U.S. patent application number 12/902001 was filed with the patent office on 2011-01-27 for human receptor proteins; related reagents and methods.
This patent application is currently assigned to Schering Corporation. Invention is credited to J. Fernando Bazan, Kevin W. Moore, Nicholas J. Murgolo, Christi L. Parham.
Application Number | 20110021412 12/902001 |
Document ID | / |
Family ID | 26759149 |
Filed Date | 2011-01-27 |
United States Patent
Application |
20110021412 |
Kind Code |
A1 |
Parham; Christi L. ; et
al. |
January 27, 2011 |
Human Receptor Proteins; Related Reagents and Methods
Abstract
Nucleic acids encoding mammalian, e.g., primate or rodent
receptors, purified receptor proteins and fragments thereof.
Antibodies, both polyclonal and monoclonal, are also provided.
Methods of using the compositions for both diagnostic and
therapeutic utilities are provided.
Inventors: |
Parham; Christi L.; (College
Station, TX) ; Moore; Kevin W.; (Palo Alto, CA)
; Murgolo; Nicholas J.; (Millington, NJ) ; Bazan;
J. Fernando; (Menlo Park, CA) |
Correspondence
Address: |
MERCK;C/O DNAX
LEGAL DEPARTMENT, 901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation
Kenilworth
NJ
|
Family ID: |
26759149 |
Appl. No.: |
12/902001 |
Filed: |
October 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12042165 |
Mar 4, 2008 |
7812126 |
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12902001 |
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10412749 |
Apr 10, 2003 |
7342103 |
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12042165 |
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09265540 |
Mar 8, 1999 |
6586228 |
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10412749 |
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60077329 |
Mar 9, 1998 |
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Current U.S.
Class: |
514/1.1 ;
435/252.3; 435/254.2; 435/348; 435/354; 435/363; 435/366; 435/375;
514/44R; 530/324; 530/350; 530/391.1; 530/391.3; 536/23.5 |
Current CPC
Class: |
C07K 14/7056 20130101;
A61P 43/00 20180101; Y10S 930/142 20130101 |
Class at
Publication: |
514/1.1 ;
435/348; 435/354; 435/363; 435/366; 435/375; 435/252.3; 435/254.2;
514/44.R; 530/324; 530/350; 530/391.1; 530/391.3; 536/23.5 |
International
Class: |
A61K 38/02 20060101
A61K038/02; C12N 5/10 20060101 C12N005/10; C12N 5/071 20100101
C12N005/071; C12N 1/21 20060101 C12N001/21; C12N 1/19 20060101
C12N001/19; A61K 31/7088 20060101 A61K031/7088; C07K 14/435
20060101 C07K014/435; C07K 16/28 20060101 C07K016/28; C07H 21/00
20060101 C07H021/00 |
Claims
1. A composition of matter selected from: a) a substantially pure
or recombinant DIRS1 polypeptide comprising at least three distinct
nonoverlapping segments of at least four amino acids identical to
segments of SEQ ID NO: 2; b) a substantially pure or recombinant
DIRS1 polypeptide comprising at least two distinct nonoverlapping
segments of at least five amino acids identical to segments of SEQ
ID NO: 2; c) a natural sequence DIRS1 comprising mature SEQ ID NO:
2; d) a fusion polypeptide comprising DIRS1 sequence; e) a
substantially pure or recombinant DIRS2 polypeptide comprising at
least three distinct nonoverlapping segments of at least ten amino
acids identical to segments of SEQ ID NO: 4; f) a substantially
pure or recombinant DIRS2 polypeptide comprising at least two
distinct nonoverlapping segments of at least eleven amino acids
identical to segments of SEQ ID NO: 4; g) a natural sequence DIRS2
comprising SEQ ID NO: 4; or h) a fusion polypeptide comprising
DIRS2 sequence.
2. The substantially pure or isolated antigenic: A) DIRS1
polypeptide of claim 1, wherein said distinct nonoverlapping
segments of identity: a) include one of at least eight amino acids;
b) include one of at least four amino acids and a second of at
least five amino acids; c) include at least three segments of at
least four, five, and six amino acids, or d) include one of at
least twelve amino acids; or B) DIRS2 polypeptide of claim 1,
wherein said distinct nonoverlapping segments of identity: a)
include one of at least thirteen amino acids; b) include one of at
least eleven amino acids and a second of at least thirteen amino
acids; c) include at least three segments of at least ten, eleven,
and twelve amino acids; or d) include one of at least twenty-five
amino acids.
3. The composition of matter of claim 1, wherein said: a) DIRS1
polypeptide: i) comprises a mature sequence of Table 1; ii) is an
unglycosylated form of DIRS1; iii) is from a primate, such as a
human; iv) comprises at least seventeen amino acids of SEQ ID NO:
2; v) exhibits at least four nonoverlapping segments of at least
seven amino acids of SEQ ID NO: 2; vi) is a natural allelic variant
of DIRS1; vii) has a length at least about 30 amino acids; viii)
exhibits at least two non-overlapping epitopes which are specific
for a primate DIRS1; ix) is glycosylated; x) has a molecular weight
of at least 30 kD with natural glycosylation; xi) is a synthetic
polypeptide; xii) is attached to a solid substrate; xiii) is
conjugated to another chemical moiety; xiv) is a 5-fold or less
substitution from natural sequence; or xv) is a deletion or
insertion variant from a natural sequence; or b) DIRS2 polypeptide:
i) comprises a mature sequence of Table 2; ii) is an unglycosylated
form of DIRS2; iii) is from a primate, such as a human; iv)
comprises at thirty-five amino acids of SEQ ID NO: 4; v) exhibits
at least four nonoverlapping segments of at least twelve amino
acids of SEQ ID NO: 4; vi) is a natural allelic variant of DIRS2;
vii) has a length at least about 30 amino acids; viii) exhibits at
least two non-overlapping epitopes which are specific for a primate
DIRS2; ix) is glycosylated; x) has a molecular weight of at least
30 kD with natural glycosylation; xi) is a synthetic polypeptide;
xii) is attached to a solid substrate; xiii) is conjugated to
another chemical moiety; xiv) is a 5-fold or less substitution from
natural sequence; or xv) is a deletion or insertion variant from a
natural sequence.
4. A composition comprising: a) a substantially pure DIRS1 and
another Interferon Receptor family member; b) a substantially pure
DIRS2 and another Interferon Receptor family member; c) a sterile
DIRS1 polypeptide of claim 1; d) a sterile DIRS2 polypeptide of
claim 1; e) said DIRS1 polypeptide of claim 1 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration; or f) said DIRS2
polypeptide of claim 1 and a carrier, wherein said carrier is: i)
an aqueous compound, including water, saline, and/or buffer; and/or
ii) formulated for oral, rectal, nasal, topical, or parenteral
administration.
5. The fusion polypeptide of claim 1, comprising: a) mature protein
sequence of Table 1; b) mature protein sequence of Table 2; c) a
detection or purification tag, including a FLAG, His 6, or Ig
sequence; or d) sequence of another interferon receptor
protein.
6. A kit comprising a polypeptide of claim 1, and: a) a compartment
comprising said protein or polypeptide; or b) instructions for use
or disposal of reagents in said kit.
7. A binding compound comprising an antigen binding site from an
antibody, which specifically binds to a natural: A) DIRS1
polypeptide of claim 1, wherein: a) said binding compound is in a
container; b) said DIRS1 polypeptide is from a human; c) said
binding compound is an Fv, Fab, or Fab2 fragment; d) said binding
compound is conjugated to another chemical moiety; or e) said
antibody: i) is raised against a peptide sequence of a mature
polypeptide of Table 1; ii) is raised against a mature DIRS1; iii)
is raised to a purified human DIRS1; iv) is immunoselected; v) is a
polyclonal antibody; vi) binds to a denatured DIRS1; vii) exhibits
a Kd to antigen of at least 30 .mu.M; viii) is attached to a solid
substrate, including a bead or plastic membrane; ix) is in a
sterile composition; or x) is detectably labeled, including a
radioactive or fluorescent label; or B) DIRS2 polypeptide of claim
1, wherein: a) said binding compound is in a container; b) said
DIRS2 protein is from a human; c) said binding compound is an Fv,
Fab, or Fab2 fragment; d) said binding compound is conjugated to
another chemical moiety; or e) said antibody: i) is raised against
a peptide sequence of a mature polypeptide of Table 2; ii) is
raised against a mature DIRS2; iii) is raised to a purified human
DIRS2; iv) is immunoselected; v) is a polyclonal antibody; vi)
binds to a denatured DIRS2; vii) exhibits a Kd to antigen of at
least 30 .mu.M; viii) is attached to a solid substrate, including a
bead or plastic membrane; ix) is in a sterile composition; or x) is
detectably labeled, including a radioactive or fluorescent
label.
8. A kit comprising said binding compound of claim 7, and: a) a
compartment comprising said binding compound; or b) instructions
for use or disposal of reagents in said kit.
9. A method of producing an antigen:antibody complex, comprising
contacting under appropriate conditions: a) a primate DIRS1
polypeptide with an antibody of claim 7A; or b) a primate DIRS2
polypeptide with an antibody of claim 7B; thereby allowing said
complex to form.
10. The method of claim 9, wherein: a) said complex is purified
from other interferon receptors; b) said complex is purified from
other antibody; c) said contacting is with a sample comprising an
interferon; d) said contacting allows quantitative detection of
said antigen; e) said contacting is with a sample comprising said
antibody; or f) said contacting allows quantitative detection of
said antibody.
11. A composition comprising: a) a sterile binding compound of
claim 7; or b) said binding compound of claim 7 and a carrier,
wherein said carrier is: i) an aqueous compound, including water,
saline, and/or buffer; and/or ii) formulated for oral, rectal,
nasal, topical, or parenteral administration.
12. An isolated or recombinant nucleic acid encoding said: A) DIRS1
polypeptide of claim 1, wherein said: a) DIRS1 is from a human; or
b) said nucleic acid: i) encodes an antigenic peptide sequence of
Table 1; ii) encodes a plurality of antigenic peptide sequences of
Table 1; iii) exhibits identity over at least thirteen nucleotides
to a natural cDNA encoding said segment; iv) is an expression
vector; v) further comprises an origin of replication; vi) is from
a natural source; vii) comprises a detectable label; viii)
comprises synthetic nucleotide sequence; ix) is less than 6 kb,
preferably less than 3 kb; x) is from a primate; xi) comprises a
natural full length coding sequence; xii) is a hybridization probe
for a gene encoding said DIRS1; or xiii) is a PCR primer, PCR
product, or mutagenesis primer; or B) DIRS2 polypeptide of claim 1,
wherein said: a) DIRS2 is from a human; or b) said nucleic acid: i)
encodes an antigenic peptide sequence of Table 2; ii) encodes a
plurality of antigenic peptide sequences of Table 2; iii) exhibits
identity over at least 30 nucleotides to a natural cDNA encoding
said segment; iv) is an expression vector; v) further comprises an
origin of replication; vi) is from a natural source; vii) comprises
a detectable label; viii) comprises synthetic nucleotide sequence;
ix) is less than 6 kb, preferably less than 3 kb; x) is from a
primate; xi) comprises a natural full length coding sequence; xii)
is a hybridization probe for a gene encoding said DIRS2; or xiii)
is a PCR primer, PCR product, or mutagenesis primer.
13. A cell or tissue comprising said recombinant nucleic acid of
claim 12.
14. The cell of claim 13, wherein said cell is: a) a prokaryotic
cell; b) a eukaryotic cell; c) a bacterial cell; d) a yeast cell;
e) an insect cell; f) a mammalian cell; g) a mouse cell; h) a
primate cell; or i) a human cell.
15. A kit comprising said nucleic acid of claim 12, and: a) a
compartment comprising said nucleic acid; b) a compartment further
comprising a primate DIRS1 polypeptide; c) a compartment further
comprising a primate DIRS2 polypeptide; or d) instructions for use
or disposal of reagents in said kit.
16. A nucleic acid which: a) hybridizes under wash conditions of 30
minutes at 30.degree. C. and less than 2M salt to the coding
portion of SEQ ID NO: 1; b) hybridizes under wash conditions of 30
minutes at 30.degree. C. and less than 2M salt to the coding
portion of SEQ ID NO: 3; c) exhibits identity over a stretch of at
least about 30 nucleotides to a primate DIRS1; or d) exhibits
identity over a stretch of at least about 30 nucleotides to a
primate DIRS2.
17. The nucleic acid of claim 16, wherein: a) said wash conditions
are at 45.degree. C. and/or 500 mM salt; or b) said stretch is at
least 55 nucleotides.
18. The nucleic acid of claim 16, wherein: a) said wash conditions
are at 55.degree. C. and/or 150 mM salt; or b) said stretch is at
least 75 nucleotides.
19. A method of modulating physiology or development of a cell or
tissue culture cells comprising contacting said cell with an
agonist or antagonist of a mammalian DIRS1 or DIRS2.
20. The method of claim 19, wherein said cell is transformed with a
nucleic acid encoding a DIRS1 or DIRS2 and another cytokine
receptor subunit.
Description
[0001] This filing is a conversion of U.S. Provisional Patent
Application 60/077,329, filed Mar. 9, 1998, which is incorporated
herein by reference, to a U.S. Utility Patent Application.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods
for affecting mammalian physiology, including morphogenesis or
immune system function. In particular, it provides nucleic acids,
proteins, and antibodies which regulate development and/or the
immune system. Diagnostic and therapeutic uses of these materials
are also disclosed.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology refers generally to techniques of
integrating genetic information from a donor source into vectors
for subsequent processing, such as through introduction into a
host, whereby the transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired protein product. The
carrier is frequently a plasmid having the capacity to incorporate
cDNA for later replication in a host and, in some cases, actually
to control expression of the cDNA and thereby direct synthesis of
the encoded product in the host. See, e.g., Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH
Press, NY.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network". Recent research has provided new
insights into the inner workings of this network. While it remains
clear that much of the immune response does, in fact, revolve
around the network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play critical roles in controlling these cellular
interactions. The interferons are generally considered to be
members of the cytokine family. Thus, there is considerable
interest in the isolation, characterization, and mechanisms of
action of cell modulatory factors, an understanding of which will
lead to significant advancements in the diagnosis and therapy of
numerous medical abnormalities, e.g., immune system disorders.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. See, e.g., Paul (ed. 1996) Fundamental Immunology
3d ed., Raven Press, New York; and Thomson (ed. 1994) The Cytokine
Handbook 2d ed., Academic Press, San Diego. They have been shown to
support the proliferation, growth, and/or differentiation of
pluripotential hematopoietic stem cells into vast numbers of
progenitors comprising diverse cellular lineages which make up a
complex immune system. Proper and balanced interactions between the
cellular components are necessary for a healthy immune response.
The different cellular lineages often respond in a different manner
when lymphokines are administered in conjunction with other
agents.
[0006] Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce and
secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its removal),
and T-cells of various subsets that secrete lymphokines and induce
or suppress the B-cells and various other cells (including other
T-cells) making up the immune network. These lymphocytes interact
with many other cell types.
[0007] One means to modulate the effect of a cytokine upon binding
to its receptor, and therefore potentially useful in treating
inappropriate immune responses, e.g., autoimmune, inflammation,
sepsis, and cancer situations, is to inhibit the receptor signal
transduction. Unfortunately, finding reagents capable of serving as
an antagonist or agonist has been severely hampered by the failure
to fully identify all of the components within the signaling
systems. In order to characterize the structural properties of a
cytokine receptor in greater detail and to understand the mechanism
of action at the molecular level, purified receptor will be very
useful. The receptors provided herein, by comparison to other
receptors or by combining structural components, will provide
further understanding of signal transduction induced by ligand
binding.
[0008] The isolated receptor gene should provide means to generate
an economical source of the receptor, allow expression of more
receptors on a cell leading to increased assay sensitivity, promote
characterization of various receptor subtypes and variants, and
allow correlation of activity with receptor structures. Moreover,
fragments of the receptor may be useful as agonists or antagonists
of ligand binding. See, e.g., Harada, et al. (1992) J. Biol. Chem.
267:22752-22758. Often, there are at least two critical subunits in
the functional receptor. See, e.g., Gonda and D'Andrea (1997) Blood
89:355-369; Presky, et al. (1996) Proc. Nat'l Acad. Sci. USA
93:14002-14007; Drachman and Kaushansky (1995) Curr. Opin. Hematol.
2:22-28; Theze (1994) Eur. Cytokine Netw. 5:353-368; and Lemmon and
Schlessinger (1994) Trends Biochem. Sci. 19:459-463.
[0009] From the foregoing, it is evident that the discovery and
development of new soluble proteins and their receptors, including
ones similar to lymphokines, should contribute to new therapies for
a wide range of degenerative or abnormal conditions which directly
or indirectly involve development, differentiation, or function,
e.g., of the immune system and/or hematopoietic cells. In
particular, the discovery and understanding of novel receptors for
lymphokine-like molecules which enhance or potentiate the
beneficial activities of other lymphokines would be highly
advantageous. The present invention provides new receptors for
ligands exhibiting similarity to cytokine like compositions and
related compounds, and methods for their use.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to novel receptors related
to cytokine receptors, e.g., primate or rodent, cytokine receptor
like molecular structures, designated DNAX Interferon-like Receptor
Subunits (DIRS), and their biological activities. In particular, it
provides description of two different subunits, designated DIRS1
and DIRS2. It includes nucleic acids coding for the polypeptides
themselves and methods for their production and use. The nucleic
acids of the invention are characterized, in part, by their
homology to cloned complementary DNA (cDNA) sequences enclosed
herein.
[0011] The present invention provides, in polypeptide embodiments:
a substantially pure or recombinant DIRS1 polypeptide comprising at
least three distinct nonoverlapping segments of at least four amino
acids identical to segments of SEQ ID NO: 2; a substantially pure
or recombinant DIRS1 polypeptide comprising at least two distinct
nonoverlapping segments of at least five amino acids identical to
segments of SEQ ID NO: 2; a natural sequence DIRS1 comprising
mature SEQ ID NO: 2; a fusion polypeptide comprising DIRS1
sequence; a substantially pure or recombinant DIRS2 polypeptide
comprising at least three distinct nonoverlapping segments of at
least ten amino acids identical to segments of SEQ ID NO: 4; a
substantially pure or recombinant DIRS2 polypeptide comprising at
least two distinct nonoverlapping segments of at least eleven amino
acids identical to segments of SEQ ID NO: 4; a natural sequence
DIRS2 comprising SEQ ID NO: 4; or a fusion polypeptide comprising
DIRS2 sequence. Preferred embodiments include, e.g., the
substantially pure or isolated antigenic: DIRS1 polypeptide,
wherein the distinct nonoverlapping segments of identity: include
one of at least eight amino acids; include one of at least four
amino acids and a second of at least five amino acids; include at
least three segments of at least four, five, and six amino acids,
or include one of at least twelve amino acids; or DIRS2
polypeptide, wherein the distinct nonoverlapping segments of
identity: include one of at least thirteen amino acids; include one
of at least eleven amino acids and a second of at least thirteen
amino acids; include at least three segments of at least ten,
eleven, and twelve amino acids; or include one of at least
twenty-five amino acids. Other embodiments include compositions
where: the DIRS1 polypeptide: comprises a mature sequence of Table
1; is an unglycosylated form of DIRS1; is from a primate, such as a
human; comprises at least seventeen amino acids of SEQ ID NO: 2;
exhibits at least four nonoverlapping segments of at least seven
amino acids of SEQ ID NO: 2; is a natural allelic variant of DIRS1;
has a length at least about 30 amino acids; exhibits at least two
non-overlapping epitopes which are specific for a primate DIRS1; is
glycosylated; has a molecular weight of at least 30 kD with natural
glycosylation; is a synthetic polypeptide; is attached to a solid
substrate; is conjugated to another chemical moiety; is a 5-fold or
less substitution from natural sequence; or is a deletion or
insertion variant from a natural sequence; or the DIRS2
polypeptide: comprises a mature sequence of Table 2; is an
unglycosylated form of DIRS2; or is from a primate, such as a
human; comprises at thirty-five amino acids of SEQ ID NO: 4;
exhibits at least four nonoverlapping segments of at least twelve
amino acids of SEQ ID NO: 4; is a natural allelic variant of DIRS2;
has a length at least about 30 amino acids; exhibits at least two
non-overlapping epitopes which are specific for a primate DIRS2; is
glycosylated; has a molecular weight of at least 30 kD with natural
glycosylation; is a synthetic polypeptide; is attached to a solid
substrate; is conjugated to another chemical moiety; is a 5-fold or
less substitution from natural sequence; or is a deletion or
insertion variant from a natural sequence. Various combination
compositions include those comprising: a substantially pure DIRS1
and another Interferon Receptor family member; a substantially pure
DIRS2 and another Interferon Receptor family member; a sterile
DIRS1 polypeptide; a sterile DIRS2 polypeptide; the DIRS1
polypeptide and a carrier, wherein the carrier is: an aqueous
compound, including water, saline, and/or buffer; and/or formulated
for oral, rectal, nasal, topical, or parenteral administration; or
the DIRS2 polypeptide and a carrier, wherein the carrier is: an
aqueous compound, including water, saline, and/or buffer; and/or
formulated for oral, rectal, nasal, topical, or parenteral
administration.
[0012] Fusion polypeptide embodiments include those comprising:
mature protein sequence of Table 1; mature protein sequence of
Table 2; a detection or purification tag, including a FLAG, His6,
or Ig sequence; or sequence of another interferon receptor protein.
Kit embodiments are provided, e.g., a kit comprising such a
polypeptide, and: a compartment comprising the protein or
polypeptide; or instructions for use or disposal of reagents in the
kit.
[0013] The invention also provides a binding compound comprising an
antigen binding site from an antibody, which specifically binds to
a: natural DIRS1 polypeptide, wherein: the binding compound is in a
container; the DIRS1 polypeptide is from a human; the binding
compound is an Fv, Fab, or Fab2 fragment; the binding compound is
conjugated to another chemical moiety; or the antibody: is raised
against a peptide sequence of a mature polypeptide of Table 1; is
raised against a mature DIRS1; is raised to a purified human DIRS1;
is immunoselected; is a polyclonal antibody; binds to a denatured
DIRS1; exhibits a Kd to antigen of at least 30 .mu.M; is attached
to a solid substrate, including a bead or plastic membrane; is in a
sterile composition; or is detectably labeled, including a
radioactive or fluorescent label; or a natural DIRS2 polypeptide,
wherein: the binding compound is in a container; the DIRS2 protein
is from a human; the binding compound is an Fv, Fab, or Fab2
fragment; the binding compound is conjugated to another chemical
moiety; or the antibody: is raised against a peptide sequence of a
mature polypeptide of Table 2; is raised against a mature DIRS2; is
raised to a purified human DIRS2; is immunoselected; is a
polyclonal antibody; binds to a denatured DIRS2; exhibits a Kd to
antigen of at least 30 .mu.M; is attached to a solid substrate,
including a bead or plastic membrane; is in a sterile composition;
or is detectably labeled, including a radioactive or fluorescent
label. Kit embodiments include, e.g., those comprising the binding
compound, and: a compartment comprising the binding compound; or
instructions for use or disposal of reagents in the kit.
[0014] Various methods are provided, e.g., of producing an
antigen:antibody complex, comprising contacting under appropriate
conditions: a primate DIRS1 polypeptide with a described antibody;
or a primate DIRS2 polypeptide with a described antibody; thereby
allowing the complex to form. In certain situations, the method is
used wherein: the complex is purified from other interferon
receptors; the complex is purified from other antibody; the
contacting is with a sample comprising an interferon; the
contacting allows quantitative detection of the antigen; the
contacting is with a sample comprising the antibody; or the
contacting allows quantitative detection of the antibody.
[0015] Other compositions comprise: a sterile binding compound as
described, or the described binding compound and a carrier, wherein
the carrier is: an aqueous compound, including water, saline,
and/or buffer; and/or formulated for oral, rectal, nasal, topical,
or parenteral administration.
[0016] Nucleic acid embodiments include, e.g., an isolated or
recombinant nucleic acid encoding the: described DIRS1 polypeptide,
wherein the: DIRS1 is from a human; or the nucleic acid: encodes an
antigenic peptide sequence of Table 1; encodes a plurality of
antigenic peptide sequences of Table 1; exhibits identity over at
least thirteen nucleotides to a natural cDNA encoding the segment;
is an expression vector; further comprises an origin of
replication; is from a natural source; comprises a detectable
label; comprises synthetic nucleotide sequence; is less than 6 kb,
preferably less than 3 kb; is from a primate; comprises a natural
full length coding sequence; is a hybridization probe for a gene
encoding the DIRS1; or is a PCR primer, PCR product, or mutagenesis
primer; or the described DIRS2 polypeptide, wherein the: DIRS2 is
from a human; or the nucleic acid: encodes an antigenic peptide
sequence of Table 2; encodes a plurality of antigenic peptide
sequences of Table 2; exhibits identity over at least 30
nucleotides to a natural cDNA encoding the segment; is an
expression vector; further comprises an origin of replication; is
from a natural source; comprises a detectable label; comprises
synthetic nucleotide sequence; is less than 6 kb, preferably less
than 3 kb; is from a primate; comprises a natural full length
coding sequence; is a hybridization probe for a gene encoding the
DIRS2; or is a PCR primer, PCR product, or mutagenesis primer.
[0017] The invention further provides a cell or tissue comprising
the described recombinant nucleic acid. Certain embodiments include
wherein the cell is: a prokaryotic cell; a eukaryotic cell; a
bacterial cell; a yeast cell; an insect cell; a mammalian cell; a
mouse cell; a primate cell; or a human cell. Kits are also
provided, e.g., the described nucleic acid and: a compartment
comprising the nucleic acid; a compartment further comprising a
primate DIRS1 polypeptide; a compartment further comprising a
primate DIRS2 polypeptide; or instructions for use or disposal of
reagents in the kit.
[0018] In other embodiments, the invention provides a nucleic acid
which: hybridizes under wash conditions of 30 minutes at 30.degree.
C. and less than 2M salt to the coding portion of SEQ ID NO: 1;
hybridizes under wash conditions of 30 minutes at 30.degree. C. and
less than 2M salt to the coding portion of SEQ ID NO: 3; exhibits
identity over a stretch of at least about 30 nucleotides to a
primate DIRS1 sequence; or exhibits identity over a stretch of at
least about 30 nucleotides to a primate DIRS2 sequence. Preferred
embodiments include those nucleic acids wherein: the wash
conditions are at 45.degree. C. and/or 500 mM salt; or the stretch
is at least 55 nucleotides. Other embodiments include those nucleic
acids wherein: the wash conditions are at 55.degree. C. and/or 150
mM salt; or the stretch is at least 75 nucleotides.
[0019] The invention further provides a method of modulating
physiology or development of a cell or tissue culture cells
comprising contacting the cell with an agonist or antagonist of a
mammalian DIRS1 or DIRS2. The method may involve where the cell is
transformed with a nucleic acid encoding a DIRS1 or DIRS2 and
another cytokine receptor subunit.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Outline
I. General
II. Activities
[0020] III. Nucleic acids
[0021] A. encoding fragments, sequence, probes
[0022] B. mutations, chimeras, fusions
[0023] C. making nucleic acids
[0024] D. vectors, cells comprising
IV. Proteins, Peptides
[0025] A. fragments, sequence, immunogens, antigens
[0026] B. muteins
[0027] C. agonists/antagonists, functional equivalents
[0028] D. making proteins
V. Making nucleic acids, proteins
[0029] A. synthetic
[0030] B. recombinant
[0031] C. natural sources
VI. Antibodies
[0032] A. polyclonals
[0033] B. monoclonal
[0034] C. fragments; Kd
[0035] D. anti-idiotypic antibodies
[0036] E. hybridoma cell lines
VII. Kits and Methods to quantify DIRS
[0037] A. ELISA
[0038] B. assay mRNA encoding
[0039] C. qualitative/quantitative
[0040] D. kits
VIII. Therapeutic compositions, methods
[0041] A. combination compositions
[0042] B. unit dose
[0043] C. administration
IX. Screening
X. Ligands
I. General
[0044] The present invention provides the amino acid sequences and
DNA sequences of mammalian, herein primate, interferon
receptor-like subunit molecules, these ones designated DNAX
Interferon Receptor family Subunit 1 (DIRS1) and DNAX Interferon
Receptor family Subunit 2, having particular defined properties,
both structural and biological. Various cDNAs encoding these
molecules were obtained from primate, e.g., human, cDNA sequence
libraries. Other primate or other mammalian counterparts would also
be desired. Descriptions, methods, and manipulations directed to
DIRS1 may be applied, as appropriate, to DIRS2.
[0045] Some of the standard methods applicable are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor Press; Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY; Ausubel, et
al., Biology, Greene Publishing Associates, Brooklyn, N.Y.; or
Ausubel, et al. (1987 and periodic supplements) Current Protocols
in Molecular Biology, Greene/Wiley, New York; each of which is
incorporated herein by reference.
[0046] A partial nucleotide (SEQ ID NO: 1) and corresponding amino
acid sequence (SEQ ID NO: 2) of a human DIRS1 coding segment is
shown in Table 1. Partial human DIRS2 sequence is provided (SEQ ID
NO: 3 and 4).
TABLE-US-00001 TABLE 1 Nucleotide and amino acid sequences of DNAX
IFN Receptor Subunit like embodiments (DIRS1), originally
designated HKAEF92. Primate, e.g., human embodiment (see SEQ ID NO:
1 and 2). Nucleotides 567, 573, 1336, 1342, and 1369 are designated
C, but may be A, C, G, or T; nucleotides 643, 1287, and 1290 are
designated C, but may be C or G; nucleotides 772, 806, and 1261 are
designated G, but may be A or G; nucleotides 1236, 1260, 1282, and
1289 are designated U, but may be G or T; residues 1247, 1257,
1293, and 1302 are designated C, but may be C or T; and nucleotides
1266 and 1298 are designated T, but may be A or T. Additional
sequencing indicates that nucleotide 567 is A; 574 is G; 640 is G;
742 is G; and 806 is G. Predicted signal cleavage is about between
thr29 and asp30. TCGACCCACG CGTCCGCGCT GCGACTCAGA CCTCAGCTCC
AACATATGCA TTCTGAAGAA 60 AGATGGCTGA GATGGACAGA ATGCTTTATT
TTGGAAAGAA ACAATGTTCT AGGTCAAACT 120 GAGTCTACCA A ATG CAG ACT TTC
ACA ATG GTT CTA GAA GAA ATC TGG ACA 170 Met Gln Thr Phe Thr Met Val
Leu Glu Glu Ile Trp Thr 1 5 10 AGT CTT TTC ATG TGG TTT TTC TAC GCA
TTG ATT CCA TGT TTG CTC ACA 218 Ser Leu Phe Met Trp Phe Phe Tyr Ala
Leu Ile Pro Cys Leu Leu Thr 15 20 25 GAT GAA GTG GCC ATT CTG CCT
GCC CCT CAG AAC CTC TCT GTA CTC TCA 266 Asp Glu Val Ala Ile Leu Pro
Ala Pro Gln Asn Leu Ser Val Leu Ser 30 35 40 45 ACC AAC ATG AAG CAT
CTC TTG ATG TGG AGC CCA GTG ATC GCG CCT GGA 314 Thr Asn Met Lys His
Leu Leu Met Trp Ser Pro Val Ile Ala Pro Gly 50 55 60 GAA ACA GTG
TAC TAT TCT GTC GAA TAC CAG GGG GAG TAC GAG AGC CTG 362 Glu Thr Val
Tyr Tyr Ser Val Glu Tyr Gln Gly Glu Tyr Glu Ser Leu 65 70 75 TAC
ACG AGC CAC ATC TGG ATC CCC AGC AGC TGG TGC TCA CTC ACT GAA 410 Tyr
Thr Ser His Ile Trp Ile Pro Ser Ser Trp Cys Ser Leu Thr Glu 80 85
90 GGT CCT GAG TGT GAT GTC ACT GAT GAC ATC ACG GCC ACT GTG CCA TAC
458 Gly Pro Glu Cys Asp Val Thr Asp Asp Ile Thr Ala Thr Val Pro Tyr
95 100 105 AAC CTT CGT GTC AGG GCC ACA TTG GGC TCA CAG ACC TCA GCC
TGG AGC 506 Asn Leu Arg Val Arg Ala Thr Leu Gly Ser Gln Thr Ser Ala
Trp Ser 110 115 120 125 ATC CTG AAG CAT CCC TTT AAT AGA AAC TCA ACC
ATC CTT ACC CGA CCT 554 Ile Leu Lys His Pro Phe Asn Arg Asn Ser Thr
Ile Leu Thr Arg Pro 130 135 140 GGG ATG GAG ATC CCC AAA CAT GGC TTC
CAC CTG GTT ATT GAG CTG GAG 602 Gly Met Glu Ile Pro Lys His Gly Phe
His Leu Val Ile Glu Leu Glu 145 150 155 GAC CTG GGG CCC CAG TTT GAG
TTC CTT GTG GCC TAC TGG ACG AGG GAG 650 Asp Leu Gly Pro Gln Phe Glu
Phe Leu Val Ala Tyr Trp Thr Arg Glu 160 165 170 CCT GGT GCC GAG GAA
CAT GTC AAA ATG GTG AGG AGT GGG GGT ATT CCA 698 Pro Gly Ala Glu Glu
His Val Lys Met Val Arg Ser Gly Gly Ile Pro 175 180 185 GTG CAC CTA
GAA ACC ATG GAG CCA GGG GCT GCA TAC TGT GTG AAG GCC 746 Val His Leu
Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val Lys Ala 190 195 200 205
CAG ACA TTC GTG AAG GCC ATT GGG AGG TAC AGC GCC TTC AGC CAG ACA 794
Gln Thr Phe Val Lys Ala Ile Gly Arg Tyr Ser Ala Phe Ser Gln Thr 210
215 220 GAA TGT GTG GAG GTG CAA GGA GAG GCC ATT CCC CTG GTA CTG GCC
CTG 842 Glu Cys Val Glu Val Gln Gly Glu Ala Ile Pro Leu Val Leu Ala
Leu 225 230 235 TTT GCC TTT GTT GGC TTC ATG CTG ATC CTT GTG GTC GTG
CCA CTG TTC 890 Phe Ala Phe Val Gly Phe Met Leu Ile Leu Val Val Val
Pro Leu Phe 240 245 250 GTC TGG AAA ATG GGC CGG CTG CTC CAG TAC TCC
TGT TGC CCC GTG GTG 938 Val Trp Lys Met Gly Arg Leu Leu Gln Tyr Ser
Cys Cys Pro Val Val 255 260 265 GTC CTC CCA GAC ACC TTG AAA ATA ACC
AAT TCA CCC CAG AAG TTA ATC 986 Val Leu Pro Asp Thr Leu Lys Ile Thr
Asn Ser Pro Gln Lys Leu Ile 270 275 280 285 AGC TGC AGA AGG GAG GAG
GTG GAT GCC TGT GCC ACG GCT GTG ATG TCT 1034 Ser Cys Arg Arg Glu
Glu Val Asp Ala Cys Ala Thr Ala Val Met Ser 290 295 300 CCT GAG GAA
CTC CTC AGG GCC TGG ATC TCA TAGGTTTGCG GAAGGGCCCA 1084 Pro Glu Glu
Leu Leu Arg Ala Trp Ile Ser 305 310 GGTGAAGCCG AGAACCTGGT
CTGCATGACA TGGAAACCAT GAGGGGACAA GTTGTGTTTC 1144 TGTTTTCCGC
CACGGACAAG GGATGAGAGA AGTAGGAAGA GCCTGTTGTC TACAAGTCTA 1204
GAAGCAACCA TCAGAGGCAG GGTGGTTTGT CTAACAGAAC AACTGACTGA GGCTATGGGG
1264 GTTGTGACCT CTAGACTTTG GGCTTCCACT TGCTTGGCTG AGCAACCCTG
GGAAAAGTGA 1324 CTTCATCCCT TCGGTCCCAA GTTTTCTCAT CTGTAATGGG
GGATCCCTAC AAAACTG 1381
TABLE-US-00002 TABLE 2 Partial nucleotide and amino acid sequences
of DNAX IFN Receptor Subunit like embodiments (DIRS2), originally
designated HOFNY28 (SEQ ID NO: 3 and 4). Nucleotide 193 designated
C, may be C or T; additional sequencing indicates that nucleotide
is C. C CGG GTC GAC CCA CGC GTC CGC CTG GTT TCC CCC TGG CTG ACA GTG
46 Arg Val Asp Pro Arg Val Arg Leu Val Ser Pro Trp Leu Thr Val 1 5
10 15 CCT TGG TTC CTG TCC TGT TGG AAT GTT ACC ATT GGG CCT CCT GAG
AGC 94 Pro Trp Phe Leu Ser Cys Trp Asn Val Thr Ile Gly Pro Pro Glu
Ser 20 25 30 ATC TGG GTG ACG CCG GGA GAA GCC TCC CTC ATC ATC AGG
TTC TCC TCT 142 Ile Trp Val Thr Pro Gly Glu Ala Ser Leu Ile Ile Arg
Phe Ser Ser 35 40 45 CCC TTC GAC GTC CCT CCC AAC CTG GGC TAT TTC
CAG TAC TAT GTC CAT 190 Pro Phe Asp Val Pro Pro Asn Leu Gly Tyr Phe
Gln Tyr Tyr Val His 50 55 60 TAC TGG GAA AAG GCG GGA ATC CAA AAG
GTT AAA GGT CCT TTC AAG AGC 238 Tyr Trp Glu Lys Ala Gly Ile Gln Lys
Val Lys Gly Pro Phe Lys Ser 65 70 75 AAC TCC ATC GTG TTG GAT GGC
TTG AGA CCC TTA AGA GAA TAC TGT TTA 286 Asn Ser Ile Val Leu Asp Gly
Leu Arg Pro Leu Arg Glu Tyr Cys Leu 80 85 90 95 CAA GTG AAG GCG CAT
CTC TTT CGC ACA TCC TGC AAC ACC TCT AGG CCC 334 Gln Val Lys Ala His
Leu Phe Arg Thr Ser Cys Asn Thr Ser Arg Pro 100 105 110 GGC CGC TTA
AGC AAC ATA ACT TGC TAC GAA ACA ATG ATG GAT GCC ACT 382 Gly Arg Leu
Ser Asn Ile Thr Cys Tyr Glu Thr Met Met Asp Ala Thr 115 120 125 ACG
AAG CTT CAA CAA GTC ATC CTC ATC GCC GTG GGA GTC TTT CTG TCG 430 Thr
Lys Leu Gln Gln Val Ile Leu Ile Ala Val Gly Val Phe Leu Ser 130 135
140 CTG GCG GCG CTG GCG GGG GGC TGT TTC TTC CTG GTG CTG AGA TAC AAA
478 Leu Ala Ala Leu Ala Gly Gly Cys Phe Phe Leu Val Leu Arg Tyr Lys
145 150 155 GGC CTG GTG AAA TAC TGG TTT CAC TCT CCG CCA AGC ATC CCA
TCA CAA 526 Gly Leu Val Lys Tyr Trp Phe His Ser Pro Pro Ser Ile Pro
Ser Gln 160 165 170 175 ATC GAA GAG TAT CTG AAG GAC CCG AGC CAG CCT
ATC CTA GAG GCC CTG 574 Ile Glu Glu Tyr Leu Lys Asp Pro Ser Gln Pro
Ile Leu Glu Ala Leu 180 185 190 GAC AAG GAC ACG TCA CCA ACA GAT GAT
GCC TGG GAC TTG GTG TCT GTT 622 Asp Lys Asp Thr Ser Pro Thr Asp Asp
Ala Trp Asp Leu Val Ser Val 195 200 205 GTT GCA TTT CCA GCA AAG GAG
CAA GAA GAT GTT CCC CAA AGC ACT TTG 670 Val Ala Phe Pro Ala Lys Glu
Gln Glu Asp Val Pro Gln Ser Thr Leu 210 215 220 ACC CAA AAC TCT GGT
GCG GTC TGC TAGCCTGTGG GGTAAGGGCT CTGAGCCGAG 724 Thr Gln Asn Ser
Gly Ala Val Cys 225 230 GAAGCTGCTG ATGTCCATGT CAGCACTTTA TGGAATCCGG
TCCTCCATTT TCCTGTCCCC 784 AAAAGGCCCG TCAGTGCCTG TGAAGATGTA
ACGGGTCTCA TGGGGGCGAC AAGCTTATTG 844 ATTTTTTTCT TCAAACTAAG
AGTTTTCTAA TCATACGCGT TTTTAGAATA ATTCTACAGA 904 TATGTCCCCG
AAAGATTAAG ATTTCTCTTA AACACTAAAA AGACATGTAA TTATTTGTTA 964
GCAAATGGGC GTCTGGCACG CCTCTGACAC TTTTTCGTCA GCAGCCAGGA CACGAGGTCC
1024 CCTCCTTGAT GAAGCCCCTC GGGCAGACCA TGTCACCTGT CCCAGCCTGC
CCCAAGAAGG 1084 GACATTAAGT GGCCCTTCTT CATATCCAAA CACCTGGCTT
GAAATGTGAT TAGCCCTGTA 1144 AATAGTTTCA CAGAGATTAA GCCTTTTTTT
CCCCCAAGTT AGGAATAAAA GACTATAATT 1204 AACTTTTTAA AAAAAAAAAA
AAAAAAAAAA AAAAAAAAAA 1244
TABLE-US-00003 TABLE 3 Sequence alignment of related IFN receptor
family members. DR1 is a primate DIRS1 protein sequence; DR2 is a
primate DIRS2 protein sequence; the IR.beta. is the human
IFN-.gamma. receptor beta subunit (SEQ ID NO: 5), see Soh, et al.
(1994) Cell 76: 793-802; and CRF is the crf2-4 protein (SEQ ID NO:
6), see Lutfalla, et al. (1993) Genomics 16: 366-373: DR2
---------- ---------- ---------- -------RVD PRVRLV---- ----------
DR1 MQTFTMVLEE IWTSLFMWFF YALIPCLLTD EVAILPAPQN LSVLSTNMKH
LLMWSPVIAP IR.beta. -----MRPTL LWSLLLLLGV FAAAAAAPPD PLSQLPAPQH
PKIRLYNAEQ VLSWEPVALS crf ---------M AWSLGSWLGG CLLVSALG--
---MVPPPEN VRMNSVNFKN ILQWESPAFA DR2 ---------- ----------
---------- ---------- ---------- --------SP DR1 GETVYYSVEY
QGEYES--LY TSHIWIPSSW CSLTEGPECD VTDDITAT-- ---VPYNLRV IR.beta.
NSTRPVVYRV QFKYTDSKWF TADIMSIGVN CTQITATECD FTAASPSAGF PMDFNVTLRL
crf KGNLTFTAQY LSYR------ -----IFQDK CMNTTLTECD FSSLSKYG--
----DHTLRV DR2 WLTVPWFLSC WNVTIGPPES IWVTPGEASL IIRFSSPFDV
PPN------- -LGYFQYYVH DR1 RATLGSQTSA WSILK-HPFN RNSTILTRPG
MEIXKXGFHL VIELE---DL GPQ------- IR.beta. RAELGALHSA WVTMPWFQHY
RNVTVGPPEN IEVTPGEGSL IIRFSSPFDI ADTS------ crf RAEFADEHSD
WVNIT-FCPV DDTIIGPP-G MQVEVLADSL HMRFLAPKIE NEYETWTMKN DR2
YW--EKAGIQ KVKGPFKSNS -IVLDGLRPL REYCLQVKAH LFRTSCNTSR PGRLSNITCY
DR1 ----FEFLVA YWXREPGAEE HVKMVRSGGI PVHLETMEPG AAYCVKAQT-
-FVKAIGX-- IR.beta. -TAFFCYYVH Y--WEKGGIQ QVKGPFRSNS -ISLDNLKPS
RVYCLQVQAQ LLWNKSNIFR crf VYNSWTYNVQ YW--KNGTDE KFQITPQYDF
-EVLRNLEPW TTYCVQVRG- -FLPDRNK-- DR2 ETMMDATTKL QQVILIAVGV
FLSLAALAGG CFFLVLRYKG LVKYWFHSPP SIPSQIEEYL DR1 YSAFSQTECV
EVQG-EAIPL VLALFAFVG- -FMLILVVVP LF--VWKMGR LLQYSCCPVV IR.beta.
VGHLSNISCY ETMADASTEL QQVILISVGT FSLLSVLAGA CFFLVLKYRG LIKYWFHTPP
crf AGEWSEPVCE QTTHDETVPS WMVAVILMAS VFMVCLALLG CFSLLWCVYK
KTKYAFSPRN DR2 KDPSQPILEA LDKDTSPTDD AWDLVSVVAF PAK--EQE--
DVPQSTLTQN DR1 VLPDTLKITN S-P-QKLISC R----REEVD AC--ATAVMS
PEE------- IR.beta. SIPLQIEEYL KDPTQPILEA LDKDSSPKDD VWDSVSIISF
PEK--EQE-- crf SLPQHLKEFL GHPHHNTLLF FSFPLSDEND VFDKLSVIAE
DSESGKQNPG DR2 SGAVC DR1 -LLRAWIS IR.beta. DVLQTL crf DSCSLGTPPG
QGPQS
[0047] Table 3 shows comparison of the available sequences of
primate embodiments of DIRS1, DIRS2, and two related interferon
receptor family members. Both of the new DIRS appear to exhibit
sequence similarity to beta interferon receptor subunits.
[0048] Structural features of the human DIRS1, and similarly for
the other receptors as aligned in Table 3, include characteristic
transmembrane segments of the IR.beta. and crf from 261-273, and
correspond to: from about val1 to pro133; fibronectin domains
corresponding to the DIRS1 sequence from about gly134 to pro232,
gly233 to gly306, and pro307 to lys403; a transmembrane segment
from about val404 to gly427; and an intracellular domain from about
arg428 to the carboxy terminus. Of particular interest is the WGEWS
motif corresponding to residues trp104 to ser108.
[0049] As used herein, the term DIRS1 shall be used to describe a
protein comprising a protein or peptide segment having or sharing
the amino acid sequence shown in Table 1, or a substantial fragment
thereof. The invention also includes a protein variation of the
respective DIRS1 allele whose sequence is provided, e.g., a mutein
or soluble extracellular construct. Typically, such agonists or
antagonists will exhibit less than about 10% sequence differences,
and thus will often have between 1- and 11-fold substitutions,
e.g., 2-, 3-, 5-, 7-fold, and others. It also encompasses allelic
and other variants, e.g., natural polymorphic, of the protein
described. Typically, it will bind to its corresponding biological
ligand, perhaps in a dimerized state with an alpha receptor
subunit, with high affinity, e.g., at least about 100 nM, usually
better than about 30 nM, preferably better than about 10 nM, and
more preferably at better than about 3 nM. The term shall also be
used herein to refer to related naturally occurring forms, e.g.,
alleles, polymorphic variants, and metabolic variants of the
mammalian protein.
[0050] This invention also encompasses proteins or peptides having
substantial amino acid sequence identity with the amino acid
sequence in Table 1. It will include sequence variants with
relatively few substitutions, e.g., preferably less than about 3-5.
Other embodiments include forms in association with an alpha
subunit, e.g., a DSRS1, and/or with ligand, e.g., DIL-30.
[0051] A substantial polypeptide "fragment", or "segment", is a
stretch of amino acid residues of at least about 8 amino acids,
generally at least 10 amino acids, more generally at least 12 amino
acids, often at least 14 amino acids, more often at least 16 amino
acids, typically at least 18 amino acids, more typically at least
20 amino acids, usually at least 22 amino acids, more usually at
least 24 amino acids, preferably at least 26 amino acids, more
preferably at least 28 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 35, 40,
50, 70, 90, 110, etc. Specific ends may be at all possible or
appropriate combinations, or at proline residues. Sequences of
segments of different proteins can be compared to one another over
appropriate length stretches.
[0052] The invention provides polypeptides exhibiting a plurality
of distinct, e.g., nonoverlapping, segments of the specified
length. Typically, the plurality will be at least two, more usually
at least three, and preferably 5, 7, or even more. While the length
minima are provided, longer lengths, of various sizes, may be
appropriate, e.g., one of length 7, and two of length 12.
[0053] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches. In some comparisons, gaps
may be introduces, as required. See, e.g., Needleham, et al. (1970)
J. Mol. Biol. 48:443-453; Sankoff, et al., (1983) chapter one in
Time Warps, String Edits, and Macromolecules: The Theory and
Practice of Sequence Comparison, Addison-Wesley, Reading, Mass.;
and software packages from NCBI, NIH; and the University of
Wisconsin Genetics Computer Group (GCG), Madison, Wis.; each of
which is incorporated herein by reference. This changes when
considering conservative substitutions as matches. Conservative
substitutions typically include substitutions within the following
groups: glycine, alanine; valine, isoleucine, leucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. Homologous amino
acid sequences are intended to include natural allelic and
interspecies variations in the cytokine sequence. Typical
homologous proteins or peptides will have from 50-100% homology (if
gaps can be introduced), to 60-100% homology (if conservative
substitutions are included) with an amino acid sequence segment of
Table 1. Homology measures will be at least about 70%, generally at
least 76%, more generally at least 81%, often at least 85%, more
often at least 88%, typically at least 90%, more typically at least
92%, usually at least 94%, more usually at least 95%, preferably at
least 96%, and more preferably at least 97%, and in particularly
preferred embodiments, at least 98% or more. The degree of homology
will vary with the length of the compared segments. Homologous
proteins or peptides, such as the allelic variants, will share most
biological activities with the embodiments described in Table
1.
[0054] As used herein, the term "biological activity" is used to
describe, without limitation, effects on inflammatory responses,
innate immunity, and/or morphogenic development by cytokine-like
ligands. For example, these receptors should mediate phosphatase or
phosphorylase activities, which activities are easily measured by
standard procedures. See, e.g., Hardie, et al. (eds. 1995) The
Protein Kinase FactBook vols. I and II, Academic Press, San Diego,
Calif.; Hanks, et al. (1991) Meth. Enzymol. 200:38-62; Hunter, et
al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et
al. (1991) Cold Spring Harbor Symp. Quant. Biol. 56:449-463; and
Parker, et al. (1993) Nature 363:736-738. The receptors, or
portions thereof, may be useful as phosphate labeling enzymes to
label general or specific substrates.
[0055] The terms ligand, agonist, antagonist, and analog of, e.g.,
a DIRS1, include molecules that modulate the characteristic
cellular responses to cytokine ligand proteins, as well as
molecules possessing the more standard structural binding
competition features of ligand-receptor interactions, e.g., where
the receptor is a natural receptor or an antibody. The cellular
responses likely are typically mediated through receptor tyrosine
kinase pathways.
[0056] Also, a ligand is a molecule which serves either as a
natural ligand to which said receptor, or an analog thereof, binds,
or a molecule which is a functional analog of the natural ligand.
The functional analog may be a ligand with structural
modifications, or may be a wholly unrelated molecule which has a
molecular shape which interacts with the appropriate ligand binding
determinants. The ligands may serve as agonists or antagonists,
see, e.g., Goodman, et al. (eds. 1990) Goodman & Gilman's: The
Pharmacological Bases of Therapeutics, Pergamon Press, New
York.
[0057] Rational drug design may also be based upon structural
studies of the molecular shapes of a receptor or antibody and other
effectors or ligands. See, e.g., Herz, et al. (1997) J. Recept.
Signal Transduct. Res. 17:671-776; and Chaiken, et al. (1996)
Trends Biotechnol. 14:369-375. Effectors may be other proteins
which mediate other functions in response to ligand binding, or
other proteins which normally interact with the receptor. One means
for determining which sites interact with specific other proteins
is a physical structure determination, e.g., x-ray crystallography
or 2 dimensional NMR techniques. These will provide guidance as to
which amino acid residues form molecular contact regions. For a
detailed description of protein structural determination, see,
e.g., Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York, which is hereby incorporated herein by
reference.
II. Activities
[0058] The cytokine receptor-like proteins will have a number of
different biological activities, e.g., modulating cell
proliferation, or in phosphate metabolism, being added to or
removed from specific substrates, typically proteins. Such will
generally result in modulation of an inflammatory function, other
innate immunity response, or a morphological effect. The subunit
will probably have a specific low affinity binding to the
ligand.
[0059] The DIRS1 has the characteristic motifs of a receptor
signaling through the JAK pathway. See, e.g., Ihle, et al. (1997)
Stem Cells 15 (suppl. 1):105-111; Silvennoinen, et al. (1997) APMIS
105:497-509; Levy (1997) Cytokine Growth Factor Review 8:81-90;
Winston and Hunter (1996) Current Biol. 6:668-671; Barrett (1996)
Baillieres Clin. Gastroenterol. 10:1-15; and Briscoe, et al. (1996)
Philos. Trans. R. Soc. Lond. B. Biol. Sci. 351:167-171.
[0060] The biological activities of the cytokine receptor subunits
will be related to addition or removal of phosphate moieties to
substrates, typically in a specific manner, but occasionally in a
non specific manner. Substrates may be identified, or conditions
for enzymatic activity may be assayed by standard methods, e.g., as
described in Hardie, et al. (eds. 1995) The Protein Kinase FactBook
vols. I and II, Academic Press, San Diego, Calif.; Hanks, et al.
(1991) Meth. Enzymol. 200:38-62; Hunter, et al. (1992) Cell
70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al. (1991) Cold
Spring Harbor Symp. Quant. Biol. 56:449-463; and Parker, et al.
(1993) Nature 363:736-738.
III. Nucleic Acids
[0061] This invention contemplates use of isolated nucleic acid or
fragments, e.g., which encode these or closely related proteins, or
fragments thereof, e.g., to encode a corresponding polypeptide,
preferably one which is biologically active. In addition, this
invention covers isolated or recombinant DNAs which encode such
proteins or polypeptides having characteristic sequences of the
DIRS1s. Typically, the nucleic acid is capable of hybridizing,
under appropriate conditions, with a nucleic acid sequence segment
shown in Table 1, but preferably not with a corresponding segment
of other receptors described in Table 3. Said biologically active
protein or polypeptide can be a full length protein, or fragment,
and will typically have a segment of amino acid sequence highly
homologous, e.g., exhibiting significant stretches of identity, to
one shown in Table 1. Further, this invention covers the use of
isolated or recombinant nucleic acid, or fragments thereof, which
encode proteins having fragments which are equivalent to the DIRS1
proteins. The isolated nucleic acids can have the respective
regulatory sequences in the 5' and 3' flanks, e.g., promoters,
enhancers, poly-A addition signals, and others from the natural
gene.
[0062] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially pure, e.g.,
separated from other components which naturally accompany a native
sequence, such as ribosomes, polymerases, and flanking genomic
sequences from the originating species. The term embraces a nucleic
acid sequence which has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates, which
are thereby distinguishable from naturally occurring compositions,
and chemically synthesized analogs or analogs biologically
synthesized by heterologous systems. A substantially pure molecule
includes isolated forms of the molecule, either completely or
substantially pure.
[0063] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
heterogeneity, preferably minor. This heterogeneity is typically
found at the polymer ends or portions not critical to a desired
biological function or activity.
[0064] A "recombinant" nucleic acid is typically defined either by
its method of production or its structure. In reference to its
method of production, e.g., a product made by a process, the
process is use of recombinant nucleic acid techniques, e.g.,
involving human intervention in the nucleotide sequence. Typically
this intervention involves in vitro manipulation, although under
certain circumstances it may involve more classical animal breeding
techniques. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants as found in
their natural state. Thus, for example, products made by
transforming cells with an unnaturally occurring vector is
encompassed, as are nucleic acids comprising sequence derived using
any synthetic oligonucleotide process. Such a process is often done
to replace a codon with a redundant codon encoding the same or a
conservative amino acid, while typically introducing or removing a
restriction enzyme sequence recognition site. Alternatively, the
process is performed to join together nucleic acid segments of
desired functions to generate a single genetic entity comprising a
desired combination of functions not found in the commonly
available natural forms, e.g., encoding a fusion protein.
Restriction enzyme recognition sites are often the target of such
artificial manipulations, but other site specific targets, e.g.,
promoters, DNA replication sites, regulation sequences, control
sequences, or other useful features may be incorporated by design.
A similar concept is intended for a recombinant, e.g., fusion,
polypeptide. This will include a dimeric repeat. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode equivalent polypeptides to fragments of DIRS1
and fusions of sequences from various different related molecules,
e.g., other cytokine receptor family members.
[0065] A "fragment" in a nucleic acid context is a contiguous
segment of at least about 17 nucleotides, generally at least 21
nucleotides, more generally at least 25 nucleotides, ordinarily at
least 30 nucleotides, more ordinarily at least 35 nucleotides,
often at least 39 nucleotides, more often at least 45 nucleotides,
typically at least 50 nucleotides, more typically at least 55
nucleotides, usually at least 60 nucleotides, more usually at least
66 nucleotides, preferably at least 72 nucleotides, more preferably
at least 79 nucleotides, and in particularly preferred embodiments
will be at least 85 or more nucleotides. Typically, fragments of
different genetic sequences can be compared to one another over
appropriate length stretches, particularly defined segments such as
the domains described below.
[0066] A nucleic acid which codes for a DIRS1 will be particularly
useful to identify genes, mRNA, and cDNA species which code for
itself or closely related proteins, as well as DNAs which code for
polymorphic, allelic, or other genetic variants, e.g., from
different individuals or related species. Preferred probes for such
screens are those regions of the interleukin which are conserved
between different polymorphic variants or which contain nucleotides
which lack specificity, and will preferably be full length or
nearly so. In other situations, polymorphic variant specific
sequences will be more useful.
[0067] This invention further covers recombinant nucleic acid
molecules and fragments having a nucleic acid sequence identical to
or highly homologous to the isolated DNA set forth herein. In
particular, the sequences will often be operably linked to DNA
segments which control transcription, translation, and DNA
replication. These additional segments typically assist in
expression of the desired nucleic acid segment.
[0068] Homologous, or highly identical, nucleic acid sequences,
when compared to one another, e.g., DIRS1 sequences, exhibit
significant similarity. The standards for homology in nucleic acids
are either measures for homology generally used in the art by
sequence comparison or based upon hybridization conditions.
Comparative hybridization conditions are described in greater
detail below.
[0069] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0070] Optical alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needlman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by visual inspection (see generally Ausubel
et al., supra).
[0071] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendrogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used
is similar to the method described by Higgins and Sharp (1989)
CABIOS 5:151-153. The program can align up to 300 sequences, each
of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0072] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described Altschul, et al. (1990) J.
Mol. Biol. 215:403-410. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http:www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul, et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a
wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and
Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915) alignments (B)
of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
[0073] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul
(1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0074] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below. Hybridization under
stringent conditions should give a background of at least 2-fold
over background, preferably at least 3-5 or more.
[0075] Substantial identity in the nucleic acid sequence comparison
context means either that the segments, or their complementary
strands, when compared, are identical when optimally aligned, with
appropriate nucleotide insertions or deletions, in at least about
60% of the nucleotides, generally at least 66%, ordinarily at least
71%, often at least 76%, more often at least 80%, usually at least
84%, more usually at least 88%, typically at least 91%, more
typically at least about 93%, preferably at least about 95%, more
preferably at least about 96 to 98% or more, and in particular
embodiments, as high at about 99% or more of the nucleotides,
including, e.g., segments encoding structural domains such as the
segments described below. Alternatively, substantial identity will
exist when the segments will hybridize under selective
hybridization conditions, to a strand or its complement, typically
using a sequence derived from Table 1. Typically, selective
hybridization will occur when there is at least about 55% homology
over a stretch of at least about 14 nucleotides, more typically at
least about 65%, preferably at least about 75%, and more preferably
at least about 90%. See, Kanehisa (1984) Nucl. Acids Res.
12:203-213, which is incorporated herein by reference. The length
of homology comparison, as described, may be over longer stretches,
and in certain embodiments will be over a stretch of at least about
17 nucleotides, generally at least about 20 nucleotides, ordinarily
at least about 24 nucleotides, usually at least about 28
nucleotides, typically at least about 32 nucleotides, more
typically at least about 40 nucleotides, preferably at least about
50 nucleotides, and more preferably at least about 75 to 100 or
more nucleotides. This includes, e.g., 125, 150, 175, 200, 225,
246, 273, and other lengths.
[0076] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters typically
controlled in hybridization reactions. Stringent temperature
conditions will usually include temperatures in excess of about
30.degree. C., more usually in excess of about 37.degree. C.,
typically in excess of about 45.degree. C., more typically in
excess of about 55.degree. C., preferably in excess of about
65.degree. C., and more preferably in excess of about 70.degree. C.
Stringent salt conditions will ordinarily be less than about 500
mM, usually less than about 400 mM, more usually less than about
300 mM, typically less than about 200 mM, preferably less than
about 100 mM, and more preferably less than about 80 mM, even down
to less than about 20 mM. However, the combination of parameters is
much more important than the measure of any single parameter. See,
e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which is
hereby incorporated herein by reference.
[0077] The isolated DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result in
novel DNA sequences which encode this protein or its derivatives.
These modified sequences can be used to produce mutant proteins
(muteins) or to enhance the expression of variant species. Enhanced
expression may involve gene amplification, increased transcription,
increased translation, and other mechanisms. Such mutant DIRS1-like
derivatives include predetermined or site-specific mutations of the
protein or its fragments, including silent mutations using genetic
code degeneracy. "Mutant DIRS1" as used herein encompasses a
polypeptide otherwise falling within the homology definition of the
DIRS1 as set forth above, but having an amino acid sequence which
differs from that of other cytokine receptor-like proteins as found
in nature, whether by way of deletion, substitution, or insertion.
In particular, "site specific mutant DIRS1" encompasses a protein
having substantial sequence identity with a protein of Table 1, and
typically shares most of the biological activities or effects of
the forms disclosed herein.
[0078] Although site specific mutation sites are predetermined,
mutants need not be site specific. Mammalian DIRS1 mutagenesis can
be achieved by making amino acid insertions or deletions in the
gene, coupled with expression. Substitutions, deletions,
insertions, or many combinations may be generated to arrive at a
final construct. Insertions include amino- or carboxy-terminal
fusions. Random mutagenesis can be conducted at a target codon and
the expressed mammalian DIRS1 mutants can then be screened for the
desired activity, providing some aspect of a structure-activity
relationship. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis. See also Sambrook, et
al. (1989) and Ausubel, et al. (1987 and periodic Supplements).
[0079] The mutations in the DNA normally should not place coding
sequences out of reading frames and preferably will not create
complementary regions that could hybridize to produce secondary
mRNA structure such as loops or hairpins.
[0080] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0081] Polymerase chain reaction (PCR) techniques can often be
applied in mutagenesis. Alternatively, mutagenesis primers are
commonly used methods for generating defined mutations at
predetermined sites. See, e.g., Innis, et al. (eds. 1990) PCR
Protocols: A Guide to Methods and Applications Academic Press, San
Diego, Calif.; and Dieffenbach and Dveksler (eds. 1995) PCR Primer:
A Laboratory Manual Cold Spring Harbor Press, CSH, NY.
IV. Proteins, Peptides
[0082] As described above, the present invention encompasses
primate DIRS1, e.g., whose sequences are disclosed in Table 1, and
described above. Allelic and other variants are also contemplated,
including, e.g., fusion proteins combining portions of such
sequences with others, including epitope tags and functional
domains.
[0083] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these rodent
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
Thus, the fusion product of a DIRS1 with another cytokine receptor
is a continuous protein molecule having sequences fused in a
typical peptide linkage, typically made as a single translation
product and exhibiting properties, e.g., sequence or antigenicity,
derived from each source peptide. A similar concept applies to
heterologous nucleic acid sequences.
[0084] In addition, new constructs may be made from combining
similar functional or structural domains from other related
proteins, e.g., cytokine receptors or Toll-like receptors,
including species variants. For example, ligand-binding or other
segments may be "swapped" between different new fusion polypeptides
or fragments. See, e.g., Cunningham, et al. (1989) Science
243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992, each of which is incorporated herein by reference.
Thus, new chimeric polypeptides exhibiting new combinations of
specificities will result from the functional linkage of
receptor-binding specificities. For example, the ligand binding
domains from other related receptor molecules may be added or
substituted for other domains of this or related proteins. The
resulting protein will often have hybrid function and properties.
For example, a fusion protein may include a targeting domain which
may serve to provide sequestering of the fusion protein to a
particular subcellular organelle.
[0085] Candidate fusion partners and sequences can be selected from
various sequence data bases, e.g., GenBank; NCBI, NIH; and BCG,
University of Wisconsin Biotechnology Computing Group, Madison,
Wis., which are each incorporated herein by reference.
[0086] The present invention particularly provides muteins which
bind cytokine-like ligands, and/or which are affected in signal
transduction. Structural alignment of human DIRS1 with other
members of the cytokine receptor family show conserved
features/residues. See Table 3. Alignment of the human DIRS1
sequence with other members of the cytokine receptor family
indicates various structural and functionally shared features. See
also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994)
Science 263:1762-1766; Sayle and Milner-White (1995) TIBS
20:374-376; and Gronenberg, et al. (1991) Protein Engineering
4:263-269.
[0087] Substitutions with either mouse sequences or human sequences
are particularly preferred. Conversely, conservative substitutions
away from the ligand binding interaction regions will probably
preserve most signaling activities; and conservative substitutions
away from the intracellular domains will probably preserve most
ligand binding properties.
[0088] "Derivatives" of the primate DIRS1 include amino acid
sequence mutants, glycosylation variants, metabolic derivatives and
covalent or aggregative conjugates with other chemical moieties.
Covalent derivatives can be prepared by linkage of functionalities
to groups which are found in the DIRS1 amino acid side chains or at
the N- or C-termini, e.g., by means which are well known in the
art. These derivatives can include, without limitation, aliphatic
esters or amides of the carboxyl terminus, or of residues
containing carboxyl side chains, O-acyl derivatives of hydroxyl
group-containing residues, and N-acyl derivatives of the amino
terminal amino acid or amino-group containing residues, e.g.,
lysine or arginine. Acyl groups are selected from the group of
alkyl-moieties, including C3 to C18 normal alkyl, thereby forming
alkanoyl aroyl species.
[0089] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., mammalian
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0090] A major group of derivatives are covalent conjugates of the
receptors or fragments thereof with other proteins of polypeptides.
These derivatives can be synthesized in recombinant culture such as
N- or C-terminal fusions or by the use of agents known in the art
for their usefulness in cross-linking proteins through reactive
side groups. Preferred derivatization sites with cross-linking
agents are at free amino groups, carbohydrate moieties, and
cysteine residues.
[0091] Fusion polypeptides between the receptors and other
homologous or heterologous proteins are also provided. Homologous
polypeptides may be fusions between different receptors, resulting
in, for instance, a hybrid protein exhibiting binding specificity
for multiple different cytokine ligands, or a receptor which may
have broadened or weakened specificity of substrate effect.
Likewise, heterologous fusions may be constructed which would
exhibit a combination of properties or activities of the derivative
proteins. Typical examples are fusions of a reporter polypeptide,
e.g., luciferase, with a segment or domain of a receptor, e.g., a
ligand-binding segment, so that the presence or location of a
desired ligand may be easily determined. See, e.g., Dull, et al.,
U.S. Pat. No. 4,859,609, which is hereby incorporated herein by
reference. Other gene fusion partners include
glutathione-S-transferase (GST), bacterial .beta.-galactosidase,
trpE, Protein A, .beta.-lactamase, alpha amylase, alcohol
dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski,
et al. (1988) Science 241:812-816.
[0092] The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable
synthetic DNA fragments. A double stranded fragment will often be
obtained either by synthesizing the complementary strand and
annealing the strand together under appropriate conditions or by
adding the complementary strand using DNA polymerase with an
appropriate primer sequence.
[0093] Such polypeptides may also have amino acid residues which
have been chemically modified by phosphorylation, sulfonation,
biotinylation, or the addition or removal of other moieties,
particularly those which have molecular shapes similar to phosphate
groups. In some embodiments, the modifications will be useful
labeling reagents, or serve as purification targets, e.g., affinity
ligands.
[0094] Fusion proteins will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, for example, in Sambrook, et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, and Ausubel, et al. (eds. 1987 and
periodic supplements) Current Protocols in Molecular Biology,
Greene/Wiley, New York, which are each incorporated herein by
reference. Techniques for synthesis of polypeptides are described,
for example, in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156;
Merrifield (1986) Science 232: 341-347; and Atherton, et al. (1989)
Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,
Oxford; each of which is incorporated herein by reference. See also
Dawson, et al. (1994) Science 266:776-779 for methods to make
larger polypeptides.
[0095] This invention also contemplates the use of derivatives of a
DIRS1 other than variations in amino acid sequence or
glycosylation. Such derivatives may involve covalent or aggregative
association with chemical moieties. These derivatives generally
fall into three classes: (1) salts, (2) side chain and terminal
residue covalent modifications, and (3) adsorption complexes, for
example with cell membranes. Such covalent or aggregative
derivatives are useful as immunogens, as reagents in immunoassays,
or in purification methods such as for affinity purification of a
receptor or other binding molecule, e.g., an antibody. For example,
a cytokine ligand can be immobilized by covalent bonding to a solid
support such as cyanogen bromide-activated Sepharose, by methods
which are well known in the art, or adsorbed onto polyolefin
surfaces, with or without glutaraldehyde cross-linking, for use in
the assay or purification of an cytokine receptor, antibodies, or
other similar molecules. The ligand can also be labeled with a
detectable group, for example radioiodinated by the chloramine T
procedure, covalently bound to rare earth chelates, or conjugated
to another fluorescent moiety for use in diagnostic assays.
[0096] An DIRS1 of this invention can be used as an immunogen for
the production of antisera or antibodies specific, e.g., capable of
distinguishing between other cytokine receptor family members, for
the DIRS1 or various fragments thereof. The purified DIRS1 can be
used to screen monoclonal antibodies or antigen-binding fragments
prepared by immunization with various forms of impure preparations
containing the protein. Antibodies can typically be substituted
with antigen binding fragments of natural antibodies, e.g., Fab,
Fab2, Fv, etc. The purified DIRS1 can also be used as a reagent to
detect antibodies generated in response to the presence of elevated
levels of expression, or immunological disorders which lead to
antibody production to the endogenous receptor. Additionally, DIRS1
fragments may also serve as immunogens to produce the antibodies of
the present invention, as described immediately below. For example,
this invention contemplates antibodies having binding affinity to
or being raised against the amino acid sequences shown in Table 1,
fragments thereof, or various homologous peptides. In particular,
this invention contemplates antibodies having binding affinity to,
or having been raised against, specific fragments which are
predicted to be, or actually are, exposed at the exterior protein
surface of the native DIRS1.
[0097] The blocking of physiological response to the receptor
ligands may result from the inhibition of binding of the ligand to
the receptor, likely through competitive inhibition. Thus, in vitro
assays of the present invention will often use antibodies or
antigen binding segments of these antibodies, or fragments attached
to solid phase substrates. These assays will also allow for the
diagnostic determination of the effects of either ligand binding
region mutations and modifications, or other mutations and
modifications, e.g., which affect signaling or enzymatic
function.
[0098] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
receptor or fragments compete with a test compound for binding to a
ligand or other antibody. In this manner, the neutralizing
antibodies or fragments can be used to detect the presence of a
polypeptide which shares one or more binding sites to a receptor
and can also be used to occupy binding sites on a receptor that
might otherwise bind a ligand.
V. Making Nucleic Acids and Protein
[0099] DNA which encodes the protein or fragments thereof can be
obtained by chemical synthesis, screening cDNA libraries, or by
screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. Natural sequences can be isolated using
standard methods and the sequences provided herein, e.g., in Table
1. Other species counterparts can be identified by hybridization
techniques, or by various PCR techniques, combined with or by
searching in sequence databases, e.g., GenBank.
[0100] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length receptor or fragments which can
in turn, for example, be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified ligand binding or kinase/phosphatase domains; and for
structure/function studies. Variants or fragments can be expressed
in host cells that are transformed or transfected with appropriate
expression vectors. These molecules can be substantially free of
protein or cellular contaminants, other than those derived from the
recombinant host, and therefore are particularly useful in
pharmaceutical compositions when combined with a pharmaceutically
acceptable carrier and/or diluent. The protein, or portions
thereof, may be expressed as fusions with other proteins.
[0101] Expression vectors are typically self-replicating DNA or RNA
constructs containing the desired receptor gene or its fragments,
usually operably linked to suitable genetic control elements that
are recognized in a suitable host cell. These control elements are
capable of effecting expression within a suitable host. The
specific type of control elements necessary to effect expression
will depend upon the eventual host cell used. Generally, the
genetic control elements can include a prokaryotic promoter system
or a eukaryotic promoter expression control system, and typically
include a transcriptional promoter, an optional operator to control
the onset of transcription, transcription enhancers to elevate the
level of mRNA expression, a sequence that encodes a suitable
ribosome binding site, and sequences that terminate transcription
and translation. Expression vectors also usually contain an origin
of replication that allows the vector to replicate independently of
the host cell.
[0102] The vectors of this invention include those which contain
DNA which encodes a protein, as described, or a fragment thereof
encoding a biologically active equivalent polypeptide. The DNA can
be under the control of a viral promoter and can encode a selection
marker. This invention further contemplates use of such expression
vectors which are capable of expressing eukaryotic cDNA coding for
such a protein in a prokaryotic or eukaryotic host, where the
vector is compatible with the host and where the eukaryotic cDNA
coding for the receptor is inserted into the vector such that
growth of the host containing the vector expresses the cDNA in
question. Usually, expression vectors are designed for stable
replication in their host cells or for amplification to greatly
increase the total number of copies of the desirable gene per cell.
It is not always necessary to require that an expression vector
replicate in a host cell, e.g., it is possible to effect transient
expression of the protein or its fragments in various hosts using
vectors that do not contain a replication origin that is recognized
by the host cell. It is also possible to use vectors that cause
integration of the protein encoding portion or its fragments into
the host DNA by recombination.
[0103] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. Expression vectors are specialized vectors which contain
genetic control elements that effect expression of operably linked
genes. Plasmids are the most commonly used form of vector but all
other forms of vectors which serve an equivalent function and which
are, or become, known in the art are suitable for use herein. See,
e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors: A
Laboratory Manual, Elsevier, N.Y., and Rodriguez, et al. (eds.
1988) Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Buttersworth, Boston, which are incorporated herein by
reference.
[0104] Transformed cells are cells, preferably mammalian, that have
been transformed or transfected with receptor vectors constructed
using recombinant DNA techniques. Transformed host cells usually
express the desired protein or its fragments, but for purposes of
cloning, amplifying, and manipulating its DNA, do not need to
express the subject protein. This invention further contemplates
culturing transformed cells in a nutrient medium, thus permitting
the receptor to accumulate in the cell membrane. The protein can be
recovered, either from the culture or, in certain instances, from
the culture medium.
[0105] For purposes of this invention, nucleic sequences are
operably linked when they are functionally related to each other.
For example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression.
[0106] Suitable host cells include prokaryotes, lower eukaryotes,
and higher eukaryotes. Prokaryotes include both gram negative and
gram positive organisms, e.g., E. coli and B. subtilis. Lower
eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and
species of the genus Dictyostelium. Higher eukaryotes include
established tissue culture cell lines from animal cells, both of
non-mammalian origin, e.g., insect cells, and birds, and of
mammalian origin, e.g., human, primates, and rodents.
[0107] Prokaryotic host-vector systems include a wide variety of
vectors for many different species. As used herein, E. coli and its
vectors will be used generically to include equivalent vectors used
in other prokaryotes. A representative vector for amplifying DNA is
pBR322 or many of its derivatives. Vectors that can be used to
express the receptor or its fragments include, but are not limited
to, such vectors as those containing the lac promoter (pUC-series);
trp promoter (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP
or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540).
See Brosius, et al. (1988) "Expression Vectors Employing Lambda-,
trp-, lac-, and Ipp-derived Promoters", in Vectors: A Survey of
Molecular Cloning Vectors and Their Uses, (eds. Rodriguez and
Denhardt), Buttersworth, Boston, Chapter 10, pp. 205-236, which is
incorporated herein by reference.
[0108] Lower eukaryotes, e.g., yeasts and Dictyostelium, may be
transformed with DIRS1 sequence containing vectors. For purposes of
this invention, the most common lower eukaryotic host is the
baker's yeast, Saccharomyces cerevisiae. It will be used to
generically represent lower eukaryotes although a number of other
strains and species are also available. Yeast vectors typically
consist of a replication origin (unless of the integrating type), a
selection gene, a promoter, DNA encoding the receptor or its
fragments, and sequences for translation termination,
polyadenylation, and transcription termination. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic enzyme gene
promoters or such inducible promoters as the alcohol dehydrogenase
2 promoter or metallothionine promoter. Suitable vectors include
derivatives of the following types: self-replicating low copy
number (such as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the YCp-series).
[0109] Higher eukaryotic tissue culture cells are normally the
preferred host cells for expression of the functionally active
interleukin protein. In principle, many higher eukaryotic tissue
culture cell lines are workable, e.g., insect baculovirus
expression systems, whether from an invertebrate or vertebrate
source. However, mammalian cells are preferred. Transformation or
transfection and propagation of such cells has become a routine
procedure. Examples of useful cell lines include HeLa cells,
Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell
lines, insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually include an
origin of replication, a promoter, a translation initiation site,
RNA splice sites (if genomic DNA is used), a polyadenylation site,
and a transcription termination site. These vectors also usually
contain a selection gene or amplification gene. Suitable expression
vectors may be plasmids, viruses, or retroviruses carrying
promoters derived, e.g., from such sources as from adenovirus,
SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
Representative examples of suitable expression vectors include
pcDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo PolyA, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC
610.
[0110] For secreted proteins, an open reading frame usually encodes
a polypeptide that consists of a mature or secreted product
covalently linked at its N-terminus to a signal peptide. The signal
peptide is cleaved prior to secretion of the mature, or active,
polypeptide. The cleavage site can be predicted with a high degree
of accuracy from empirical rules, e.g., von-Heijne (1986) Nucleic
Acids Research 14:4683-4690 and Nielsen, et al. (1997) Protein Eng.
10:1-12, and the precise amino acid composition of the signal
peptide often does not appear to be critical to its function, e.g.,
Randall, et al. (1989) Science 243:1156-1159; Kaiser et al. (1987)
Science 235:312-317.
[0111] It will often be desired to express these polypeptides in a
system which provides a specific or defined glycosylation pattern.
In this case, the usual pattern will be that provided naturally by
the expression system. However, the pattern will be modifiable by
exposing the polypeptide, e.g., an unglycosylated form, to
appropriate glycosylating proteins introduced into a heterologous
expression system. For example, the receptor gene may be
co-transformed with one or more genes encoding mammalian or other
glycosylating enzymes. Using this approach, certain mammalian
glycosylation patterns will be achievable in prokaryote or other
cells.
[0112] The source of DIRS1 can be a eukaryotic or prokaryotic host
expressing recombinant DIRS1, such as is described above. The
source can also be a cell line such as mouse Swiss 3T3 fibroblasts,
but other mammalian cell lines are also contemplated by this
invention, with the preferred cell line being from the human
species.
[0113] Now that the sequences are known, the primate DIRS1,
fragments, or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include processes such
as are described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; and Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; all of each which are
incorporated herein by reference. For example, an azide process, an
acid chloride process, an acid anhydride process, a mixed anhydride
process, an active ester process (for example, p-nitrophenyl ester,
N-hydroxysuccinimide ester, or cyanomethyl ester), a
carbodiimidazole process, an oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid
phase and solution phase syntheses are both applicable to the
foregoing processes. Similar techniques can be used with partial
DIRS1 sequences.
[0114] The DIRS1 proteins, fragments, or derivatives are suitably
prepared in accordance with the above processes as typically
employed in peptide synthesis, generally either by a so-called
stepwise process which comprises condensing an amino acid to the
terminal amino acid, one by one in sequence, or by coupling peptide
fragments to the terminal amino acid. Amino groups that are not
being used in the coupling reaction typically must be protected to
prevent coupling at an incorrect location.
[0115] If a solid phase synthesis is adopted, the C-terminal amino
acid is bound to an insoluble carrier or support through its
carboxyl group. The insoluble carrier is not particularly limited
as long as it has a binding capability to a reactive carboxyl
group. Examples of such insoluble carriers include halomethyl
resins, such as chloromethyl resin or bromomethyl resin,
hydroxymethyl resins, phenol resins,
tert-alkyloxycarbonylhydrazidated resins, and the like.
[0116] An amino group-protected amino acid is bound in sequence
through condensation of its activated carboxyl group and the
reactive amino group of the previously formed peptide or chain, to
synthesize the peptide step by step. After synthesizing the
complete sequence, the peptide is split off from the insoluble
carrier to produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc. 85:2149-2156, which is incorporated herein by reference.
[0117] The prepared protein and fragments thereof can be isolated
and purified from the reaction mixture by means of peptide
separation, for example, by extraction, precipitation,
electrophoresis, various forms of chromatography, and the like. The
receptors of this invention can be obtained in varying degrees of
purity depending upon desired uses. Purification can be
accomplished by use of the protein purification techniques
disclosed herein, see below, or by the use of the antibodies herein
described in methods of immunoabsorbant affinity chromatography.
This immunoabsorbant affinity chromatography is carried out by
first linking the antibodies to a solid support and then contacting
the linked antibodies with solubilized lysates of appropriate
cells, lysates of other cells expressing the receptor, or lysates
or supernatants of cells producing the protein as a result of DNA
techniques, see below.
[0118] Generally, the purified protein will be at least about 40%
pure, ordinarily at least about 50% pure, usually at least about
60% pure, typically at least about 70% pure, more typically at
least about 80% pure, preferable at least about 90% pure and more
preferably at least about 95% pure, and in particular embodiments,
97%-99% or more. Purity will usually be on a weight basis, but can
also be on a molar basis. Different assays will be applied as
appropriate.
VI. Antibodies
[0119] Antibodies can be raised to the various mammalian, e.g.,
primate DIRS1 proteins and fragments thereof, both in naturally
occurring native forms and in their recombinant forms, the
difference being that antibodies to the active receptor are more
likely to recognize epitopes which are only present in the native
conformations. Denatured antigen detection can also be useful in,
e.g., Western analysis. Anti-idiotypic antibodies are also
contemplated, which would be useful as agonists or antagonists of a
natural receptor or an antibody.
[0120] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the protein can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective protein, or screened
for agonistic or antagonistic activity. These monoclonal antibodies
will usually bind with at least a K.sub.D of about 1 mM, more
usually at least about 300 .mu.M, typically at least about 100
.mu.M, more typically at least about 30 .mu.M, preferably at least
about 10 .mu.M, and more preferably at least about 3 .mu.M or
better.
[0121] The antibodies, including antigen binding fragments, of this
invention can have significant diagnostic or therapeutic value.
They can be potent antagonists that bind to the receptor and
inhibit binding to ligand or inhibit the ability of the receptor to
elicit a biological response, e.g., act on its substrate. They also
can be useful as non-neutralizing antibodies and can be coupled to
toxins or radionuclides to bind producing cells, or cells localized
to the source of the interleukin. Further, these antibodies can be
conjugated to drugs or other therapeutic agents, either directly or
indirectly by means of a linker.
[0122] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they might bind to the receptor without inhibiting ligand or
substrate binding. As neutralizing antibodies, they can be useful
in competitive binding assays. They will also be useful in
detecting or quantifying ligand. They may be used as reagents for
Western blot analysis, or for immunoprecipitation or
immunopurification of the respective protein.
[0123] Protein fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. Mammalian cytokine receptors
and fragments may be fused or covalently linked to a variety of
immunogens, such as keyhole limpet hemocyanin, bovine serum
albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical
Division, Harper and Row, 1969; Landsteiner (1962) Specificity of
Serological Reactions, Dover Publications, New York; and Williams,
et al. (1967) Methods in Immunology and Immunochemistry, Vol. 1,
Academic Press, New York; each of which are incorporated herein by
reference, for descriptions of methods of preparing polyclonal
antisera. A typical method involves hyperimmunization of an animal
with an antigen. The blood of the animal is then collected shortly
after the repeated immunizations and the gamma globulin is
isolated.
[0124] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites, et al. (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York; and particularly in Kohler and
Milstein (1975) in Nature 256: 495-497, which discusses one method
of generating monoclonal antibodies. Each of these references is
incorporated herein by reference. Summarized briefly, this method
involves injecting an animal with an immunogen. The animal is then
sacrificed and cells taken from its spleen, which are then fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secrete a
single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0125] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546, each of
which is hereby incorporated herein by reference. The polypeptides
and antibodies of the present invention may be used with or without
modification, including chimeric or humanized antibodies.
Frequently, the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents, teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant or chimeric
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; or made in transgenic mice, see, e.g., Mendez, et al.
(1997) Nature Genetics 15:146-156. These references are
incorporated herein by reference.
[0126] The antibodies of this invention can also be used for
affinity chromatography in isolating the DIRS1 proteins or
peptides. Columns can be prepared where the antibodies are linked
to a solid support, e.g., particles, such as agarose, Sephadex, or
the like, where a cell lysate may be passed through the column, the
column washed, followed by increasing concentrations of a mild
denaturant, whereby the purified protein will be released. The
protein may be used to purify antibody. Conversely, the antibodies
may be immunoselected or immunodepleted to provide binding
compositions of defined specificities.
[0127] The antibodies may also be used to screen expression
libraries for particular expression products. Usually the
antibodies used in such a procedure will be labeled with a moiety
allowing easy detection of presence of antigen by antibody
binding.
[0128] Antibodies raised against a cytokine receptor will also be
used to raise anti-idiotypic antibodies. These will be useful in
detecting or diagnosing various immunological conditions related to
expression of the protein or cells which express the protein. They
also will be useful as agonists or antagonists of the ligand, which
may be competitive inhibitors or substitutes for naturally
occurring ligands.
[0129] A cytokine receptor protein that specifically binds to or
that is specifically immunoreactive with an antibody generated
against a defined immunogen, such as an immunogen consisting of the
amino acid sequence of SEQ ID NO: 2, is typically determined in an
immunoassay. The immunoassay typically uses a polyclonal antiserum
which was raised, e.g., to a protein of SEQ ID NO: 2. This
antiserum is selected to have low crossreactivity against other
cytokine receptor family members, e.g., IL-12 receptor beta or
gp130, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption prior to use in the
immunoassay.
[0130] In order to produce antisera for use in an immunoassay, the
protein, e.g., of SEQ ID NO: 2, is isolated as described herein.
For example, recombinant protein may be produced in a mammalian
cell line. An appropriate host, e.g., an inbred strain of mice such
as Balb/c, is immunized with the selected protein, typically using
a standard adjuvant, such as Freund's adjuvant, and a standard
mouse immunization protocol (see Harlow and Lane, supra).
Alternatively, a synthetic peptide derived from the sequences
disclosed herein and conjugated to a carrier protein can be used an
immunogen. Polyclonal sera are collected and titered against the
immunogen protein in an immunoassay, e.g., a solid phase
immunoassay with the immunogen immobilized on a solid support.
Polyclonal antisera with a titer of 10.sup.4 or greater are
selected and tested for their cross reactivity against other
cytokine receptor family members, e.g., IL-12 receptor beta and/or
gp130, using a competitive binding immunoassay such as the one
described in Harlow and Lane, supra, at pages 570-573. Preferably
at least two cytokine receptor family members are used in this
determination. These cytokine receptor family members can be
produced as recombinant proteins and isolated using standard
molecular biology and protein chemistry techniques as described
herein.
[0131] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the protein of
SEQ ID NO: 2 can be immobilized to a solid support. Proteins added
to the assay compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the antisera to the immobilized protein is
compared to the proteins of IL-12 receptor beta or gp130. The
percent crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
crossreactivity with each of the proteins listed above are selected
and pooled. The cross-reacting antibodies are then removed from the
pooled antisera by immunoabsorption with the above-listed
proteins.
[0132] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen protein (e.g., the DIRS1 like
protein of SEQ ID NO: 2). In order to make this comparison, the two
proteins are each assayed at a wide range of concentrations and the
amount of each protein required to inhibit 50% of the binding of
the antisera to the immobilized protein is determined. If the
amount of the second protein required is less than twice the amount
of the protein of the selected protein or proteins that is
required, then the second protein is said to specifically bind to
an antibody generated to the immunogen.
[0133] It is understood that these cytokine receptor proteins are
members of a family of homologous proteins that comprise at least 6
so far identified genes. For a particular gene product, such as the
DIRS1, the term refers not only to the amino acid sequences
disclosed herein, but also to other proteins that are allelic,
non-allelic, or species variants. It is also understood that the
terms include nonnatural mutations introduced by deliberate
mutation using conventional recombinant technology such as single
site mutation, or by excising short sections of DNA encoding the
respective proteins, or by substituting new amino acids, or adding
new amino acids. Such minor alterations typically will
substantially maintain the immunoidentity of the original molecule
and/or its biological activity. Thus, these alterations include
proteins that are specifically immunoreactive with a designated
naturally occurring DIRS1 protein. The biological properties of the
altered proteins can be determined by expressing the protein in an
appropriate cell line and measuring the appropriate effect, e.g.,
upon transfected lymphocytes. Particular protein modifications
considered minor would include conservative substitution of amino
acids with similar chemical properties, as described above for the
cytokine receptor family as a whole. By aligning a protein
optimally with the protein of the cytokine receptors and by using
the conventional immunoassays described herein to determine
immunoidentity, one can determine the protein compositions of the
invention.
VII. Kits and Quantitation
[0134] Both naturally occurring and recombinant forms of the
cytokine receptor like molecules of this invention are particularly
useful in kits and assay methods. For example, these methods would
also be applied to screening for binding activity, e.g., ligands
for these proteins. Several methods of automating assays have been
developed in recent years so as to permit screening of tens of
thousands of compounds per year. See, e.g., a BIOMEK automated
workstation, Beckman Instruments, Palo Alto, Calif., and Fodor, et
al. (1991) Science 251:767-773, which is incorporated herein by
reference. The latter describes means for testing binding by a
plurality of defined polymers synthesized on a solid substrate. The
development of suitable assays to screen for a ligand or
agonist/antagonist homologous proteins can be greatly facilitated
by the availability of large amounts of purified, soluble cytokine
receptors in an active state such as is provided by this
invention.
[0135] Purified DIRS1 can be coated directly onto plates for use in
the aforementioned ligand screening techniques. However,
non-neutralizing antibodies to these proteins can be used as
capture antibodies to immobilize the respective receptor on the
solid phase, useful, e.g., in diagnostic uses.
[0136] This invention also contemplates use of DIRS1, fragments
thereof, peptides, and their fusion products in a variety of
diagnostic kits and methods for detecting the presence of the
protein or its ligand. Alternatively, or additionally, antibodies
against the molecules may be incorporated into the kits and
methods. Typically the kit will have a compartment containing
either a DIRS1 peptide or gene segment or a reagent which
recognizes one or the other. Typically, recognition reagents, in
the case of peptide, would be a receptor or antibody, or in the
case of a gene segment, would usually be a hybridization probe.
[0137] A preferred kit for determining the concentration of DIRS1
in a sample would typically comprise a labeled compound, e.g.,
ligand or antibody, having known binding affinity for DIRS1, a
source of DIRS1 (naturally occurring or recombinant) as a positive
control, and a means for separating the bound from free labeled
compound, for example a solid phase for immobilizing the DIRS1 in
the test sample. Compartments containing reagents, and
instructions, will normally be provided.
[0138] Antibodies, including antigen binding fragments, specific
for mammalian DIRS1 or a peptide fragment, or receptor fragments
are useful in diagnostic applications to detect the presence of
elevated levels of ligand and/or its fragments. Diagnostic assays
may be homogeneous (without a separation step between free reagent
and antibody-antigen complex) or heterogeneous (with a separation
step). Various commercial assays exist, such as radioimmunoassay
(RIA), enzyme-linked immunosorbent assay (ELISA), enzyme
immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),
substrate-labeled fluorescent immunoassay (SLFIA) and the like. For
example, unlabeled antibodies can be employed by using a second
antibody which is labeled and which recognizes the antibody to a
cytokine receptor or to a particular fragment thereof. These assays
have also been extensively discussed in the literature. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH., and
Coligan (ed. 1991 and periodic supplements) Current Protocols In
Immunology Greene/Wiley, New York.
[0139] Anti-idiotypic antibodies may have similar use to serve as
agonists or antagonists of cytokine receptors. These should be
useful as therapeutic reagents under appropriate circumstances.
[0140] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody, or
labeled ligand is provided. This is usually in conjunction with
other additives, such as buffers, stabilizers, materials necessary
for signal production such as substrates for enzymes, and the like.
Preferably, the kit will also contain instructions for proper use
and disposal of the contents after use. Typically the kit has
compartments for each useful reagent, and will contain instructions
for proper use and disposal of reagents. Desirably, the reagents
are provided as a dry lyophilized powder, where the reagents may be
reconstituted in an aqueous medium having appropriate
concentrations for performing the assay.
[0141] The aforementioned constituents of the diagnostic assays may
be used without modification or may be modified in a variety of
ways. For example, labeling may be achieved by covalently or
non-covalently joining a moiety which directly or indirectly
provides a detectable signal. In many of these assays, a test
compound, cytokine receptor, or antibodies thereto can be labeled
either directly or indirectly. Possibilities for direct labeling
include label groups: radiolabels such as .sup.125I, enzymes (U.S.
Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase,
and fluorescent labels (U.S. Pat. No. 3,940,475) capable of
monitoring the change in fluorescence intensity, wavelength shift,
or fluorescence polarization. Both of the patents are incorporated
herein by reference. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0142] There are also numerous methods of separating the bound from
the free ligand, or alternatively the bound from the free test
compound. The cytokine receptor can be immobilized on various
matrixes followed by washing. Suitable matrices include plastic
such as an ELISA plate, filters, and beads. Methods of immobilizing
the receptor to a matrix include, without limitation, direct
adhesion to plastic, use of a capture antibody, chemical coupling,
and biotin-avidin. The last step in this approach involves the
precipitation of antibody/antigen complex by any of several methods
including those utilizing, e.g., an organic solvent such as
polyethylene glycol or a salt such as ammonium sulfate. Other
suitable separation techniques include, without limitation, the
fluorescein antibody magnetizable particle method described in
Rattle, et al. (1984) Clin. Chem. 30(9):1457-1461, and the double
antibody magnetic particle separation as described in U.S. Pat. No.
4,659,678, each of which is incorporated herein by reference.
[0143] The methods for linking protein or fragments to various
labels have been extensively reported in the literature and do not
require detailed discussion here. Many of the techniques involve
the use of activated carboxyl groups either through the use of
carbodiimide or active esters to form peptide bonds, the formation
of thioethers by reaction of a mercapto group with an activated
halogen such as chloroacetyl, or an activated olefin such as
maleimide, for linkage, or the like. Fusion proteins will also find
use in these applications.
[0144] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an cytokine receptor. These sequences can be used as probes for
detecting levels of the respective cytokine receptor in patients
suspected of having an immunological disorder. The preparation of
both RNA and DNA nucleotide sequences, the labeling of the
sequences, and the preferred size of the sequences has received
ample description and discussion in the literature. Normally an
oligonucleotide probe should have at least about 14 nucleotides,
usually at least about 18 nucleotides, and the polynucleotide
probes may be up to several kilobases. Various labels may be
employed, most commonly radionuclides, particularly .sup.32P.
However, other techniques may also be employed, such as using
biotin modified nucleotides for introduction into a polynucleotide.
The biotin then serves as the site for binding to avidin or
antibodies, which may be labeled with a wide variety of labels,
such as radionuclides, fluorescers, enzymes, or the like.
Alternatively, antibodies may be employed which can recognize
specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA
hybrid duplexes, or DNA-protein duplexes. The antibodies in turn
may be labeled and the assay 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. The
use of probes to the novel anti-sense RNA may be carried out in
conventional techniques such as nucleic acid hybridization, plus
and minus screening, recombinational probing, hybrid released
translation (HRT), and hybrid arrested translation (HART). This
also includes amplification techniques such as polymerase chain
reaction (PCR).
[0145] Diagnostic kits which also test for the qualitative or
quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet, et al. (1989) Progress in Growth
Factor Res. 1:89-97.
VIII. Therapeutic Utility
[0146] This invention provides reagents with significant
therapeutic value. See, e.g., Levitzki (1996) Curr. Opin. Cell
Biol. 8:239-244. The cytokine receptors (naturally occurring or
recombinant), fragments thereof, mutein receptors, and antibodies,
along with compounds identified as having binding affinity to the
receptors or antibodies, should be useful in the treatment of
conditions exhibiting abnormal expression of the receptors of their
ligands. Such abnormality will typically be manifested by
immunological disorders. Additionally, this invention should
provide therapeutic value in various diseases or disorders
associated with abnormal expression or abnormal triggering of
response to the ligand. For example, the IL-1 ligands have been
suggested to be involved in morphologic development, e.g.,
dorso-ventral polarity determination, and immune responses,
particularly the primitive innate responses. See, e.g., Sun, et al.
(1991) Eur. J. Biochem. 196:247-254; and Hultmark (1994) Nature
367:116-117.
[0147] Recombinant cytokine receptors, muteins, agonist or
antagonist antibodies thereto, or antibodies can be purified and
then administered to a patient. These reagents can be combined for
therapeutic use with additional active ingredients, e.g., in
conventional pharmaceutically acceptable carriers or diluents,
along with physiologically innocuous stabilizers and excipients.
These combinations can be sterile, e.g., filtered, and placed into
dosage forms as by lyophilization in dosage vials or storage in
stabilized aqueous preparations. This invention also contemplates
use of antibodies or binding fragments thereof which are not
complement binding.
[0148] Ligand screening using cytokine receptor or fragments
thereof can be performed to identify molecules having binding
affinity to the receptors. Subsequent biological assays can then be
utilized to determine if a putative ligand can provide competitive
binding, which can block intrinsic stimulating activity. Receptor
fragments can be used as a blocker or antagonist in that it blocks
the activity of ligand. Likewise, a compound having intrinsic
stimulating activity can activate the receptor and is thus an
agonist in that it simulates the activity of ligand, e.g., inducing
signaling. This invention further contemplates the therapeutic use
of antibodies to cytokine receptors as antagonists.
[0149] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, reagent physiological life,
pharmacological life, physiological state of the patient, and other
medicants administered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages used in vitro
may provide useful guidance in the amounts useful for in situ
administration of these reagents. Animal testing of effective doses
for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds. 1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa.; each of which is hereby
incorporated herein by reference. Methods for administration are
discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers will
include water, saline, buffers, and other compounds described,
e.g., in the Merck Index, Merck & Co., Rahway, N.J. Because of
the likely high affinity binding, or turnover numbers, between a
putative ligand and its receptors, low dosages of these reagents
would be initially expected to be effective. And the signaling
pathway suggests extremely low amounts of ligand may have effect.
Thus, dosage ranges would ordinarily be expected to be in amounts
lower than 1 mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 pM (picomolar), and most preferably less than about 1
fM (femtomolar), with an appropriate carrier. Slow release
formulations, or slow release apparatus will often be utilized for
continuous administration.
[0150] Cytokine receptors, fragments thereof, and antibodies or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations comprise
at least one active ingredient, as defined above, together with one
or more acceptable carriers thereof. Each carrier must be both
pharmaceutically and physiologically acceptable in the sense of
being compatible with the other ingredients and not injurious to
the patient. Formulations include those suitable for oral, rectal,
nasal, or parenteral (including subcutaneous, intramuscular,
intravenous and intradermal) administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by methods well known in the art of pharmacy. See, e.g., Gilman, et
al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of
Therapeutics, 8th Ed., Pergamon Press; and Remington's
Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co.,
Easton, Pa.; Avis, et al. (eds. 1993) Pharmaceutical Dosage Forms:
Parenteral Medications Dekker, NY; Lieberman, et al. (eds. 1990)
Pharmaceutical Dosage Forms: Tablets Dekker, NY; and Lieberman, et
al. (eds. 1990) Pharmaceutical Dosage Forms Disperse Systems
Dekker, NY. The therapy of this invention may be combined with or
used in association with other therapeutic agents, particularly
agonists or antagonists of other cytokine receptor family
members.
IX. Screening
[0151] Drug screening using DIRS1 or fragments thereof can be
performed to identify compounds having binding affinity to the
receptor subunit, including isolation of associated components.
Subsequent biological assays can then be utilized to determine if
the compound has intrinsic stimulating activity and is therefore a
blocker or antagonist in that it blocks the activity of the ligand.
Likewise, a compound having intrinsic stimulating activity can
activate the receptor and is thus an agonist in that it simulates
the activity of a cytokine ligand. This invention further
contemplates the therapeutic use of antibodies to the receptor as
cytokine agonists or antagonists.
[0152] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing the DIRS1. Cells may be
isolated which express a receptor in isolation from other
functional receptors. Such cells, either in viable or fixed form,
can be used for standard ligand/receptor binding assays. See also,
Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)
Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive
methods to detect cellular responses. Competitive assays are
particularly useful, where the cells (source of putative ligand)
are contacted and incubated with a labeled receptor or antibody
having known binding affinity to the ligand, such as
.sup.125I-antibody, and a test sample whose binding affinity to the
binding composition is being measured. The bound and free labeled
binding compositions are then separated to assess the degree of
ligand binding. The amount of test compound bound is inversely
proportional to the amount of labeled receptor binding to the known
source. Any one of numerous techniques can be used to separate
bound from free ligand to assess the degree of ligand binding. This
separation step could typically involve a procedure such as
adhesion to filters followed by washing, adhesion to plastic
followed by washing, or centrifugation of the cell membranes.
Viable cells could also be used to screen for the effects of drugs
on cytokine mediated functions, e.g., second messenger levels,
i.e., Ca.sup.++; cell proliferation; inositol phosphate pool
changes; and others. Some detection methods allow for elimination
of a separation step, e.g., a proximity sensitive detection system.
Calcium sensitive dyes will be useful for detecting Ca.sup.++
levels, with a fluorimeter or a fluorescence cell sorting
apparatus.
X. Ligands
[0153] The descriptions of the DIRS1 herein provide means to
identify ligands, as described above. Such ligand should bind
specifically to the respective receptor with reasonably high
affinity. Various constructs are made available which allow either
labeling of the receptor to detect its ligand. For example,
directly labeling cytokine receptor, fusing onto it markers for
secondary labeling, e.g., FLAG or other epitope tags, etc., will
allow detection of receptor. This can be histological, as an
affinity method for biochemical purification, or labeling or
selection in an expression cloning approach. A two-hybrid selection
system may also be applied making appropriate constructs with the
available cytokine receptor sequences. See, e.g., Fields and Song
(1989) Nature 340:245-246.
[0154] Generally, descriptions of cytokine receptors will be
analogously applicable to individual specific embodiments directed
to DIRS1 reagents and compositions.
[0155] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
I. General Methods
[0156] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene/Wiley,
New York. Methods for protein purification include such methods as
ammonium sulfate precipitation, column chromatography,
electrophoresis, centrifugation, crystallization, and others. See,
e.g., Ausubel, et al. (1987 and periodic supplements); Coligan, et
al. (ed. 1996) and periodic supplements, Current Protocols In
Protein Science Greene/Wiley, New York; Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology, vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, N.J.,
or Bio-Rad, Richmond, Calif. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) QIAexpress: The High Level Expression
& Protein Purification System QUIAGEN, Inc., Chatsworth,
Calif.
[0157] Computer sequence analysis is performed, e.g., using
available software programs, including those from the GCG (U.
Wisconsin) and GenBank sources. Public sequence databases were also
used, e.g., from GenBank and others.
[0158] Many techniques applicable to IL-10 or IL-12 receptors may
be applied to the DIRS1, as described, e.g., in U.S. Ser. No.
08/110,683 (IL-10 receptor), which is incorporated herein by
reference.
II. Computational Analysis
[0159] Human sequences related to cytokine receptors were
identified from genomic sequence database using, e.g., the BLAST
server (Altschul, et al. (1994) Nature Genet. 6:119-129). Standard
analysis programs may be used to evaluate structure, e.g., PHD
(Rost and Sander (1994) Proteins 19:55-72) and DSC (King and
Sternberg (1996) Protein Sci. 5:2298-2310). Standard comparison
software includes, e.g., Altschul, et al. (1990) J. Mol. Biol.
215:403-10; Waterman (1995) Introduction to Computational Biology:
Maps, Sequences, and Genomes Chapman & Hall; Lander and
Waterman (eds. 1995) Calculating the Secrets of Life: Applications
of the Mathematical Sciences in Molecular Biology National Academy
Press; and Speed and Waterman (eds. 1996) Genetic Mapping and DNA
Sequencing (Ima Volumes in Mathematics and Its Applications, Vol
81) Springer Verlag.
III. Cloning of Full-Length DIRS cDNAs; Chromosomal
Localization
[0160] PCR primers derived from the DIRS sequences are used to
probe a human cDNA library. Full length cDNAs for primate, rodent,
or other species DIRS1 are cloned, e.g., by DNA hybridization
screening of .lamda.gt10 phage. PCR reactions are conducted using
T. aquaticus Taqplus DNA polymerase (Stratagene) under appropriate
conditions.
[0161] Chromosome spreads are prepared. In situ hybridization is
performed on chromosome preparations obtained from
phytohemagglutinin-stimulated human lymphocytes cultured for 72 h.
5-bromodeoxyuridine was added for the final seven hours of culture
(60 .mu.g/ml of medium), to ensure a posthybridization chromosomal
banding of good quality.
[0162] A PCR fragment, amplified with the help of primers, is
cloned into an appropriate vector. The vector is labeled by
nick-translation with .sup.3H. The radiolabeled probe is hybridized
to metaphase spreads at final concentration of 200 ng/ml of
hybridization solution as described in Mattei, et al. (1985) Hum.
Genet. 69:327-331.
[0163] After coating with nuclear track emulsion (KODAK NTB.sub.2),
slides are exposed. To avoid any slipping of silver grains during
the banding procedure, chromosome spreads are first stained with
buffered Giemsa solution and metaphase photographed. R-banding is
then performed by the fluorochrome-photolysis-Giemsa (FPG) method
and metaphases rephotographed before analysis. Alternatively,
mapped sequence tags may be searched in a database.
[0164] Similar appropriate methods are used for other species.
IV. Localization of DIRS1 or DIRS2 mRNA
[0165] Human multiple tissue (Cat# 1, 2) and cancer cell line blots
(Cat# 7757-1), containing approximately 2 .mu.g of poly(A).sup.+
RNA per lane, are purchased from Clontech (Palo Alto, Calif.).
Probes are radiolabeled with [.alpha.-.sup.32P] dATP, e.g., using
the Amersham Rediprime random primer labeling kit (RPN1633).
Prehybridization and hybridizations are performed at 65.degree. C.
in 0.5 M Na.sub.2HPO.sub.4, 7% SDS, 0.5 M EDTA (pH 8.0). High
stringency washes are conducted, e.g., at 65.degree. C. with two
initial washes in 2.times.SSC, 0.1% SDS for 40 min followed by a
subsequent wash in 0.1.times.SSC, 0.1% SDS for 20 min. Membranes
are then exposed at -70.degree. C. to X-Ray film (Kodak) in the
presence of intensifying screens. More detailed studies by cDNA
library Southerns are performed with selected human DIRS1 clones to
examine their expression in hemopoietic or other cell subsets.
[0166] Alternatively, two appropriate primers are selected from
Table 1 or 2. RT-PCR is used on an appropriate mRNA sample selected
for the presence of message to produce a cDNA, e.g., a sample which
expresses the gene.
[0167] Full length clones may be isolated by hybridization of cDNA
libraries from appropriate tissues pre-selected by PCR signal.
Northern blots can be performed.
[0168] Message for genes encoding DIRS1 will be assayed by
appropriate technology, e.g., PCR, immunoassay, hybridization, or
otherwise. Tissue and organ cDNA preparations are available, e.g.,
from Clontech, Mountain View, Calif. Identification of sources of
natural expression are useful, as described. And the identification
of functional receptor subunit pairings will allow for prediction
of what cells express the combination of receptor subunits which
will result in a physiological responsiveness to each of the
cytokine ligands.
[0169] For mouse distribution, e.g., Southern Analysis can be
performed: DNA (5 .mu.g) from a primary amplified cDNA library was
digested with appropriate restriction enzymes to release the
inserts, run on a 1% agarose gel and transferred to a nylon
membrane (Schleicher and Schuell, Keene, N.H.).
[0170] Samples for mouse mRNA isolation may include: resting mouse
fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogen
receptor) transfected cells, control (C201); T cells, TH1 polarized
(Mel14 bright, CD4+ cells from spleen, polarized for 7 days with
IFN-.gamma. and anti IL-4; T200); T cells, TH2 polarized (Mel14
bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and
anti-IFN-.gamma.; T201); T cells, highly TH1 polarized (see
Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367; activated with
anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highly TH2
polarized (see Openshaw, et al. (1995) J. Exp. Med. 182:1357-1367;
activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44- CD25+
pre T cells, sorted from thymus (T204); TH1 T cell clone D1.1,
resting for 3 weeks after last stimulation with antigen (T205); TH1
T cell clone D1.1, 10 .mu.g/ml ConA stimulated 15 h (T206); TH2 T
cell clone CDC35, resting for 3 weeks after last stimulation with
antigen (T207); TH2 T cell clone CDC35, 10 .mu.g/ml ConA stimulated
15 h (T208); Mel14+ naive T cells from spleen, resting (T209);
Mel14+ T cells, polarized to Th1 with IFN-.gamma./IL-12/anti-IL-4
for 6, 12, 24 h pooled (T210); Mel14+ T cells, polarized to Th2
with IL-4/anti-IFN-.gamma. for 6, 13, 24 h pooled (T211);
unstimulated mature B cell leukemia cell line A20 (B200);
unstimulated B cell line CH12 (B201); unstimulated large B cells
from spleen (B202); B cells from total spleen, LPS activated
(B203); metrizamide enriched dendritic cells from spleen, resting
(D200); dendritic cells from bone marrow, resting (D201); monocyte
cell line RAW 264.7 activated with LPS 4 h (M200); bone-marrow
macrophages derived with GM and M-CSF (M201); macrophage cell line
J774, resting (M202); macrophage cell line J774+LPS+anti-IL-10 at
0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line
J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 h pooled (M204); aerosol
challenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7,
14, 23 h pooled (see Garlisi, et al. (1995) Clinical Immunology and
Immunopathology 75:75-83; X206); Nippostrongulus-infected lung
tissue (see Coffman, et al. (1989) Science 245:308-310; X200);
total adult lung, normal (O200); total lung, rag-1 (see Schwarz, et
al. (1993) Immunodeficiency 4:249-252; 0205); IL-10 K.O. spleen
(see Kuhn, et al. (1991) Cell 75:263-274; X201); total adult
spleen, normal (O201); total spleen, rag-1 (O207); IL-10 K.O.
Peyer's patches (O202); total Peyer's patches, normal (O210); IL-10
K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes,
normal (O211); IL-10 K.O. colon (X203); total colon, normal (O212);
NOD mouse pancreas (see Makino, et al. (1980) Jikken Dobutsu
29:1-13; X205); total thymus, rag-1 (O208); total kidney, rag-1
(O209); total heart, rag-1 (O202); total brain, rag-1 (O203); total
testes, rag-1 (O204); total liver, rag-1 (O206); rat normal joint
tissue (O300); and rat arthritic joint tissue (X300).
[0171] Samples for human mRNA isolation may include: peripheral
blood mononuclear cells (monocytes, T cells, NK cells,
granulocytes, B cells), resting (T100); peripheral blood
mononuclear cells, activated with anti-CD3 for 2, 6, 12 h pooled
(T101); T cell, TH0 clone Mot 72, resting (T102); T cell, TH0 clone
Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled
(T103); T cell, TH0 clone Mot 72, anergic treated with specific
peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06,
resting (T107); T cell, TH1 clone HY06, activated with anti-CD28
and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,
anergic treated with specific peptide for 2, 6, 12 h pooled (T109);
T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,
activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111);
T cells CD4+CD45RO- T cells polarized 27 days in anti-CD28, IL-4,
and anti IFN-.gamma., TH2 polarized, activated with anti-CD3 and
anti-CD28 4 h (T116); T cell tumor lines Jurkat and Hut78, resting
(T117); T cell clones, pooled AD130.2, Tc783.12, Tc783.13,
Tc783.58, Tc782.69, resting (T118); T cell random .gamma..delta. T
cell clones, resting (T119); Splenocytes, resting (B100);
Splenocytes, activated with anti-CD40 and IL-4 (B101); B cell EBV
lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting
(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h
pooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones
pooled, activated with PMA and ionomycin for 6 h (K101); NKL clone,
derived from peripheral blood of LGL leukemia patient, IL-2 treated
(K106); NK cytotoxic clone 640-A30-1, resting (K107); hematopoietic
precursor line TF1, activated with PMA and ionomycin for 1, 6 h
pooled (C100); U937 premonocytic line, resting (M100); U937
premonocytic line, activated with PMA and ionomycin for 1, 6 h
pooled (M101); elutriated monocytes, activated with LPS,
IFN.gamma., anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102);
elutriated monocytes, activated with LPS, IFN.gamma., IL-10 for 1,
2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated with
LPS, IFN.gamma., anti-IL-10 for 4, 16 h pooled (M106); elutriated
monocytes, activated with LPS, IFN.gamma., IL-10 for 4, 16 h pooled
(M107); elutriated monocytes, activated LPS for 1 h (M108);
elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+,
from CD34+ GM-CSF, TNF.alpha. 12 days, resting (D101); DC 70%
CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days, activated with PMA
and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+ GM-CSF,
TNF.alpha. 12 days, activated with PMA and ionomycin for 6 hr
(D103); DC 95% CD1a+, from CD34+ GM-CSF, TNF.alpha. 12 days FACS
sorted, activated with PMA and ionomycin for 1, 6 h pooled (D104);
DC 95% CD14+, ex CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted,
activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+
CD86+, from CD34+ GM-CSF, TNF.alpha. 12 days FACS sorted, activated
with PMA and ionomycin for 1, 6 h pooled (D106); DC from monocytes
GM-CSF, IL-4 5 days, resting (D107); DC from monocytes GM-CSF, IL-4
5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5 days,
activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4
5 days, activated TNF.alpha., monocyte supe for 4, 16 h pooled
(D110); leiomyoma L11 benign tumor (X101); normal myometrium M5
(O115); malignant leiomyosarcoma GS1 (X103); lung fibroblast
sarcoma line MRC5, activated with PMA and ionomycin for 1, 6 h
pooled (C101); kidney epithelial carcinoma cell line CHA, activated
with PMA and ionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk
male (O100); lung fetal 28 wk male (O101); liver fetal 28 wk male
(O102); heart fetal 28 wk male (O103); brain fetal 28 wk male
(O104); gallbladder fetal 28 wk male (O106); small intestine fetal
28 wk male (O107); adipose tissue fetal 28 wk male (O108); ovary
fetal 25 wk female (O109); uterus fetal 25 wk female (O110); testes
fetal 28 wk male (O111); spleen fetal 28 wk male (O112); adult
placenta 28 wk (O113); and tonsil inflamed, from 12 year old
(X100).
[0172] With a cDNA Southern, the human DIRS1 was found in LPS
activated dendritic cells ("DC LPS"); monokine activated dendritic
cells ("DC mix"); normal skin; Psoriasis skin; inflamed tonsil;
fetal liver; fetal small intestine; fetal ovary; resting "70%
dendritic cells"; 6 hr activated 70% dendritic cells; and LPS
activated monocytes. A signal was also detected in normal monkey
lung and Ascaris-challenged monkey lung (24 h), which indicates
cross species hybridization. The following libraries had weaker
expression of DIRS1: smoker lung pool; fetal spleen CD4+ T cells
(TH2 polarized); gamma delta T cells; activated splenocytes; and B
cells.
[0173] HOFNy28 (DIRS2) is expressed in U937 (a premonocytic cell
line) cells, both resting and activated; activated A549 cells
(epithelial cells, IL-1.beta. activated); fetal uterus; fetal
testes; and fetal spleen. This data is from PCR on these cDNA
libraries followed by Southern hybridization.
[0174] Similar samples may isolated in other species for
evaluation.
V. Cloning of Species Counterparts of DIRS1 or DIRS2
[0175] Various strategies are used to obtain species counterparts
of, e.g., the DIRS1, preferably from other primates or rodents. One
method is by cross hybridization using closely related species DNA
probes. It may be useful to go into evolutionarily similar species
as intermediate steps. Another method is by using specific PCR
primers based on the identification of blocks of similarity or
difference between genes, e.g., areas of highly conserved or
nonconserved polypeptide or nucleotide sequence. Database sequence
searches may also identify species counterparts.
VI. Production of Mammalian DIRS1 or DIRS2 Protein
[0176] An appropriate, e.g., GST, fusion construct is engineered
for expression, e.g., in E. coli. For example, a mouse IGIF pGex
plasmid is constructed and transformed into E. coli. Freshly
transformed cells are grown, e.g., in LB medium containing 50
.mu.g/ml ampicillin and induced with IPTG (Sigma, St. Louis, Mo.).
After overnight induction, the bacteria are harvested and the
pellets containing the DIRS1 protein are isolated. The pellets are
homogenized, e.g., in TE buffer (50 mM Tris-base pH 8.0, 10 mM EDTA
and 2 mM pefabloc) in 2 liters. This material is passed through a
microfluidizer (Microfluidics, Newton, Mass.) three times. The
fluidized supernatant is spun down on a Sorvall GS-3 rotor for 1 h
at 13,000 rpm. The resulting supernatant containing the cytokine
receptor protein is filtered and passed over a
glutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH
8.0. The fractions containing the DIRS1-GST fusion protein are
pooled and cleaved, e.g., with thrombin (Enzyme Research
Laboratories, Inc., South Bend, Ind.). The cleaved pool is then
passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base.
Fractions containing DIRS1 are pooled and diluted in cold distilled
H.sub.2O, to lower the conductivity, and passed back over a fresh
Q-Sepharose column, alone or in succession with an immunoaffinity
antibody column. Fractions containing the DIRS1 protein are pooled,
aliquoted, and stored in the -70.degree. C. freezer.
[0177] Comparison of the CD spectrum with cytokine receptor protein
may suggest that the protein is correctly folded. See Hazuda, et
al. (1969) J. Biol. Chem. 264:1689-1693.
VII. Determining Physiological Forms of Receptors
[0178] The cellular forms of receptors for ligands can be tested
with the various ligands and receptor subunits provided, e.g.,
IL-10 related sequences. In particular, multiple cytokine receptor
like ligands have been identified, see, e.g., U.S. Ser. No.
60/027,368, 08/934,959, and 08/842,659, which are incorporated
herein by reference.
[0179] Cotransformation of the DIRS1 with putative other receptor
subunit genes may be performed. In particular, the DSRS1 is
suggested to be a second receptor subunit needed for functional
receptor signaling. Such cells may be used to screen putative
cytokine ligands, such as the DIL-30, for signaling. A cell
proliferation assay may be used.
[0180] In addition, it has been known that many cytokine receptors
function as heterodimers. The IL-1.alpha. and IL-1.beta. ligands
bind an IL-1R1 as the primary receptor and this complex then forms
a high affinity receptor complex with the IL-1R3. As indicated
above, the sequence similarity to IL-12 receptor subunits suggests
functional similarity of the functional receptor, e.g., a soluble
alpha subunit, and transmembrane beta subunit.
[0181] These subunit combinations can be tested now with the
provided reagents. In particular, appropriate constructs can be
made for transformation or transfection of subunits into cells.
Constructs for the alpha chains, e.g., DSRS1 forms, can be made.
Likewise for the beta subunit DIRS1. Combinatorial transfections of
transformations can make cells expressing defined subunits, which
can be tested for response to the predicted ligands. Appropriate
cell types can be used, e.g., 293 T cells, with, e.g., an
NF.kappa.b reporter construct.
[0182] Biological assays will generally be directed to the ligand
binding feature of the protein or to the kinase/phosphatase
activity of the receptor. The activity will typically be
reversible, as are many other enzyme reactions, and may mediate
phosphatase or phosphorylase activities, which activities are
easily measured by standard procedures. See, e.g., Hardie, et al.
(eds. 1995) The Protein Kinase FactBook vols. I and II, Academic
Press, San Diego, Calif.; Hanks, et al. (1991) Meth. Enzymol.
200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell
61:743-752; Pines, et al. (1991) Cold Spring Harbor Symp. Quant.
Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738.
[0183] The family of cytokines contains molecules which are
important mediators of hematopoiesis or inflammatory disease. See,
e.g., Thomson (ed. 1994) The Cytokine Handbook Academic Press, San
Diego; and Dinarello (1996) Blood 87:2095-2147.
VIII. Antibodies Specific for DIRS1 or DIRS2
[0184] Inbred Balb/c mice are immunized intraperitoneally with
recombinant forms of the protein, e.g., purified DIRS1 or stable
transfected N1H-3T3 cells. Animals are boosted at appropriate time
points with protein, with or without additional adjuvant, to
further stimulate antibody production. Serum is collected, or
hybridomas produced with harvested spleens.
[0185] Alternatively, Balb/c mice are immunized with cells
transformed with the gene or fragments thereof, either endogenous
or exogenous cells, or with isolated membranes enriched for
expression of the antigen. Serum is collected at the appropriate
time, typically after numerous further administrations. Various
gene therapy techniques may be useful, e.g., in producing protein
in situ, for generating an immune response. Serum may be
immunoselected or depleted to prepare substantially purified
antibodies of defined specificity and high affinity. Preparations
which specifically bind particular segments or defined epitopes may
be made.
[0186] Monoclonal antibodies may be made. For example, splenocytes
are fused with an appropriate fusion partner and hybridomas are
selected in growth medium by standard procedures. Hybridoma
supernatants are screened for the presence of antibodies which bind
to the DIRS1, e.g., by ELISA or other assay. Antibodies which
specifically recognize specific DIRS1 embodiments may also be
selected or prepared.
[0187] In another method, synthetic peptides or purified protein
are presented to an immune system to generate monoclonal or
polyclonal antibodies. See, e.g., Coligan (ed. 1991) Current
Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press. In
appropriate situations, the binding reagent is either labeled as
described above, e.g., fluorescence or otherwise, or immobilized to
a substrate for panning methods. Nucleic acids may also be
introduced into cells in an animal to produce the antigen, which
serves to elicit an immune response. See, e.g., Wang, et al. (1993)
Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994)
BioTechniques 16:616-619; and Xiang, et al. (1995) Immunity 2:
129-135.
[0188] Moreover, antibodies which may be useful to determine the
combination of the DIRS1 with a functional alpha subunit may be
generated. Thus, e.g., epitopes characteristic of a particular
functional alpha/beta combination may be identified with
appropriate antibodies.
IX. Production of Fusion Proteins with DIRS1 or DIRS2
[0189] Various fusion constructs are made with DIRS1 or DIRS2. A
portion of the appropriate gene is fused to an epitope tag, e.g., a
FLAG tag, or to a two hybrid system construct. See, e.g., Fields
and Song (1989) Nature 340:245-246.
[0190] The epitope tag may be used in an expression cloning
procedure with detection with anti-FLAG antibodies to detect a
binding partner, e.g., ligand for the respective cytokine receptor.
The two hybrid system may also be used to isolate proteins which
specifically bind to DIRS1.
X. Structure Activity Relationship
[0191] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the positions
identified above, and evaluating biological activities of the
variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0192] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analyzed,
e.g., by PCR analysis and sequencing. This allows evaluation of
population polymorphisms.
XI. Isolation of a Ligand for DIRS1 or DIRS2
[0193] A cytokine receptor can be used as a specific binding
reagent to identify its binding partner, by taking advantage of its
specificity of binding, much like an antibody would be used.
Typically, the binding receptor is a heterodimer of receptor
subunits. A binding reagent is either labeled as described above,
e.g., fluorescence or otherwise, or immobilized to a substrate for
panning methods.
[0194] The binding composition is used to screen an expression
library made from a cell line which expresses a binding partner,
i.e., ligand, preferably membrane associated. Standard staining
techniques are used to detect or sort surface expressed ligand, or
surface expressing transformed cells are screened by panning.
Screening of intracellular expression is performed by various
staining or immunofluorescence procedures. See also McMahan, et al.
(1991) EMBO J. 10:2821-2832.
[0195] For example, on day 0, precoat 2-chamber permanox slides
with 1 ml per chamber of fibronectin, 10 ng/ml in PBS, for 30 min
at room temperature. Rinse once with PBS. Then plate COS cells at
2-3.times.10.sup.5 cells per chamber in 1.5 ml of growth media.
Incubate overnight at 37.degree. C.
[0196] On day 1 for each sample, prepare 0.5 ml of a solution of 66
.mu.g/ml DEAE-dextran, 66 .mu.M chloroquine, and 4 .mu.g DNA in
serum free DME. For each set, a positive control is prepared, e.g.,
of DIRS1-FLAG cDNA at 1 and 1/200 dilution, and a negative mock.
Rinse cells with serum free DME. Add the DNA solution and incubate
5 hr at 37.degree. C. Remove the medium and add 0.5 ml 10% DMSO in
DME for 2.5 min. Remove and wash once with DME. Add 1.5 ml growth
medium and incubate overnight.
[0197] On day 2, change the medium. On days 3 or 4, the cells are
fixed and stained. Rinse the cells twice with Hank's Buffered
Saline Solution (HBSS) and fix in 4% paraformaldehyde (PFA)/glucose
for 5 min. Wash 3.times. with HBSS. The slides may be stored at
-80.degree. C. after all liquid is removed. For each chamber, 0.5
ml incubations are performed as follows. Add HBSS/saponin (0.1%)
with 32 .mu.l/ml of 1 M NaN.sub.3 for 20 min. Cells are then washed
with HBSS/saponin 1.times.. Add appropriate DIRS1 or DIRS1/antibody
complex to cells and incubate for 30 min. Wash cells twice with
HBSS/saponin. If appropriate, add first antibody for 30 min. Add
second antibody, e.g., Vector anti-mouse antibody, at 1/200
dilution, and incubate for 30 min. Prepare ELISA solution, e.g.,
Vector Elite ABC horseradish peroxidase solution, and preincubate
for 30 min. Use, e.g., 1 drop of solution A (avidin) and 1 drop
solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with
HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash
cells twice with HBSS, second wash for 2 min, which closes cells.
Then add Vector diaminobenzoic acid (DAB) for 5 to 10 min. Use 2
drops of buffer plus 4 drops DAB plus 2 drops of H.sub.2O.sub.2 per
5 ml of glass distilled water. Carefully remove chamber and rinse
slide in water. Air dry for a few minutes, then add 1 drop of
Crystal Mount and a cover slip. Bake for 5 min at 85-90.degree.
C.
[0198] Evaluate positive staining of pools and progressively
subclone to isolation of single genes responsible for the
binding.
[0199] Alternatively, receptor reagents are used to affinity purify
or sort out cells expressing a putative ligand. See, e.g.,
Sambrook, et al. or Ausubel, et al.
[0200] Another strategy is to screen for a membrane bound receptor
by panning. The receptor cDNA is constructed as described above.
The ligand can be immobilized and used to immobilize expressing
cells. Immobilization may be achieved by use of appropriate
antibodies which recognize, e.g., a FLAG sequence of a DIRS1 fusion
construct, or by use of antibodies raised against the first
antibodies. Recursive cycles of selection and amplification lead to
enrichment of appropriate clones and eventual isolation of receptor
expressing clones.
[0201] Phage expression libraries can be screened by mammalian
DIRS1. Appropriate label techniques, e.g., anti-FLAG antibodies,
will allow specific labeling of appropriate clones.
[0202] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0203] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
Sequence CWU 1
1
611381DNAHomo
sapiensCDS(132)..(1064)misc_feature(567)..(567)unknown nucleotide
1tcgacccacg cgtccgcgct gcgactcaga cctcagctcc aacatatgca ttctgaagaa
60agatggctga gatggacaga atgctttatt ttggaaagaa acaatgttct aggtcaaact
120gagtctacca a atg cag act ttc aca atg gtt cta gaa gaa atc tgg aca
170 Met Gln Thr Phe Thr Met Val Leu Glu Glu Ile Trp Thr 1 5 10agt
ctt ttc atg tgg ttt ttc tac gca ttg att cca tgt ttg ctc aca 218Ser
Leu Phe Met Trp Phe Phe Tyr Ala Leu Ile Pro Cys Leu Leu Thr 15 20
25gat gaa gtg gcc att ctg cct gcc cct cag aac ctc tct gta ctc tca
266Asp Glu Val Ala Ile Leu Pro Ala Pro Gln Asn Leu Ser Val Leu
Ser30 35 40 45acc aac atg aag cat ctc ttg atg tgg agc cca gtg atc
gcg cct gga 314Thr Asn Met Lys His Leu Leu Met Trp Ser Pro Val Ile
Ala Pro Gly 50 55 60gaa aca gtg tac tat tct gtc gaa tac cag ggg gag
tac gag agc ctg 362Glu Thr Val Tyr Tyr Ser Val Glu Tyr Gln Gly Glu
Tyr Glu Ser Leu 65 70 75tac acg agc cac atc tgg atc ccc agc agc tgg
tgc tca ctc act gaa 410Tyr Thr Ser His Ile Trp Ile Pro Ser Ser Trp
Cys Ser Leu Thr Glu 80 85 90ggt cct gag tgt gat gtc act gat gac atc
acg gcc act gtg cca tac 458Gly Pro Glu Cys Asp Val Thr Asp Asp Ile
Thr Ala Thr Val Pro Tyr 95 100 105aac ctt cgt gtc agg gcc aca ttg
ggc tca cag acc tca gcc tgg agc 506Asn Leu Arg Val Arg Ala Thr Leu
Gly Ser Gln Thr Ser Ala Trp Ser110 115 120 125atc ctg aag cat ccc
ttt aat aga aac tca acc atc ctt acc cga cct 554Ile Leu Lys His Pro
Phe Asn Arg Asn Ser Thr Ile Leu Thr Arg Pro 130 135 140ggg atg gag
atc ncc aaa nat ggc ttc cac ctg gtt att gag ctg gag 602Gly Met Glu
Ile Xaa Lys Xaa Gly Phe His Leu Val Ile Glu Leu Glu 145 150 155gac
ctg ggg ccc cag ttt gag ttc ctt gtg gcc tac tgg asg agg gag 650Asp
Leu Gly Pro Gln Phe Glu Phe Leu Val Ala Tyr Trp Xaa Arg Glu 160 165
170cct ggt gcc gag gaa cat gtc aaa atg gtg agg agt ggg ggt att cca
698Pro Gly Ala Glu Glu His Val Lys Met Val Arg Ser Gly Gly Ile Pro
175 180 185gtg cac cta gaa acc atg gag cca ggg gct gca tac tgt gtg
aag gcc 746Val His Leu Glu Thr Met Glu Pro Gly Ala Ala Tyr Cys Val
Lys Ala190 195 200 205cag aca ttc gtg aag gcc att ggg arg tac agc
gcc ttc agc cag aca 794Gln Thr Phe Val Lys Ala Ile Gly Xaa Tyr Ser
Ala Phe Ser Gln Thr 210 215 220gaa tgt gtg gar gtg caa gga gag gcc
att ccc ctg gta ctg gcc ctg 842Glu Cys Val Glu Val Gln Gly Glu Ala
Ile Pro Leu Val Leu Ala Leu 225 230 235ttt gcc ttt gtt ggc ttc atg
ctg atc ctt gtg gtc gtg cca ctg ttc 890Phe Ala Phe Val Gly Phe Met
Leu Ile Leu Val Val Val Pro Leu Phe 240 245 250gtc tgg aaa atg ggc
cgg ctg ctc cag tac tcc tgt tgc ccc gtg gtg 938Val Trp Lys Met Gly
Arg Leu Leu Gln Tyr Ser Cys Cys Pro Val Val 255 260 265gtc ctc cca
gac acc ttg aaa ata acc aat tca ccc cag aag tta atc 986Val Leu Pro
Asp Thr Leu Lys Ile Thr Asn Ser Pro Gln Lys Leu Ile270 275 280
285agc tgc aga agg gag gag gtg gat gcc tgt gcc acg gct gtg atg tct
1034Ser Cys Arg Arg Glu Glu Val Asp Ala Cys Ala Thr Ala Val Met Ser
290 295 300cct gag gaa ctc ctc agg gcc tgg atc tca taggtttgcg
gaagggccca 1084Pro Glu Glu Leu Leu Arg Ala Trp Ile Ser 305
310ggtgaagccg agaacctggt ctgcatgaca tggaaaccat gaggggacaa
gttgtgtttc 1144tgttttccgc cacggacaag ggatgagaga agtaggaaga
gcctgttgtc tacaagtcta 1204gaagcaacca tcagaggcag ggtggtttgt
ckaacagaac aaytgactga ggytakrggg 1264gwtgtgacct ctagactktg
ggstkscayt tgcwtggytg agcaaccctg ggaaaagtga 1324cttcatccct
tnggtccnaa gttttctcat ctgtaatggg ggatncctac aaaactg 13812311PRTHomo
sapiensmisc_feature(146)..(146)The 'Xaa' at location 146 stands for
Thr, Ala, Pro, or Ser. 2Met Gln Thr Phe Thr Met Val Leu Glu Glu Ile
Trp Thr Ser Leu Phe1 5 10 15Met Trp Phe Phe Tyr Ala Leu Ile Pro Cys
Leu Leu Thr Asp Glu Val 20 25 30Ala Ile Leu Pro Ala Pro Gln Asn Leu
Ser Val Leu Ser Thr Asn Met 35 40 45Lys His Leu Leu Met Trp Ser Pro
Val Ile Ala Pro Gly Glu Thr Val 50 55 60Tyr Tyr Ser Val Glu Tyr Gln
Gly Glu Tyr Glu Ser Leu Tyr Thr Ser65 70 75 80His Ile Trp Ile Pro
Ser Ser Trp Cys Ser Leu Thr Glu Gly Pro Glu 85 90 95Cys Asp Val Thr
Asp Asp Ile Thr Ala Thr Val Pro Tyr Asn Leu Arg 100 105 110Val Arg
Ala Thr Leu Gly Ser Gln Thr Ser Ala Trp Ser Ile Leu Lys 115 120
125His Pro Phe Asn Arg Asn Ser Thr Ile Leu Thr Arg Pro Gly Met Glu
130 135 140Ile Xaa Lys Xaa Gly Phe His Leu Val Ile Glu Leu Glu Asp
Leu Gly145 150 155 160Pro Gln Phe Glu Phe Leu Val Ala Tyr Trp Xaa
Arg Glu Pro Gly Ala 165 170 175Glu Glu His Val Lys Met Val Arg Ser
Gly Gly Ile Pro Val His Leu 180 185 190Glu Thr Met Glu Pro Gly Ala
Ala Tyr Cys Val Lys Ala Gln Thr Phe 195 200 205Val Lys Ala Ile Gly
Xaa Tyr Ser Ala Phe Ser Gln Thr Glu Cys Val 210 215 220Glu Val Gln
Gly Glu Ala Ile Pro Leu Val Leu Ala Leu Phe Ala Phe225 230 235
240Val Gly Phe Met Leu Ile Leu Val Val Val Pro Leu Phe Val Trp Lys
245 250 255Met Gly Arg Leu Leu Gln Tyr Ser Cys Cys Pro Val Val Val
Leu Pro 260 265 270Asp Thr Leu Lys Ile Thr Asn Ser Pro Gln Lys Leu
Ile Ser Cys Arg 275 280 285Arg Glu Glu Val Asp Ala Cys Ala Thr Ala
Val Met Ser Pro Glu Glu 290 295 300Leu Leu Arg Ala Trp Ile Ser305
31031244DNAHomo sapiensCDS(2)..(694) 3c cgg gtc gac cca cgc gtc cgc
ctg gtt tcc ccc tgg ctg aca gtg cct 49 Arg Val Asp Pro Arg Val Arg
Leu Val Ser Pro Trp Leu Thr Val Pro 1 5 10 15tgg ttc ctg tcc tgt
tgg aat gtt acc att ggg cct cct gag agc atc 97Trp Phe Leu Ser Cys
Trp Asn Val Thr Ile Gly Pro Pro Glu Ser Ile 20 25 30tgg gtg acg ccg
gga gaa gcc tcc ctc atc atc agg ttc tcc tct ccc 145Trp Val Thr Pro
Gly Glu Ala Ser Leu Ile Ile Arg Phe Ser Ser Pro 35 40 45ttc gac gtc
cct ccc aac ctg ggc tat ttc cag tac tat gtc cat tay 193Phe Asp Val
Pro Pro Asn Leu Gly Tyr Phe Gln Tyr Tyr Val His Tyr 50 55 60tgg gaa
aag gcg gga atc caa aag gtt aaa ggt cct ttc aag agc aac 241Trp Glu
Lys Ala Gly Ile Gln Lys Val Lys Gly Pro Phe Lys Ser Asn65 70 75
80tcc atc gtg ttg gat ggc ttg aga ccc tta aga gaa tac tgt tta caa
289Ser Ile Val Leu Asp Gly Leu Arg Pro Leu Arg Glu Tyr Cys Leu Gln
85 90 95gtg aag gcg cat ctc ttt cgc aca tcc tgc aac acc tct agg ccc
ggc 337Val Lys Ala His Leu Phe Arg Thr Ser Cys Asn Thr Ser Arg Pro
Gly 100 105 110cgc tta agc aac ata act tgc tac gaa aca atg atg gat
gcc act acg 385Arg Leu Ser Asn Ile Thr Cys Tyr Glu Thr Met Met Asp
Ala Thr Thr 115 120 125aag ctt caa caa gtc atc ctc atc gcc gtg gga
gtc ttt ctg tcg ctg 433Lys Leu Gln Gln Val Ile Leu Ile Ala Val Gly
Val Phe Leu Ser Leu 130 135 140gcg gcg ctg gcg ggg ggc tgt ttc ttc
ctg gtg ctg aga tac aaa ggc 481Ala Ala Leu Ala Gly Gly Cys Phe Phe
Leu Val Leu Arg Tyr Lys Gly145 150 155 160ctg gtg aaa tac tgg ttt
cac tct ccg cca agc atc cca tca caa atc 529Leu Val Lys Tyr Trp Phe
His Ser Pro Pro Ser Ile Pro Ser Gln Ile 165 170 175gaa gag tat ctg
aag gac ccg agc cag cct atc cta gag gcc ctg gac 577Glu Glu Tyr Leu
Lys Asp Pro Ser Gln Pro Ile Leu Glu Ala Leu Asp 180 185 190aag gac
acg tca cca aca gat gat gcc tgg gac ttg gtg tct gtt gtt 625Lys Asp
Thr Ser Pro Thr Asp Asp Ala Trp Asp Leu Val Ser Val Val 195 200
205gca ttt cca gca aag gag caa gaa gat gtt ccc caa agc act ttg acc
673Ala Phe Pro Ala Lys Glu Gln Glu Asp Val Pro Gln Ser Thr Leu Thr
210 215 220caa aac tct ggt gcg gtc tgc tagcctgtgg ggtaagggct
ctgagccgag 724Gln Asn Ser Gly Ala Val Cys225 230gaagctgctg
atgtccatgt cagcacttta tggaatccgg tcctccattt tcctgtcccc
784aaaaggcccg tcagtgcctg tgaagatgta acgggtctca tgggggcgac
aagcttattg 844atttttttct tcaaactaag agttttctaa tcatacgcgt
ttttagaata attctacaga 904tatgtccccg aaagattaag atttctctta
aacactaaaa agacatgtaa ttatttgtta 964gcaaatgggc gtctggcacg
cctctgacac tttttcgtca gcagccagga cacgaggtcc 1024cctccttgat
gaagcccctc gggcagacca tgtcacctgt cccagcctgc cccaagaagg
1084gacattaagt ggcccttctt catatccaaa cacctggctt gaaatgtgat
tagccctgta 1144aatagtttca cagagattaa gccttttttt cccccaagtt
aggaataaaa gactataatt 1204aactttttaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 12444231PRTHomo sapiens 4Arg Val Asp Pro Arg Val Arg Leu
Val Ser Pro Trp Leu Thr Val Pro1 5 10 15Trp Phe Leu Ser Cys Trp Asn
Val Thr Ile Gly Pro Pro Glu Ser Ile 20 25 30Trp Val Thr Pro Gly Glu
Ala Ser Leu Ile Ile Arg Phe Ser Ser Pro 35 40 45Phe Asp Val Pro Pro
Asn Leu Gly Tyr Phe Gln Tyr Tyr Val His Tyr 50 55 60Trp Glu Lys Ala
Gly Ile Gln Lys Val Lys Gly Pro Phe Lys Ser Asn65 70 75 80Ser Ile
Val Leu Asp Gly Leu Arg Pro Leu Arg Glu Tyr Cys Leu Gln 85 90 95Val
Lys Ala His Leu Phe Arg Thr Ser Cys Asn Thr Ser Arg Pro Gly 100 105
110Arg Leu Ser Asn Ile Thr Cys Tyr Glu Thr Met Met Asp Ala Thr Thr
115 120 125Lys Leu Gln Gln Val Ile Leu Ile Ala Val Gly Val Phe Leu
Ser Leu 130 135 140Ala Ala Leu Ala Gly Gly Cys Phe Phe Leu Val Leu
Arg Tyr Lys Gly145 150 155 160Leu Val Lys Tyr Trp Phe His Ser Pro
Pro Ser Ile Pro Ser Gln Ile 165 170 175Glu Glu Tyr Leu Lys Asp Pro
Ser Gln Pro Ile Leu Glu Ala Leu Asp 180 185 190Lys Asp Thr Ser Pro
Thr Asp Asp Ala Trp Asp Leu Val Ser Val Val 195 200 205Ala Phe Pro
Ala Lys Glu Gln Glu Asp Val Pro Gln Ser Thr Leu Thr 210 215 220Gln
Asn Ser Gly Ala Val Cys225 2305337PRTHomo sapiens 5Met Arg Pro Thr
Leu Leu Trp Ser Leu Leu Leu Leu Leu Gly Val Phe1 5 10 15Ala Ala Ala
Ala Ala Ala Pro Pro Asp Pro Leu Ser Gln Leu Pro Ala 20 25 30Pro Gln
His Pro Lys Ile Arg Leu Tyr Asn Ala Glu Gln Val Leu Ser 35 40 45Trp
Glu Pro Val Ala Leu Ser Asn Ser Thr Arg Pro Val Val Tyr Arg 50 55
60Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe Thr Ala Asp Ile Met65
70 75 80Ser Ile Gly Val Asn Cys Thr Gln Ile Thr Ala Thr Glu Cys Asp
Phe 85 90 95Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met Asp Phe Asn
Val Thr 100 105 110Leu Arg Leu Arg Ala Glu Leu Gly Ala Leu His Ser
Ala Trp Val Thr 115 120 125Met Pro Trp Phe Gln His Tyr Arg Asn Val
Thr Val Gly Pro Pro Glu 130 135 140Asn Ile Glu Val Thr Pro Gly Glu
Gly Ser Leu Ile Ile Arg Phe Ser145 150 155 160Ser Pro Phe Asp Ile
Ala Asp Thr Ser Thr Ala Phe Phe Cys Tyr Tyr 165 170 175Val His Tyr
Trp Glu Lys Gly Gly Ile Gln Gln Val Lys Gly Pro Phe 180 185 190Arg
Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys Pro Ser Arg Val Tyr 195 200
205Cys Leu Gln Val Gln Ala Gln Leu Leu Trp Asn Lys Ser Asn Ile Phe
210 215 220Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr Glu Thr Met
Ala Asp225 230 235 240Ala Ser Thr Glu Leu Gln Gln Val Ile Leu Ile
Ser Val Gly Thr Phe 245 250 255Ser Leu Leu Ser Val Leu Ala Gly Ala
Cys Phe Phe Leu Val Leu Lys 260 265 270Tyr Arg Gly Leu Ile Lys Tyr
Trp Phe His Thr Pro Pro Ser Ile Pro 275 280 285Leu Gln Ile Glu Glu
Tyr Leu Lys Asp Pro Thr Gln Pro Ile Leu Glu 290 295 300Ala Leu Asp
Lys Asp Ser Ser Pro Lys Asp Asp Val Trp Asp Ser Val305 310 315
320Ser Ile Ile Ser Phe Pro Glu Lys Glu Gln Glu Asp Val Leu Gln Thr
325 330 335Leu6325PRTHomo sapiens 6Met Ala Trp Ser Leu Gly Ser Trp
Leu Gly Gly Cys Leu Leu Val Ser1 5 10 15Ala Leu Gly Met Val Pro Pro
Pro Glu Asn Val Arg Met Asn Ser Val 20 25 30Asn Phe Lys Asn Ile Leu
Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly 35 40 45Asn Leu Thr Phe Thr
Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp 50 55 60Lys Cys Met Asn
Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser65 70 75 80Lys Tyr
Gly Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp Glu 85 90 95His
Ser Asp Trp Val Asn Ile Thr Phe Cys Pro Val Asp Asp Thr Ile 100 105
110Ile Gly Pro Pro Gly Met Gln Val Glu Val Leu Ala Asp Ser Leu His
115 120 125Met Arg Phe Leu Ala Pro Lys Ile Glu Asn Glu Tyr Glu Thr
Trp Thr 130 135 140Met Lys Asn Val Tyr Asn Ser Trp Thr Tyr Asn Val
Gln Tyr Trp Lys145 150 155 160Asn Gly Thr Asp Glu Lys Phe Gln Ile
Thr Pro Gln Tyr Asp Phe Glu 165 170 175Val Leu Arg Asn Leu Glu Pro
Trp Thr Thr Tyr Cys Val Gln Val Arg 180 185 190Gly Phe Leu Pro Asp
Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val 195 200 205Cys Glu Gln
Thr Thr His Asp Glu Thr Val Pro Ser Trp Met Val Ala 210 215 220Val
Ile Leu Met Ala Ser Val Phe Met Val Cys Leu Ala Leu Leu Gly225 230
235 240Cys Phe Ser Leu Leu Trp Cys Val Tyr Lys Lys Thr Lys Tyr Ala
Phe 245 250 255Ser Pro Arg Asn Ser Leu Pro Gln His Leu Lys Glu Phe
Leu Gly His 260 265 270Pro His His Asn Thr Leu Leu Phe Phe Ser Phe
Pro Leu Ser Asp Glu 275 280 285Asn Asp Val Phe Asp Lys Leu Ser Val
Ile Ala Glu Asp Ser Glu Ser 290 295 300Gly Lys Gln Asn Pro Gly Asp
Ser Cys Ser Leu Gly Thr Pro Pro Gly305 310 315 320Gln Gly Pro Gln
Ser 325
* * * * *