga
gccccagtcc tagagaaaac 850 acttggctac aacatatggt actatccaga
aagcaacact aacctcacag 900 aaacaatgaa cactactaac cagcagcttg
aactgcatct gggaggcgag 950 agcttttggg tgtctatgat ttcttataat
tctcttggga agtctccagt 1000 ggccaccctg aggattccag ctattcaaga
aaaatcattt cagtgcattg 1050 aggtcatgca ggcctgcgtt gctgaggacc
agctagtggt gaagtggcaa 1100 agctctgctc tagacgtgaa cacttggatg
attgaatggt ttccggatgt 1150 ggactcagag cccaccaccc tttcctggga
atctgtgtct caggccacga 1200 actggacgat ccagcaagat aaattaaaac
ctttctggtg ctataacatc 1250 tctgtgtatc caatgttgca tgacaaagtt
ggcgagccat attccatcca 1300 ggcttatgcc aaagaaggcg ttccatcaga
aggtcctgag accaaggtgg 1350 agaacattgg cgtgaagacg gtcacgatca
catggaaaga gattcccaag 1400 agtgagagaa aggggatcat ctgcaactac
accatctttt accaagctga 1450 aggtggaaaa ggattctcca agacagtcaa
ttccagcatc ttgcagtacg 1500 gcctggagtc cctgaaacga aagacctctt
acattgttca ggtcatggcc 1550 agcaccagtg ctgggggaac caacgggacc
agcataaatt tcaagacatt 1600 gtcattcagt gtctttgaga ttatcctcat
aacttctctg attggtggag 1650 gccttcttat tctcattatc ctgacagtgg
catatggtct caaaaaaccc 1700 aacaaattga ctcatctgtg ttggcccacc
gttcccaacc ctgctgaaag 1750 tagtatagcc acatggcatg gagatgattt
caaggataag ctaaacctga 1800 aggagtctga tgactctgtg aacacagaag
acaggatctt aaaaccatgt 1850 tccaccccca gtgacaagtt ggtgattgac
aagttggtgg tgaactttgg 1900 gaatgttctg caagaaattt tcacagatga
agccagaacg ggtcaggaaa 1950 acaatttagg aggggaaaag aatgggtatg
tgacctgccc cttcaggcct 2000 gattgtcccc tggggaaaag ttttgaggag
ctcccagttt cacctgagat 2050 tccgcccaga aaatcccaat acctacgttc
gaggatgcca gaggggaccc 2100 gcccagaagc caaagagcag cttctctttt
ctggtcaaag tttagtacca 2150 gatcatctgt gtgaggaagg agccccaaat
ccatatttga aaaattcagt 2200 gacagccagg gaatttcttg tgtctgaaaa
acttccagag cacaccaagg 2250 gagaagtcta aatgcgacca tagcatgaga
ccctcggggc ctcagtgtgg 2300 atggcccttg ccagagaaga tgtcaagact
cggcatgcag cgcttgcttg 2350 gccctgccac atcctgccta ggttaaagtt
tcccctgccc cttgagctgc 2400 cagttgaact tggtcggcaa agatgcgacc
ttgtactggg aagaagggat 2450 ggtgataagc ccgagttttg taaaggaaaa a 2481
2 732 PRT Homo sapien 2 Met Met Trp Thr Trp Ala Leu Trp Met Leu Pro
Ser Leu Cys Lys 1 5 10 15 Phe Ser Leu Ala Ala Leu Pro Ala Lys Pro
Glu Asn Ile Ser Cys 20 25 30 Val Tyr Tyr Tyr Arg Lys Asn Leu Thr
Cys Thr Trp Ser Pro Gly 35 40 45 Lys Glu Thr Ser Tyr Thr Gln Tyr
Thr Val Lys Arg Thr Tyr Ala 50 55 60 Phe Gly Glu Lys His Asp Asn
Cys Thr Thr Asn Ser Ser Thr Ser 65 70 75 Glu Asn Arg Ala Ser Cys
Ser Phe Phe Leu Pro Arg Ile Thr Ile 80 85 90 Pro Asp Asn Tyr Thr
Ile Glu Val Glu Ala Glu Asn Gly Asp Gly 95 100 105 Val Ile Lys Ser
His Met Thr Tyr Trp Arg Leu Glu Asn Ile Ala 110 115 120 Lys Thr Glu
Pro Pro Lys Ile Phe Arg Val Lys Pro Val Leu Gly 125 130 135 Ile Lys
Arg Met Ile Gln Ile Glu Trp Ile Lys Pro Glu Leu Ala 140 145 150 Pro
Val Ser Ser Asp Leu Lys Tyr Thr Leu Arg Phe Arg Thr Val 155 160 165
Asn Ser Thr Ser Trp Met Glu Val Asn Phe Ala Lys Asn Arg Lys 170 175
180 Asp Lys Asn Gln Thr Tyr Asn Leu Thr Gly Leu Gln Pro Phe Thr 185
190 195 Glu Tyr Val Ile Ala Leu Arg Cys Ala Val Lys Glu Ser Lys Phe
200 205 210 Trp Ser Asp Trp Ser Gln Glu Lys Met Gly Met Thr Glu Glu
Glu 215 220 225 Ala Pro Cys Gly Leu Glu Leu Trp Arg Val Leu Lys Pro
Ala Glu 230 235 240 Ala Asp Gly Arg Arg Pro Val Arg Leu Leu Trp Lys
Lys Ala Arg 245 250 255 Gly Ala Pro Val Leu Glu Lys Thr Leu Gly Tyr
Asn Ile Trp Tyr 260 265 270 Tyr Pro Glu Ser Asn Thr Asn Leu Thr Glu
Thr Met Asn Thr Thr 275 280 285 Asn Gln Gln Leu Glu Leu His Leu Gly
Gly Glu Ser Phe Trp Val 290 295 300 Ser Met Ile Ser Tyr Asn Ser Leu
Gly Lys Ser Pro Val Ala Thr 305 310 315 Leu Arg Ile Pro Ala Ile Gln
Glu Lys Ser Phe Gln Cys Ile Glu 320 325 330 Val Met Gln Ala Cys Val
Ala Glu Asp Gln Leu Val Val Lys Trp 335 340 345 Gln Ser Ser Ala Leu
Asp Val Asn Thr Trp Met Ile Glu Trp Phe 350 355 360 Pro Asp Val Asp
Ser Glu Pro Thr Thr Leu Ser Trp Glu Ser Val 365 370 375 Ser Gln Ala
Thr Asn Trp Thr Ile Gln Gln Asp Lys Leu Lys Pro 380 385 390 Phe Trp
Cys Tyr Asn Ile Ser Val Tyr Pro Met Leu His Asp Lys 395 400 405 Val
Gly Glu Pro Tyr Ser Ile Gln Ala Tyr Ala Lys Glu Gly Val 410 415 420
Pro Ser Glu Gly Pro Glu Thr Lys Val Glu Asn Ile Gly Val Lys 425 430
435 Thr Val Thr Ile Thr Trp Lys Glu Ile Pro Lys Ser Glu Arg Lys 440
445 450 Gly Ile Ile Cys Asn Tyr Thr Ile Phe Tyr Gln Ala Glu Gly Gly
455 460 465 Lys Gly Phe Ser Lys Thr Val Asn Ser Ser Ile Leu Gln Tyr
Gly 470 475 480 Leu Glu Ser Leu Lys Arg Lys Thr Ser Tyr Ile Val Gln
Val Met 485 490 495 Ala Ser Thr Ser Ala Gly Gly Thr Asn Gly Thr Ser
Ile Asn Phe 500 505 510 Lys Thr Leu Ser Phe Ser Val Phe Glu Ile Ile
Leu Ile Thr Ser 515 520 525 Leu Ile Gly Gly Gly Leu Leu Ile Leu Ile
Ile Leu Thr Val Ala 530 535 540 Tyr Gly Leu Lys Lys Pro Asn Lys Leu
Thr His Leu Cys Trp Pro 545 550 555 Thr Val Pro Asn Pro Ala Glu Ser
Ser Ile Ala Thr Trp His Gly 560 565 570 Asp Asp Phe Lys Asp Lys Leu
Asn Leu Lys Glu Ser Asp Asp Ser 575 580 585 Val Asn Thr Glu Asp Arg
Ile Leu Lys Pro Cys Ser Thr Pro Ser 590 595 600 Asp Lys Leu Val Ile
Asp Lys Leu Val Val Asn Phe Gly Asn Val 605 610 615 Leu Gln Glu Ile
Phe Thr Asp Glu Ala Arg Thr Gly Gln Glu Asn 620 625 630 Asn Leu Gly
Gly Glu Lys Asn Gly Tyr Val Thr Cys Pro Phe Arg 635 640 645 Pro Asp
Cys Pro Leu Gly Lys Ser Phe Glu Glu Leu Pro Val Ser 650 655 660 Pro
Glu Ile Pro Pro Arg Lys Ser Gln Tyr Leu Arg Ser Arg Met 665 670 675
Pro Glu Gly Thr Arg Pro Glu Ala Lys Glu Gln Leu Leu Phe Ser 680 685
690 Gly Gln Ser Leu Val Pro Asp His Leu Cys Glu Glu Gly Ala Pro 695
700 705 Asn Pro Tyr Leu Lys Asn Ser Val Thr Ala Arg Glu Phe Leu Val
710 715 720 Ser Glu Lys Leu Pro Glu His Thr Lys Gly Glu Val 725 730
3 30 DNA Artificial sequence Artificial Sequence Full Forward PCR
Primer 3 gtcaaggagt caaagttctg gagtgactgg 30 4 27 DNA Artificial
sequence Artificial Sequence Full Reverse PCR Primer 4 cgcacatcgc
agagctatga catattc 27 5 33 DNA Artificial sequence Artificial
Sequence Full Hybridization probe 5 cgtacaacct cacggggctg
cagcctttta cag 33 6 39 DNA Artificial sequence Artificial Sequence
Full PCR Primer (sense) 6 cttttcgaaa cagcaaagga aacccaacaa
attgactca 39 7 33 DNA Artificial sequence Artificial Sequence Full
PCR Primer (antisense) 7 gggtttcctt tgctgtttcg aaaagagaaa aac 33 8
20 DNA Artificial sequence Artificial Sequence Full PCR Primer
(sense) 8 cctggagtcc ctgaaacgaa 20 9 19 DNA Artificial sequence
Artificial Sequence Full PCR Primer (antisense) 9 gttggttccc
ccagcactg 19 10 29 DNA Artificial sequence Artificial Sequence Full
PCR Primer (probe) 10 ctcttacatt gttcaggtca tggccagca 29 11 20 DNA
Artificial sequence Artificial Sequence Full PCR Primer (sense) 11
gatgccggaa aaacaccttg 20 12 19 DNA Artificial sequence Artificial
Sequence Full PCR Primer (antisense) 12 tggctgtacc cttccgctt 19 13
23 DNA Artificial sequence Artificial Sequence Full PCR Primer
(probe) 13 cctatgccca tgtgcctgcc ctt 23 14 17 DNA Artificial
sequence Artificial Sequence Full PCR Primer 14 catttcccgt aaatcat
17 15 20 DNA Artificial sequence Artificial Sequence Full PCR
Primer 15 gatttctagg aattcaatcc 20
* * * * *
References