U.S. patent application number 10/777789 was filed with the patent office on 2005-03-03 for mammalian cytokines; related reagents and methods.
This patent application is currently assigned to Schering Corporation, a New Jersey corporation.. Invention is credited to Bazan, J. Fernando, Kastelein, Robert A., Oppmann, Birgit, Timans, Jacqueline C..
Application Number | 20050048625 10/777789 |
Document ID | / |
Family ID | 32993405 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050048625 |
Kind Code |
A1 |
Oppmann, Birgit ; et
al. |
March 3, 2005 |
Mammalian cytokines; related reagents and methods
Abstract
Purified genes encoding cytokine from a mammal, reagents related
thereto including purified proteins, specific antibodies, and
nucleic acids encoding this molecule are provided. Methods of using
said reagents and diagnostic kits are also provided.
Inventors: |
Oppmann, Birgit; (Berlin,
DE) ; Timans, Jacqueline C.; (Mountain View, CA)
; Kastelein, Robert A.; (Redwood City, CA) ;
Bazan, J. Fernando; (Menlo Park, CA) |
Correspondence
Address: |
DNAX RESEARCH, INC.
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Assignee: |
Schering Corporation, a New Jersey
corporation.
|
Family ID: |
32993405 |
Appl. No.: |
10/777789 |
Filed: |
February 11, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10777789 |
Feb 11, 2004 |
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09521335 |
Mar 9, 2000 |
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6800460 |
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60124319 |
Mar 11, 1999 |
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Current U.S.
Class: |
435/69.5 ;
435/320.1; 435/325; 530/351; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/54 20130101 |
Class at
Publication: |
435/069.5 ;
435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
C07H 021/04; C12P
021/02; C07K 014/52 |
Claims
What is claimed is:
1. An isolated soluble complex comprising at least 6 amino acids of
the mature protein portion of SEQ ID NO: 2 or 4, and: a) at least 6
amino acids of the mature protein portion of SEQ ID NO: 12 or 13;
or b) at least 6 amino acids of the mature protein portion of the
CNTF-R.
2. The complex of claim 1, wherein said complex: a) comprises a
recombinant polypeptide of mature SEQ ID NO: 2 or 4; b) comprises a
recombinant polypeptide of mature SEQ ID NO: 12 or 13; c) comprises
a recombinant polypeptide of mature CNTF-R; d) comprises both a
recombinant polypeptide of mature SEQ ID NO: 2 or 4, and a
recombinant polypeptide of mature SEQ ID NO: 12 or 13; e) comprises
both a recombinant polypeptide of mature SEQ ID NO: 2 or 4, and a
recombinant polypeptide of mature CNTF-R; f) is detectably labeled;
g) is in a buffered solution; or h) is in a sterile solution.
3. The complex of claim 1, which: a) comprises a mature IL-B60
polypeptide; b) comprises a mature CLF-1 polypeptide; c) comprises
a mature CNTF-R polypeptide; d) exhibits at least four
nonoverlapping segments of at least seven amino acids of SEQ ID NO:
2 or 4; e) exhibits epitopes from both primate L-B60 and primate
CLF-1; f) exhibits epitopes from both primate L-B60 and primate
CNTF-R; g) is not glycosylated; h) is attached to a solid
substrate; i) is conjugated to another chemical moiety; or j)
comprises a detection or purification tag, including a FLAG, His6,
or Ig sequence.
4. A kit comprising said complex of claim 1, and: a) a compartment
comprising said complex; or b) instructions for use or disposal of
reagents in said kit.
5. An isolated or recombinant polypeptide comprising: a) a first
segment comprising at least seven amino acids identical to segments
of SEQ ID NO: 2 or 4, and a second segment comprising at least
seven amino acids identical to segments of mature SEQ ID NO: 12 or
13; b) at least two distinct nonoverlapping segments of at least
five amino acids identical to segments of mature SEQ ID NO: 2 or 4,
and a third segment comprising at least seven amino acids identical
to segments of mature SEQ ID NO: 12 or 13; c) at least one segment
comprising at least seven amino acids identical to segments of
mature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments
of at least five amino acids identical to segments of mature SEQ ID
NO: 12 or 13; d) a first segment comprising at least seven amino
acids identical to segments of SEQ ID NO: 2 or 4, and a second
segment comprising at least seven amino acids identical to segments
of mature primate CNTF-R; e) at least two distinct nonoverlapping
segments of at least five amino acids identical to segments of
mature SEQ ID NO: 2 or 4, and a third segment comprising at least
seven amino acids identical to segments of mature primate CNTF-R;
or f) at least one segment comprising at least seven amino acids
identical to segments of mature SEQ ID NO: 2 or 4, and two distinct
nonoverlapping segments of at least five amino acids identical to
segments of mature primate CNTF-R.
6. The polypeptide of claim 5, wherein said distinct nonoverlapping
segments of identity: a) include one of at least eight amino acids;
b) include one of at least five amino acids and a second of at
least six 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.
7. The polypeptide of claim 5, which: a) comprises a mature IL-B60
sequence; b) comprises a mature CLF-1 sequence; c) comprises a
mature CNTF-R sequence; d) exhibits at least four nonoverlapping
segments of at least seven amino acids of SEQ ID NO: 2 or 4; e) has
a length at least about 30 amino acids; f) exhibits epitopes from
both primate IL-B60 and primate CLF-1; g) exhibits epitopes from
both primate IL-B60 and primate CNTF-R; h) is not glycosylated; i)
has a molecular weight of at least 30 kD; j) is a synthetic
polypeptide; k) is attached to a solid substrate; l) is conjugated
to another chemical moiety; or m) comprises a detection or
purification tag, including a FLAG, His6, or Ig sequence.
8. A composition comprising: a) substantially pure combination of
IL-B60 and CLF-1; b) substantially pure combination of IL-B60 and
CNTF-R; c) a sterile polypeptide of claim 5; or d) said polypeptide
of claim 5 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.
9. A kit comprising a polypeptide of claim 5, and: a) a compartment
comprising said polypeptide; or b) instructions for use or disposal
of reagents in said kit.
10. A method: a) of making an antibody which recognizes a complex
of claim 1, comprising inducing an immune response in an animal
with said complex; b) of immunoselecting antibodies, comprising
contacting a population of antibodies to a complex of claim 1, and
separating antibodies that bind from those which do not bind; or c)
of formulating a composition, comprising admixing a complex of
claim 1 with a carrier.
11. A binding compound comprising an antigen binding site from an
antibody, which antibody specifically binds said complex of claim
2d or 2e, but not to any of said mature polypeptides of SEQ ID NO:
2, 4, 12, 13, or CNTF-R.
12. The binding compound of claim 11, wherein: a) said binding
compound is: i) in a container; ii) an Fv, Fab, or Fab2 fragment;
or iii) conjugated to another chemical moiety; or b) said antibody:
i) is raised against a substantially pure complex of IL-B60 with
CLF-1; ii) is raised against a substantially pure complex of IL-B60
with CNTF-R; iii) is immunoselected; iv) is a polyclonal antibody;
v) exhibits a Kd to antigen of at least 30 .mu.M; vi) is attached
to a solid substrate, including a bead or plastic membrane; vii) is
in a sterile composition; or viii) is detectably labeled, including
a radioactive or fluorescent label.
13. A composition comprising: a) a sterile binding compound of
claim 12, or b) said binding compound of claim 12 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.
14. A kit comprising said binding compound of claim 11, and: a) a
compartment comprising said binding compound; or b) instructions
for use or disposal of reagents in said kit.
15. A method of producing an antigen:antibody complex, comprising
contacting under appropriate conditions a primate complex
comprising: a) IL-B60 and CLF-1 polypeptides; or b) IL-B60 and
CNTF-R polypeptides; with an antibody of claim 11, thereby allowing
said complex to form.
16. The method of claim 15, wherein: a) said complex is purified
from other cytokines; b) said complex is purified from other
antibody; c) said contacting is with a sample comprising a
cytokine; 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.
17. An isolated or recombinant nucleic acid: a) encoding said amino
acid portions of claim 5; b) encoding said amino acid portions of
claim 5, and comprise a segment at least 30 contiguous nucleotides
from SEQ ID NO: 1 or 3; c) which will coexpress a segment of at
least seven contiguous amino acids from SEQ ID NO: 2 or 4, and a
segment of at least seven contiguous amino acids from SEQ ID NO: 12
or 13; or d) which will coexpress a segment of at least seven
contiguous amino acids from SEQ ID NO: 2 or 4, and a segment of at
least seven contiguous amino acids from CNTF-R.
18. The nucleic acid of claim 17, which: a) encodes IL-B60 from a
human; b) encodes CLF-1 from a human; c) encodes CNTF-R from a
human; d) is an expression vector; e) further comprises an origin
of replication; f) comprises a detectable label; g) comprises
synthetic nucleotide sequence; or h) is less than 6 kb, preferably
less than 3 kb.
19. A cell comprising said recombinant nucleic acid of claim
18.
20. The cell of claim 19, 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.
21. A kit comprising said nucleic acid of claim 18, and: a) a
compartment comprising said nucleic acid; b) a compartment further
comprising a primate IL-B60 polypeptide; c) a compartment further
comprising a primate CLF-1 polypeptide; d) a compartment further
comprising a primate CNTF-R polypeptide; or e) instructions for use
or disposal of reagents in said kit.
22. A method: a) of making a duplex nucleic acid, comprising
contacting a nucleic acid of claim 17 with a complementary nucleic
acid under appropriate conditions, thereby forming said duplex; b)
of expressing a polypeptide, comprising expressing said nucleic
acid of claim 17, thereby producing said polypeptide; or c) of
transfecting a cell, comprising contacting said cell under
appropriate conditions with said nucleic acid of claim 17.
23. An isolated or recombinant nucleic acid which encodes at least
5 contiguous amino acids of SEQ ID NO: 12, 13, or primate CNTF-R
and: 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; or b) exhibits identity over a stretch of at least about 30
nucleotides to a primate IL-B60.
24. The isolated nucleic acid of claim 23, wherein: a) said
contiguous amino acids number at least 8; b) said wash conditions
are at 45.degree. C. and/or 500 mM salt; or c) said stretch is at
least 55 nucleotides.
25. The recombinant nucleic acid of claim 23, wherein: a) said
contiguous amino acids number at least 12; b) said wash conditions
are at 55.degree. C. and/or 150 mM salt; or c) said stretch is at
least 75 nucleotides.
26. 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 complex comprising mammalian IL-B60 and:
a) CLF-1; or b) CNTF-R.
27. A method of: a) producing a complex of claim 1, comprising
coexpressing a recombinant IL-B60 with a recombinant CLF-1 or
CNTF-R; b) increasing the secretion of an IL-B60 polypeptide
comprising expressing said polypeptide with CLF-1; or c) increasing
the secretion of a CLF-1 polypeptide, comprising expressing said
CLF-1 with an IL-B60.
28. The method of claim 27, wherein: a) said increasing is at least
3 fold; or b) said expressing is of a recombinant nucleic acid
encoding one or both of said polypeptide and CLF-1.
29. A method of screening for a receptor which binds said complex
of claim 1, comprising contacting said complex to a cell expressing
said receptor under conditions allowing said complex to bind to
said receptor, thereby forming a detectable interaction.
30. The method of claim 29, wherein said interaction results in a
physiological response in said cell.
Description
[0001] This filing is a conversion to U.S. Utility Patent
Application of U.S. Ser. No. 60/124,319, filed Mar. 11, 1999, which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention pertains to compositions related to
proteins which function in controlling biology and physiology of
mammalian cells, e.g., cells of a mammalian immune system. In
particular, it provides purified genes, proteins, antibodies,
related reagents, and methods useful, e.g., to regulate activation,
development, differentiation, and function of various cell types,
including hematopoietic cells.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology refers generally to the technique
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.
[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". See, e.g., Paul (1998) Fundamental
Immunology (4th ed.) Raven Press, NY. Recent research has provided
new insights into the inner workings of this network. While it
remains clear that much of the 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 a critical role in controlling these cellular
interactions. 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. Some of these
factors are hematopoietic growth factors, e.g., granulocyte colony
stimulating factor (G-CSF). See, e.g., Thomson (ed. 1998) The
Cytokine Handbook (3d ed.) Academic Press, San Diego; Mire-Sluis
and Thorpe (ed. 1998) Cytokines Academic Press, San Diego; Metcalf
and Nicola (1995) The Hematopoietic Colony Stimulating Factors
Cambridge University Press; and Aggarwal and Gutterman (1991) Human
Cytokines Blackwell Pub.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. 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 making 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] Research to better understand and treat various immune
disorders has been hampered by the general inability to maintain
cells of the immune system in vitro. Immunologists have discovered
that culturing these cells can be accomplished through the use of
T-cell and other cell supernatants, which contain various growth
factors, including many of the lymphokines.
[0008] From the foregoing, it is evident that the discovery and
development of new lymphokines, e.g., related to G-CSF and/or IL-6,
could contribute to new therapies for a wide range of degenerative
or abnormal conditions which directly or indirectly involve the
immune system and/or hematopoietic cells. In particular, the
discovery and development of lymphokines which enhance or
potentiate the beneficial activities of known lymphokines would be
highly advantageous. The present invention provides new interleukin
compositions and related compounds, and methods for their use.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to mammalian, e.g.,
primate or rodent, interleukin-B60 (IL-B60) and its biological
activities. It includes nucleic acids coding for polypeptides
themselves and methods for their production and use. The nucleic
acids of the invention are characterized, in part, by their
homology to complementary DNA (cDNA) sequences disclosed herein,
and/or by functional assays for growth factor- or cytokine-like
activities, e.g., G-CSF (see Nagata (1994) in Thomson The Cytokine
Handbook 2d ed., Academic Press, San Diego) and/or IL-6 (see Hirano
(1994) in Thomson The Cytokine Handbook 2d ed., Academic Press, San
Diego). Also provided are polypeptides, antibodies, and methods of
using them, including using nucleic acid expression methods.
Methods for modulating or intervening in the control of a growth
factor dependent physiology or an immune response are provided.
[0010] The present invention is based, in part, upon the discovery
of a new cytokine sequence exhibiting significant sequence and
structural similarity to G-CSF and IL-6. In particular, it provides
primate, e.g., human, and rodent, e.g., mouse, genes encoding a
protein whose mature size is about 198 amino acids. Functional
equivalents exhibiting significant sequence homology will be
available from other mammalian, e.g., cow, horse, and rat
species.
[0011] Moreover, the present invention identifies a second
associated component of a complex. Compositions related to the
combination of components in the complex are provided, along with
methods of use.
[0012] In one embodiment, the invention provides a substantially
pure or recombinant polypeptide comprising the mature protein
portion of SEQ ID NO: 2 or 4. Preferably, the polypeptide is:
detectably labeled; unglycosylated; denatured; attached to a solid
substrate; conjugated to another chemical moiety; or in a sterile
composition. Kit forms include those comprising the polypeptide
and: a compartment comprising the polypeptide; or with instructions
for use or disposal of reagents in the kit.
[0013] Binding compounds include those comprising an antigen
binding site from an antibody that specifically binds to the
described polypeptide. The binding compound can also be in a kit
comprising: a compartment comprising the binding compound; or with
instructions for use or disposal of reagents in the kit.
[0014] The invention further provides a method of producing an
antigen:antibody complex, comprising contacting, under appropriate
conditions, a primate IL-B60 polypeptide with an antibody that
specifically or selectively binds the polypeptide of the invention,
thereby allowing the complex to form.
[0015] Nucleic acid embodiments include an isolated or recombinant
polynucleotide encoding the mature protein portion of SEQ ID NO: 2
or 4.
[0016] In other embodiments, the invention provides an isolated
soluble complex comprising the mature protein portion of SEQ ID NO:
2 or 4, and the mature protein portion of SEQ ID NO: 12 or 13.
Preferably the complex: comprises a recombinant polypeptide of SEQ
ID NO: 2, 4, 12, or 13; is detectably labeled; is in a buffered
solution; is in a sterile solution. Kits are provided containing
such a complex and: a compartment comprising the complex; or
instructions for use or disposal of reagents in the kit.
[0017] Binding compounds are provided comprising an antigen binding
site from an antibody that specifically binds to the soluble
complex but not to the mature polypeptide of SEQ ID NO: 12 or 13.
Kits are provided comprising the binding compound and: a
compartment comprising the binding compound; or instructions for
use or disposal of reagents in the kit.
[0018] Methods are provided, e.g., of producing an antigen:antibody
complex, comprising contacting, under appropriate conditions, a
primate complex comprising IL-B60 and CLF-1 polypeptides with an
antibody that selectively or specifically binds to an isolated
soluble complex comprising the mature protein portion of SEQ ID NO:
2 or 4, and the mature protein portion of SEQ ID NO: 12 or 13,
thereby allowing the complex to form.
[0019] Nucleic acid embodiments include an isolated or recombinant
nucleic acid encoding the mature protein portion of SEQ ID NO: 2 or
4, and the mature protein portion of SEQ ID NO: 12 or 13.
[0020] The invention also provides a composition of matter selected
from: an isolated polypeptide comprising at least seven amino acids
identical to segments of SEQ ID NO: 2 or 4; a substantially pure or
recombinant polypeptide comprising at least two distinct
nonoverlapping segments of at least five amino acids identical to
segments of SEQ ID NO: 2 or 4; a natural sequence polypeptide
comprising mature SEQ ID NO: 2 or 4; or a fusion polypeptide
comprising IL-B60 sequence. In certain embodiments, the distinct
nonoverlapping segments of identity include: one of at least eight
amino acids; one of at least five amino acids and a second of at
least six amino acids; at least three segments of at least four,
five, and six amino acids, or one of at least twelve amino acids.
In other embodiments the polypeptide of the composition of matter:
is the polypeptide which: comprises a mature sequence of Table 1;
is an unglycosylated form of IL-B60; is from a primate, such as a
human; comprises at least seventeen amino acids of SEQ ID NO: 2 or
4; exhibits at least four nonoverlapping segments of at least seven
amino acids of SEQ ID NO: 2 or 4; is a natural allelic variant of
IL-B60; has a length at least about 30 amino acids; exhibits at
least two non-overlapping epitopes which are specific for a primate
IL-B60; 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; is a deletion
or insertion variant from a natural sequence; or which further
comprises: at least seven amino acids identical to segments of SEQ
ID NO: 12 or 13; at least two distinct nonoverlapping segments of
at least five amino acids identical to segments of SEQ ID NO: 12 or
13; a natural sequence polypeptide comprising mature SEQ ID NO: 12
or 13; or a primate CLF-1. In additional preferred embodiments, the
composition comprises: a substantially pure IL-B60 and CLF-1; a
sterile IL-B60 polypeptide comprising the mature protein of SEQ ID
NO: 2 or 4; or the described 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. The invention provides fusion
polypeptides which comprise: mature protein sequence of Table 1; a
detection or purification tag, including a FLAG, His6, or Ig
sequence; or sequence of another cytokine receptor family protein,
including CLF-1. Kit embodiments include those comprising the
polypeptide of the composition and: a compartment comprising the
protein or polypeptide; or instructions for use or disposal of
reagents in the kit.
[0021] The invention further provides methods of using the
described polypeptides: to label the polypeptide, comprising
labeling the polypeptide with a radioactive label; to separate the
polypeptide from another polypeptide in a mixture, comprising
running the mixture on a chromatography matrix, thereby separating
the polypeptides; to identify a compound that binds selectively to
the polypeptide, comprising incubating the compound with the
polypeptide under appropriate conditions; thereby causing the
component to bind to the polypeptide; or to conjugate the
polypeptide to a matrix, comprising derivatizing the polypeptide
with a reactive reagent, and conjugating the polypeptide to the
matrix.
[0022] Related binding compounds include those comprising an
antigen binding site from an antibody that specifically or
selectively binds to a natural polypeptide, as described above,
wherein: the binding compound is in a container; the IL-B60
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 mature
polypeptide of Table 1; is raised against a mature IL-B60; is
raised to a purified human IL-B60; is immunoselected; is a
polyclonal antibody; binds to a denatured IL-B60; 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. Kits are provided comprising such a binding compound and: a
compartment comprising the binding compound; or instructions for
use or disposal of the reagents of the kit.
[0023] Methods are provided for producing an antigen:antibody
complex, comprising contacting, under appropriate conditions, a
primate IL-B60 polypeptide with a described antibody, thereby
allowing the complex to form. Preferably, in the method: the
complex is purified from other cytokines; the complex is purified
from other antibody; the contacting is with a sample comprising a
cytokine; 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.
[0024] In another embodiment the invention includes a composition
comprising: a sterile binding compound, as described, or the
binding compound and a carrier, wherein the 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.
[0025] Nucleic acid embodiments include an isolated or recombinant
nucleic acid encoding the described polypeptide, wherein: the
IL-B60 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; encodes a plurality of antigenic
peptide sequences of Table 4; 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
IL-B60; or is a PCR primer, PCR product, or mutagenesis primer.
Preferred embodiments include where the isolated or recombinant
nucleic acid is in a cell or tissue. The cell may be: 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.
[0026] Kits are provided comprising the described nucleic acid and:
a compartment comprising the nucleic acid; a compartment further
comprising a primate IL-B60 polypeptide; or instructions for use or
disposal of reagents in the kit.
[0027] The invention further provides methods for forming a duplex
with a polynucleotide described above, comprising contacting the
polynucleotide with a probe that hybridizes, under stringent
conditions, to at least 25 contiguous nucleotides of the coding
portion of SEQ ID NO: 1, 3, or encoding the nature SEQ ID NO: 12 or
13; thereby forming the duplex.
[0028] In a further aspect, 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; or
exhibits identity over a stretch of at least about 30 nucleotides
to a primate IL-B60. In preferred embodiments, the wash conditions
that are at 45.degree. C. and/or 500 mM salt; or at 55.degree. C.
and/or 150 mM salt; or the stretch is at least 55 nucleotides,
e.g., at least 75 nucleotides.
[0029] Methods are provided, e.g., of modulating physiology or
development of a cell or tissue culture cells comprising contacting
the cell with an agonist or antagonist of a mammalian IL-B60; or
contacting the cell with an agonist or antagonist of a complex
comprising mammalian IL-B60 and CLF-1. Additionally, the invention
provides a method of increasing the secretion of: an IL-B60
sequence, comprising expressing the polypeptide with CLF-1; or a
CLF-1, comprising expressing the CLF-1 with an IL-B60 sequence. In
preferred embodiments of the method, the increasing is at least 3
fold, 5.times., 7.times., 10.times., or more; or the expressing is
of a recombinant nucleic acid encoding one or both of the
polypeptide and CLF-1.
[0030] The invention further provides a method of screening for a
receptor which binds an isolated soluble complex comprising the
mature protein portion of SEQ ID NO: 2 or 4, and the mature protein
portion of SEQ ID NO: 12 or 13, comprising contacting the complex
to a cell expressing the receptor under conditions allowing the
complex to bind to the receptor, thereby forming a detectable
interaction. Preferably, the interaction results in a physiological
response in the cell.
[0031] Other embodiments of the invention include, e.g., an
isolated soluble complex comprising at least 6 amino acids of the
mature protein portion of SEQ ID NO: 2 or 4, and: at least 6 amino
acids of the mature protein portion of SEQ ID NO: 12 or 13; or at
least 6 amino acids of the mature protein portion of the CNTF-R.
Such complex may, e.g., comprise a recombinant polypeptide of
mature SEQ ID NO: 2 or 4; comprise a recombinant polypeptide of
mature SEQ ID NO: 12 or 13; comprise a recombinant polypeptide of
mature CNTF-R; comprise both a recombinant polypeptide of mature
SEQ ID NO: 2 or 4, and a recombinant polypeptide of mature SEQ ID
NO: 12 or 13; comprise both a recombinant polypeptide of mature SEQ
ID NO: 2 or 4, and a recombinant polypeptide of mature CNTF-R; be
detectably labeled; be in a buffered solution; or be in a sterile
solution. Preferred embodiments include those which: comprise a
mature IL-B60 polypeptide; comprise a mature CLF-1 polypeptide;
comprises a mature CNTF-R polypeptide; exhibit at least four
nonoverlapping segments of at least seven amino acids of SEQ ID NO:
2 or 4; exhibit epitopes from both primate L-B60 and primate CLF-1;
exhibit epitopes from both primate L-B60 and primate CNTF-R; not be
glycosylated; be attached to a solid substrate; be conjugated to
another chemical moiety; or comprise a detection or purification
tag, including a FLAG, His6, or Ig sequence.
[0032] Kits are provided, e.g., comprising the complex and: a
compartment comprising the complex, and/or instructions for use or
disposal of reagents in the kit.
[0033] Fusion polypeptides are provided, which include, e.g., an
isolated or recombinant polypeptide comprising: a first segment
comprising at least seven amino acids identical to segments of SEQ
ID NO: 2 or 4, and a second segment comprising at least seven amino
acids identical to segments of mature SEQ ID NO: 12 or 13; at least
two distinct nonoverlapping segments of at least five amino acids
identical to segments of mature SEQ ID NO: 2 or 4, and a third
segment comprising at least seven amino acids identical to segments
of mature SEQ ID NO: 12 or 13; at least one segment comprising at
least seven amino acids identical to segments of mature SEQ ID NO:
2 or 4, and two distinct nonoverlapping segments of at least five
amino acids identical to segments of mature SEQ ID NO: 12 or 13; a
first segment comprising at least seven amino acids identical to
segments of SEQ ID NO: 2 or 4, and a second segment comprising at
least seven amino acids identical to segments of mature primate
CNTF-R; at least two distinct nonoverlapping segments of at least
five amino acids identical to segments of mature SEQ ID NO: 2 or 4,
and a third segment comprising at least seven amino acids identical
to segments of mature primate CNTF-R; or at least one segment
comprising at least seven amino acids identical to segments of
mature SEQ ID NO: 2 or 4, and two distinct nonoverlapping segments
of at least five amino acids identical to segments of mature
primate CNTF-R. Certain embodiments include those wherein the
distinct nonoverlapping segments of identity: include one of at
least eight amino acids; include one of at least five amino acids
and a second of at least six 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. Other embodiments include those
which: comprise a mature IL-B60 sequence; comprise a mature CLF-1
sequence; comprise a mature CNTF-R sequence; exhibit at least four
nonoverlapping segments of at least seven amino acids of SEQ ID NO:
2 or 4; have a length at least about 30 amino acids; exhibit
epitopes from both primate IL-B60 and primate CLF-1; exhibits
epitopes from both primate IL-B60 and primate CNTF-R; are not
glycosylated; have a molecular weight of at least 30 kD; be a
synthetic polypeptide; be attached to a solid substrate; be
conjugated to another chemical moiety; or comprise a detection or
purification tag, including a FLAG, His6, or Ig sequence.
[0034] Other embodiments include a composition comprising:
substantially pure combination of IL-B60 and CLF-1; substantially
pure combination of IL-B60 and CNTF-R; a sterile polypeptide
described above; or the polypeptide described above 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. A kit is provided comprising
a polypeptide as described, and: a compartment comprising the
polypeptide; and/or instructions for use or disposal of reagents in
the kit.
[0035] Methods are also provided, e.g., of making an antibody which
recognizes a complex as described, comprising inducing an immune
response in an animal with the complex; of immunoselecting
antibodies, comprising contacting a population of antibodies to a
complex as described, and separating antibodies that bind from
those which do not bind; or of formulating a composition,
comprising admixing a complex as described with a carrier.
[0036] Binding compounds are provided, e.g., comprising an antigen
binding site from an antibody, which antibody specifically binds a
described complex, but not to any of the mature polypeptides of SEQ
ID NO: 2, 4, 12, 13, or CNTF-R. Certain embodiments include those
wherein: the binding compound is: in a container; an Fv, Fab, or
Fab2 fragment; or conjugated to another chemical moiety; or the
antibody: is raised against a substantially pure complex of IL-B60
with CLF-1; is raised against a substantially pure complex of
IL-B60 with CNTF-R; is immunoselected; is a polyclonal antibody;
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. Additional embodiments include a
composition comprising: a sterile binding compound as described, or
the binding compound as described 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.
[0037] With the binding composition are provided a kit comprising
the binding compound and: a compartment comprising the binding
compound; or instructions for use or disposal of reagents in the
kit. Also provided are methods of producing an antigen:antibody
complex, comprising contacting under appropriate conditions a
primate complex comprising: IL-B60 and CLF-1 polypeptides; or
IL-B60 and CNTF-R polypeptides; with an antibody as described,
thereby allowing the complex to form. Preferably, in the method,
the complex is purified from other cytokines; the complex is
purified from other antibody; the contacting is with a sample
comprising a cytokine; 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.
[0038] Various nucleic acids are provided, e.g., an isolated or
recombinant nucleic acid: encoding the amino acid portions
described above; encoding the amino acid portions as described, and
comprise a segment at least 30 contiguous nucleotides from SEQ ID
NO: 1 or 3; which will coexpress a segment of at least seven
contiguous amino acids from SEQ ID NO: 2 or 4, and a segment of at
least seven contiguous amino acids from SEQ ID NO: 12 or 13; or
which will coexpress a segment of at least seven contiguous amino
acids from SEQ ID NO: 2 or 4, and a segment of at least seven
contiguous amino acids from CNTF-R. Preferred nucleic acids include
those which, e.g.,: encode IL-B60 from a human; encode CLF-1 from a
human; encodes CNTF-R from a human; are an expression vector;
further comprise an origin of replication; comprise a detectable
label; comprise synthetic nucleotide sequence; or are less than 6
kb, preferably less than 3 kb. A cell comprising the recombinant
nucleic acid is provided, e.g., 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. Various nucleic acid kits are provided, e.g., comprising the
nucleic acid and: a compartment comprising the nucleic acid; a
compartment further comprising a primate IL-B60 polypeptide; a
compartment further comprising a primate CLF-1 polypeptide; a
compartment further comprising a primate CNTF-R polypeptide; or
instructions for use or disposal of reagents in the kit. Methods
are also provided, e.g., of making a duplex nucleic acid,
comprising contacting such a nucleic acid with a complementary
nucleic acid under appropriate conditions, thereby forming said
duplex; of expressing a polypeptide, comprising expressing the
nucleic acid, thereby producing the polypeptide; or of transfecting
a cell, comprising contacting said cell under appropriate
conditions with the nucleic acid, thereby transfecting the
cell.
[0039] In an alternative embodiment, the invention provides an
isolated or recombinant nucleic acid which encodes at least 5
contiguous amino acids of SEQ ID NO: 12, 13, or primate CNTF-R and:
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; or
exhibits identity over a stretch of at least about 30 nucleotides
to a primate IL-B60. Preferred embodiments include: the isolated
nucleic acid, wherein: the contiguous amino acids number at least
8; the wash conditions are at 45.degree. C. and/or 500 mM salt; or
the stretch is at least 55 nucleotides; or the recombinant nucleic
acid, wherein: the contiguous amino acids number at least 12; the
wash conditions are at 55.degree. C. and/or 150 mM salt; or the
stretch is at least 75 nucleotides.
[0040] The invention particularly provides methods of modulating
physiology or development of a cell or tissue culture cells
comprising contacting the cell with an agonist or antagonist of a
complex comprising mammalian IL-B60 and: CLF-1; or CNTF-R. It also
provides methods of producing the proteins, e.g., producing a
complex described, comprising coexpressing a recombinant IL-B60
with a recombinant CLF-1 or CNTF-R; increasing the secretion of an
IL-B60 polypeptide comprising expressing the polypeptide with CLF-1
or CNTF-R; or increasing the secretion of a CLF-1 polypeptide,
comprising expressing the CLF-1 with an IL-B60. Typically, the
increasing is at least 3 fold; or the expressing is of a
recombinant nucleic acid encoding one or both of the polypeptide
and CLF-1.
[0041] Also provided are methods of screening for a receptor which
binds the described complex, comprising contacting the complex to a
cell expressing the receptor under conditions allowing the complex
to bind to the receptor, thereby forming a detectable interaction.
Preferably, the interaction results in a physiological response in
the cell.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] All references cited 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.
[0043] Outline
[0044] I. General
[0045] II. Purified IL-B60 or complex
[0046] A. physical properties
[0047] B. biological properties
[0048] III. Physical Variants
[0049] A. sequence variants, fragments
[0050] B. post-translational variants
[0051] 1. glycosylation
[0052] 2. others
[0053] IV. Functional Variants
[0054] A. analogs, fragments
[0055] 1. agonists
[0056] 2. antagonists
[0057] B. mimetics
[0058] 1. protein
[0059] 2. chemicals
[0060] C. species variants
[0061] V. Antibodies
[0062] A. polyclonal
[0063] B. monoclonal
[0064] C. fragments, binding compositions
[0065] VI. Nucleic Acids
[0066] A. natural isolates; methods
[0067] B. synthetic genes
[0068] C. methods to isolate
[0069] VII. Making IL-B60 or complex, mimetics
[0070] A. recombinant methods
[0071] B. synthetic methods
[0072] C. natural purification
[0073] VIII. Uses
[0074] A. diagnostic
[0075] B. therapeutic
[0076] IX. Kits
[0077] A. nucleic acid reagents
[0078] B. protein reagents
[0079] C. antibody reagents
[0080] X. Isolating receptors for IL-B60 or complex
[0081] I. General
[0082] The present invention provides amino acid sequences and DNA
sequences encoding various mammalian proteins which are cytokines,
e.g., which are secreted molecules which can mediate a signal
between immune or other cells. See, e.g., Paul (1998) Fundamental
Immunology (4th ed.) Raven Press, N.Y. The full length cytokines,
and fragments, or antagonists will be useful, e.g., in
physiological modulation of cells expressing a receptor. It is
likely that IL-B60 has either stimulatory or inhibitory effects on
hematopoietic cells, including, e.g., lymphoid cells, such as
T-cells, B-cells, natural killer (NK) cells, macrophages, dendritic
cells, hematopoietic progenitors, etc. The proteins will also be
useful as antigens, e.g., immunogens, for raising antibodies to
various epitopes on the protein, both linear and conformational
epitopes.
[0083] A sequence encoding IL-B60 was identified from a human
genomic sequence. The molecule was designated huIL-B60. A rodent
sequence, e.g., from mouse, is also described.
[0084] The human gene encodes a small soluble cytokine-like
protein, of about 198 amino acids. The psort predicted signal
sequence probably is about 17 residues, and would run from the Met
to about Ala. See Table 1 and SEQ. ID. NO: 1 and 2. IL-B60 exhibits
structural motifs characteristic of a member of the long chain
cytokines. Compare, e.g., IL-B60, G-CSF, and IL-6, sequences
available from GenBank. Closest matching is with CT-1, oncostatin
M, and CNTF. See also Table 2.
1TABLE 1 Nucleic acid (SEQ ID NO: 1) encoding IL-B60 from a
primate, e.g., human. Predicted signal cleavage site is indicated.
Nucleotide 375 may be A. Translated amino acid sequence is SEQ ID
NO: 2. ccgagcgaaa aaaacctgcg agtgggcctg gcggatggga ttattaaagc
ttcgccggag 60 ccgcggctcg ccctcccact ccgccagcct ccgggagagg
agccgcaccc ggccggcccg 120 gccccagccc catggacctc cgagcagggg
actcgtgggg g atg tta gcg tgc ctg 176 Met Leu Ala Cys Leu -15 tgc
acg gtg ctc tgg cac ctc cct gca gtg cca gct ctc aat cgc aca 224 Cys
Thr Val Leu Trp His Leu Pro Ala Val Pro Ala Leu Asn Arg Thr -10 -5
-1 1 ggg gac cca ggg cct ggc ccc tcc atc cag aaa acc tat gac ctc
acc 272 Gly Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys Thr Tyr Asp Leu
Thr 5 10 15 20 cgc tac ctg gag cac caa ctc cgc agc ttg gct ggg acc
tat ctg aac 320 Arg Tyr Leu Glu His Gln Leu Arg Ser Leu Ala Gly Thr
Tyr Leu Asn 25 30 35 tac ctg ggc ccc cct ttc aac gag cca gac ttc
aac cct ccc cgc ctg 368 Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe
Asn Pro Pro Arg Leu 40 45 50 ggg gca gag act ctg ccc agg gcc act
gtt gac ttg gag gtg tgg cga 416 Gly Ala Glu Thr Leu Pro Arg Ala Thr
Val Asp Leu Glu Val Trp Arg 55 60 65 agc ctc aat gac aaa ctg cgg
ctg acc cag aac tac gag gcc tac agc 464 Ser Leu Asn Asp Lys Leu Arg
Leu Thr Gln Asn Tyr Glu Ala Tyr Ser 70 75 80 cac ctt ctg tgt tac
ttg cgt ggc ctc aac cgt cag gct gcc act gct 512 His Leu Leu Cys Tyr
Leu Arg Gly Leu Asn Arg Gln Ala Ala Thr Ala 85 90 95 100 gag ctg
cgc cgc agc ctg gcc cac ttc tgc acc agc ctc cag ggc ctg 560 Glu Leu
Arg Arg Ser Leu Ala His Phe Cys Thr Ser Leu Gln Gly Leu 105 110 115
ctg ggc agc att gcg ggc gtc atg gca gct ctg ggc tac cca ctg ccc 608
Leu Gly Ser Ile Ala Gly Val Met Ala Ala Leu Gly Tyr Pro Leu Pro 120
125 130 cag ccg ctg cct ggg act gaa ccc act tgg act cct ggc cct gcc
cac 656 Gln Pro Leu Pro Gly Thr Glu Pro Thr Trp Thr Pro Gly Pro Ala
His 135 140 145 agt gac ttc ctc cag aag atg gac gac ttc tgg ctg ctg
aag gag ctg 704 Ser Asp Phe Leu Gln Lys Met Asp Asp Phe Trp Leu Leu
Lys Glu Leu 150 155 160 cag acc tgg ctg tgg cgc tcg gcc aag gac ttc
aac cgg ctc aag aag 752 Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe
Asn Arg Leu Lys Lys 165 170 175 180 aag atg cag cct cca gca gct gca
gtc acc ctg cac ctg ggg gct cat 800 Lys Met Gln Pro Pro Ala Ala Ala
Val Thr Leu His Leu Gly Ala His 185 190 195 ggc ttc tgacttctga
ccttctcctc ttcgctcccc cttcaaaccc tgctcccact 856 Gly Phe ttgtgagagc
cagccctgta tgccaacacc tgttgagcca ggagacagaa gctgtgagcc 916
tctggccctt tcctggaccg gctgggcgtg tgatgcgatc agccctgtct cctccccacc
976 tcccaaaggt ctaccgagct ggggaggagg tacagtaggc cctgtcctgt
cctgtttcta 1036 caggaagtca tgctcgaggg agtgtgaagt ggttcaggtt
ggtgcagagg cgctcatggc 1096 ctcctgcttc ttgcctacca cttggccagt
gcccacccag cccctcaggt ggcacatctg 1156 gagggcaggg gttgaggggc
caccaccaca catgcctttc tggggtgaag ccctttggct 1216 gccccactct
ccttggatgg gtgttgctcc cttatcccca aatcactcta tacatccaat 1276
tcaggaaaca aacatggtgg caattctaca caaaaagaga tgagattaac agtgcagggt
1336 tggggtctgc attggaggtg ccctataaac cagaagagaa aatactgaaa
gcacaggggc 1396 agggacagac cagaccagac ccaggagtct ccaaagcaca
gagtggcaaa caaaacccga 1456 gctgagcatc aggaccttgc ctcgaattgt
cttccagtat tacggtgcct cttctctgcc 1516 ccctttccca gggtatctgt
gggttgccag gctggggagg gcaaccatag ccacaccaca 1576 ggatttcctg
aaagtttaca atgcagtagc attttggggt gtagggtggc agctccccaa 1636
ggccctgccc cccagcccca cccactcatg actctaagtg tgttgtatta atatttattt
1696 atttggagat gttatttatt agatgatatt tattgcagaa tttctattct
tgtattaaca 1756 aataaaatgc ttgccccaga acaaaaaaaa aaaa 1790
MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGTY-
LNYLGPPFNEPDFNPPR LGAETLPRATVDLEVWRSLNDKLRLTQNYEAYSHLLCYL-
RGLNRQAATAELRRSLAHFCTSLQGLLGSI AGVMAALGYPLPQPLPGTEPTWTPGPA-
HSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAV TLHLGAHGF Rodent, e.g.,
mouse, IL-B60 (SEQ ID NO: 3 and 4): atg tta gct tgc cta tgc acg gtg
ctg tgg cac ctc cct gca gtg cca 48 Met Leu Ala Cys Leu Cys Thr Val
Leu Trp His Leu Pro Ala Val Pro -15 -10 -5 gct ctt aat cgc aca gga
gat cca ggc cct ggc ccc tcc atc cag aaa 96 Ala Leu Asn Arg Thr Gly
Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys -1 1 5 10 15 acc tat gac
ctc acc cgc tac ctg gag cat caa ctc cgc agc tta gct 144 Thr Tyr Asp
Leu Thr Arg Tyr Leu Glu His Gln Leu Arg Ser Leu Ala 20 25 30 ggg
acc tac ctg aac tac ctg ggg ccc cct ttc aac gag cct gac ttc 192 Gly
Thr Tyr Leu Asn Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe 35 40
45 aat cct cct cga ctg ggg gca gaa act ctg ccc agg gcc acg gtc aac
240 Asn Pro Pro Arg Leu Gly Ala Glu Thr Leu Pro Arg Ala Thr Val Asn
50 55 60 ttg gaa gtg tgg cga agc ctc aat gac agg ctg cgg ctg acc
cag aac 288 Leu Glu Val Trp Arg Ser Leu Asn Asp Arg Leu Arg Leu Thr
Gln Asn 65 70 75 tat gag gcg tac agt cac ctc ctg tgt tac ttg cgt
ggc ctc aac cgt 336 Tyr Glu Ala Tyr Ser His Leu Leu Cys Tyr Leu Arg
Gly Leu Asn Arg 80 85 90 95 cag gct gcc aca gct gaa ctc cga cgt agc
ctg gcc cac ttc tgt acc 384 Gln Ala Ala Thr Ala Glu Leu Arg Arg Ser
Leu Ala His Phe Cys Thr 100 105 110 agc ctc cag ggc ctg ctg ggc agc
att gca ggt gtc atg gcg acg ctt 432 Ser Leu Gln Gly Leu Leu Gly Ser
Ile Ala Gly Val Met Ala Thr Leu 115 120 125 ggc tac cca ctg ccc cag
cct ctg cca ggg act gag cca gcc tgg gcc 480 Gly Tyr Pro Leu Pro Gln
Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala 130 135 140 cct ggc cct gcc
cac agt gac ttc ctc cag aag atg gat gac ttc tgg 528 Pro Gly Pro Ala
His Ser Asp Phe Leu Gln Lys Met Asp Asp Phe Trp 145 150 155 ctg ctg
aag gag ctg cag acc tgg cta tgg cgt tca gcc aag gac ttc 576 Leu Leu
Lys Glu Leu Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe 160 165 170
175 aac cgg ctt aag aag aag atg cag cct cca gca gct tca gtc acc ctg
624 Asn Arg Leu Lys Lys Lys Met Gln Pro Pro Ala Ala Ser Val Thr Leu
180 185 190 cac ttg gag gcc cat ggt ttc tga 648 His Leu Glu Ala His
Gly Phe 195 MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDL-
TRYLEHQLRSLAGTYLNYLGPPFNEPDFNPPR LGAETLPRATVNLEVWRSLNDRLR-
LTQNYEAYSHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSI
AGVMATLGYPLPQPLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASV
TLHLEAHGF
[0085]
2TABLE 2 Comparison of primate and rodent embodiments of IL-B60,
both the nucleotide and amino acid sequences. hIL-B60
ATGTTAGCGTGCCTGTGCACGGTGCTCTGGC- ACCTCCCTGCAGTGCCAGCTCTCAATCGC
mIL-B60 ATGTTAGCTTGCCTATGCACGGTGCTGT-
GGCACCTCCCTGCAGTGCCAGCTCTTAATCGC ******** ***** ***********
************************** ****** hIL-B60
ACAGGGGACCCAGGGCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG
mIL-B60
ACAGGAGATCCAGGCCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG *****
** ***** ********************************************* hIL-B60
GAGCACCAACTCCGCAGCTTGGCTGGGACCTATCTGAACTACCTGGGCCCCCCTTTCA- AC
mIL-B60 GAGCATCAACTCCGCAGCTTAGCTGGGACCTACCTGAACTACCTGGGGCCCCCTTT-
CAAC ***** ************** *********** ************** ************
hIL-B60 GAGCCAGACTTCAACCCTCCCCGCCTGGGGGCAGAGACTCTGCCCAGGG-
CCACTGTTGAC mIL-B60
GAGCCTGACTTCAATCCTCCTCGACTGGGGGCAGAAACTCTGCCCAG- GGCCACGGTCAAC
***** ******** ***** ** *********** ***************** ** ** hIL-B60
TTGGAGGTGTGGCGAAGCCTCAAT- GACAAACTGCGGCTGACCCAGAACTACGAGGCCTAC
mIL-B60
TTGGAAGTGTGGCGAAGCCTCAATGACAGGCTGCGGCTGACCCAGAACTATGAGGCGTAC *****
********************** ******************** ***** *** hIL-B60
AGCCACCTTCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACTGCTGAGCTGCGC
mIL-B60
AGTCACCTCCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACAGCTGAACTCCGA **
***** ************************************** ***** ** ** hIL-B60
CGCAGCCTGGCCCACTTCTGCACCAGCCTCCAGGGCCTGCTGGGCAGCATTGCGG- GCGTC
mIL-B60 CGTAGCCTGGCCCACTTCTGTACCAGCCTCCAGGGCCTGCTGGGCAGCATTGC-
AGGTGTC ** ***************** ******************************** **
*** hIL-B60 ATGGCAGCTCTGGGCTACCCACTGCCCCAGCCGCTGCCTGGGACT-
GAACCCACTTGGACT mIL-B60
ATGGCGACGCTTGGCTACCCACTGCCCCAGCCTCTGCCAGGGA- CTGAGCCAGCCTGGGCC
***** * ** ******************** ***** ******** ** * *** * hIL-B60
CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGATGG- ACGACTTCTGGCTGCTGAAGGAG
mIL-B60 CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGAT-
GGATGACTTCTGGCTGCTGAAGGAG **************************************
********************* hIL-B60 CTGCAGACCTGGCTGTGGCGCTCGGCC-
AAGGACTTCAACCGGCTCAAGAAGAAGATGCAG mIL-B60
CTGCAGACCTGGCTATGGCGTTCAG- CCAAGGACTTCAACCGGCTTAAGAAGAAGATGCAG
************** ***** ** ******************** ***************
hIL-B60 CCTCCAGCAGCTGCAGTCACCCTGCACCTGGGGGCTCATGGCTTCTGA mIL-B60
CCTCCAGCAGCTTCAGTCACCCTGCACTTGGAGGCCCATGGTTTCTGA ************
************** *** *** ***** ****** A hIL-B60
MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRY- LEHQLRSLAGT mIL-B60
MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRYLEHQLRSL- AGT
************************************************** B hIL-B60
YLNYLGPPFNEPDFNPPRLGAETLPRATVDLEVWRSLNDKLRLTQNYEAY mIL-B60
YLNYLGPPFNEPDFNPPRLGAETLPRATVNLEVWRSLNDRLRLTQNYEAY
*****************************:*********:********** C hIL-B60
SHLLCYLRGLNRQAATAELRRSLA- HFCTSLQGLLGSIAGVMAALGYPLPQ mIL-B60
SHLLCYLRGLNRQAATAELRRSLAHFCTSLQG- LLGSIAGVMATLGYPLPQ
******************************************:****- *** D hIL-B60
PLPGTEPTWTPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ mIL-B60
PLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ
*******:*:**************************************** hIL-B60
PPAAAVTLHLGAHGF mIL-B60 PPAASVTLHLEAHGF ****:***** **** Alignment
of IL-B60: underlined are proposed helices. In general, those
residues that are in helix A and D and not pointing inward toward
the core (mostly the hydrophobic residues in A and D helix) are the
most likely residues to interact with receptors.
[0086]
3TABLE 3 Comparison of various cytokines compared to IL-B60. Human
IL-B60 is SEQ ID NO: 2; mouse IL-B60 is SEQ ID NO:4; mouse LIF
(mLIF) is SEQ ID NO: 5 and Accession number X06381; human LIF
(hLIF) is SEQ ID NO: 6 and Accession numbers M63420 J05436; human
CT-1 (hCT-1) is SEQ ID NO: 7 and Accession number U43030; mouse
CT-1 (mCT-1) is SEQ ID NO: 8 and Accession number U18366; human
CNTF (hCNTF) is SEQ ID NO: 9 and Accession number X60542; mouse
CNTF (mCNTF) is SEQ ID NO: 10 and Accession number U05342; human
DNAX IL-40 (hDIL-40) is SEQ ID NO: 11. mLIF
-MKVLAAGIVPLLLLVLHWKHGAGSPLPI-TPVNATC-AIR- HPCHGNLMN hLIF
-MKVLAAGVVP-LLLVLHWKHGAGSPLPI-TPVNATC-AIRHPCHNNLMN hCT-1
--MSRREGSLE---D--PQTDSSVSLLPH-LEA-----KIRQT-HS--LA mCT-1
--MSQREGSLE---D--HQTDSSISFLPH-LEA-----KIRQT-HN--LA hIL-B60
-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT mIL-B60
-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT hCNTF
------MAFTE------HSPLTPHR-R---D-L-CSR-SIW-------LA mCNTF
------MAFAE------QSPLTLHR-R---D-L-CSR-SIW-------LA hDIL-40
MTHLSLLGPLPCVRTSQQLPETQQVTTPGKKPVSVGRREVRVP-----GT : . mLIF
QIKNQLAQLNGSANALFISYYTAQGEPF--PNNVEK-LCAPNMTDFPSFH hLIF
QIRSQLAQLNGSANALFILYYTAQGEPF--PNNLDK-LCGPNVTDFPPFH hCT-1
HLLTKYAEQ------LLQEYVQLQGDPFGLPSFSPPRLPVAGLSAPAPSH mCT-1
RLLTKYAEQ------LLEEYVQQQGEPFGLPGFSPPRLPLAGLSGPAPSH hIL-B60
RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVDL mIL-B60
RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVNL hCNTF
RKIRSDLTA------LTESYVKHQG--LNK---NINLDSADGMPVASTD- mCNTF
RKIRSDLTA------LMESYVKHQG--LNK---NISLDSVDGVPVASTD- hDIL-40
ALVPSLLSV------SVLLQLQYQGSPFSDPGFSAPELQLSSLPPATAFF * :. . :. . mLIF
---GNGTEKTKLVELYRMVAYLSASLTNITR-DQKVLNPTAVSLQVKLNA hLIF
---ANGTEKAKLVELYRIVVYLGTSLGNITR-DQKILNPSALSLHSKLNA hCT-1
---AGLPVHERLRLDAAALAALPPLLDAVCR-RQAELNPRAPRLLRRLED mCT-1
---AGLPVSERLRQDAAALSVLPALLDAVRR-RQAELNPRAPRLLRSLED hIL-B60
EVWRSLNDKLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS mIL-B60
EVWRSLNDRLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS hCNTF
-QWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHT mCNTF
-RWSEMTEAERLQENLQAYRTFQGMLTKLLEDQRVHFTPTEGDFHQAIHT hDIL-40
KTWHALDDGERLSLAQRAID---PHLQLVED-DQSDLNPGSPILPAQLGA : :* * : : :
mLIF TIDVMRGLLSNVLCRLCNKYRV--GHVDVPP-----VPDHSDKE--AFQR hLIF
TADILRGLLSNVLCRLCSKYHV--GHVDVTY-----GPDTSGKD--VFQK hCT-1
AARQARALGAAVEALLAALGAANRGPRAEPP--AATASAASATG--VFPA mCT-1
AARQVRALGAAVETVLAALGAAARGPGPEPVTVATLFTANSTAG--IFSA hIL-B60
LQGLLGSIAGVMAALGYPLPQP--LPGTEPT----WTPGPAHS---DFLQ mIL-B60
LQGLLGSIAGVMATLGYPLPQP--LPGTEPA----WAPGPAHS---DFLQ hCNTF
LLLQVAAFAYQIEELMILLEYK--IPRNEAD----GMPINVGDGG-LFEK mCNTF
LTLQVSAFAYQLEELMALLEQK--VPEKEAD----GMPVTIGDGG-LFEK hDIL-40
ARLRAQGPLGNMAAIMTALGLP--IP-PEED-----TPGLAAFGASAFER . : . * mLIF
KKLGCQLLGTYKQVIS----VVVQAF--------------------- hLIF
KKLGCQLLGKYKQIIA----VLAQAF--------------------- hCT-1
KVLGLRVCGLYREWLSRTEGDLGQLLPGGSA---------------- mCT-1
KVLGFHVCGLYGEWVSRTEGDLGQLVPGGVA---------------- hIL-B60
KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAVTLHLGAHGF-- mIL-B60
KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASVTLHLEAHGF-- hCNTF
KLWGLKVLQELSQWTVRSIHDL-RFISSHQTGIPARGSHYIANNKKM mCNTF
KLWGLKVLQELSQWTVRSIHDL-RVISSHHMGISAHESHYGA--KQM hDIL-40
KCRGYVVTREYGHWTDRAVRDLALLKAKYSA---------------- * . : . . : :.
[0087] The structural homology of IL-B60 to related cytokine
proteins suggests related function of this molecule. IL-B60 is a
long chain cytokine exhibiting sequence similarity to IL-6 and
G-CSF.
[0088] IL-B60 agonists, or antagonists, may also act as functional
or receptor antagonists, e.g., which block IL-6 or G-CSF binding to
their respective receptors, or mediating the opposite actions.
Thus, IL-B60, or its antagonists, may be useful in the treatment of
abnormal medical conditions, including immune disorders, e.g., T
cell immune deficiencies, chronic inflammation, or tissue
rejection, or in cardiovascular or neurophysiological
conditions.
[0089] The natural antigens are capable of mediating various
biochemical responses which lead to biological or physiological
responses in target cells. The preferred embodiment characterized
herein is from human, but other primate, or other species
counterparts exist in nature. Additional sequences for proteins in
other mammalian species, e.g., primates, canines, felines, and
rodents, should also be available. See below. The descriptions
below are directed, for exemplary purposes, to a human IL-B60, but
are likewise applicable to related embodiments from other
species.
[0090] In particular, the association of the IL-B60 with a partner
has been confirmed. The IL-B60 and CLF-1 molecules have likely
evolved together, reflected in their homology between species. For
example, the coevolution of their function is suggested by the
observation that the human/mouse relationship of the IL-B60 is very
close, as is the human/mouse CLF-1. If the two functionally
associate, they might act together in the fashion of IL-12. See,
e.g., Trinchieri (1998) Adv. Immunol. 70:83-243; Gately, et
al.(1998) Ann. Rev. Immunol. 16:495-521; and Trinchieri (1998) Int.
Rev. Immunol. 16:365-396.
[0091] As a complex, however, they will interact with two tall
receptors in the cytokine receptor family, e.g., gp130, LIF-R,
OSM-R, IL-12Rb1, IL-12Rb2, and NR30. These receptors will be tested
for binding to the soluble complex. A series of BAF/3 cells that
stably express various of these tall receptors have been
constructed.
[0092] The supernatants of transfectants of both IL-B60 and CLF-1
(or a single combination construct) in the same cell, will be used
to test these various BAF/3 cells to see if there is a
proliferative or other signaling response. As such, most of the
physiological effects of the cytokine may be due to the complex of
the proteins. As such, many of the descriptions below of biology
resulting from the cytokine may actually be physiologically
effected by the complex comprising the combination of the
subunits.
[0093] Table 4 provides the sequences of the IL-B60 partner, known
as CLF-1. The CNTF receptor (CNTF-R) subunit alpha was described,
e.g., by Davis, et al. (1991) Science 253:59-63. See also GenBank
accession numbers NM1001842 and M73238 (human); AF068615 (mouse);
and S54212 (rat); each of which is incorporated herein by
reference.
4TABLE 4 Alignment of human and mouse Cytokine-Like Factor 1
(CLF-1; SEQ ID NO: 12 and 13) See Elson, et al. (1998) J. Immunol.
161:1371-1379; GenBank Accession number AF059293 and NM_004750;
also described by Douglas J. Hilton (Australia) in W09920755.
Reported signal sequence of 37 amino acids in human form, cleavage
at GSG/AHT. hCLF-1
MPAGRRGPAAQSARRPPPLLPLLLLLCVLGAPRAGSGAHTAVISPQDPTL mCLF-1
--------------RPLSSLWSPLLLCVLGVPRGGSGAHTAVISPQDPTL ** . *
*******.**.**************** hCLF-1
LIGSSLLATCSVHGDPPGATAEGLYWTLNGRRLPPELSRVLNASTLALAL mCLF-1
LIGSSLQATCSIHGDTPGATAEGLYWTLNGRRLP-SLSRLLNTSTLALAL ******
****:***.****************** .***:**:******* hCLF-1
ANLNGSRQRSGDNLVCHARDGSILAGSCLYVGLPPEKPVNISCWSKNMKD mCLF-1
ANLNGSRQQSGDNLVCHARDGSILAGSCLYVGLPPEKPFNISCWSRNMKD
********:*****************************.******:**** hCLF-1
LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD mCLF-1
LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD
************************************************** hCLF-1
LALFTPYEIWVEATNRLGSARSDVLTLDILDVVTTDPPPDVHVSRVGGLE mCLF-1
LALFTPYEIWVEATNRLGSARSDVLTLDVLDVVTTDPPPDVHVSRVGGLE
****************************:********************* hCLF-1
DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG mCLF-1
DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG
************************************************** hCLF-1
LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGA mCLF-1
LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGV
*************************************************. hCLF-1
CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK mCLF-1
CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK
************************************************** hCLF-1
TRNQ---VLPDKL--------- mCLF-1 TRNQDEGILPSGRRGAARGPAG **** :**.
[0094] Standard domains of the human CLF-1 receptor sequence
correspond approximately to: signal from 1 to about 38; first
IG-like domain from about residue 39 to 130; a second domain from
about 131 to about 237; and the last from about 238 to the end.
[0095] The descriptions below may also be:applied to the CLF-1, or
to the IL-B60/CLF-1 complex. A fusion of the IL-B60 with CLF-1 may
be constructed, as, e.g., the hyper IL-6. See, e.g., Fischer, et
al. (1997) Nature Biotechnol. 15:142-145; Rakemann, et al. (1999)
J. Biol. Chem. 274:1257-1266; and Peters, et al.(1998) J. Immunol.
161:3575-3581; which are incorporated herein by reference.
[0096] The original discovery and molecular characterization of
CNTF as a potent survival factor for neuronal cells (see, e.g.,
Hughes, et al. (1988) Nature 335:70-73; and Stockli, et al. (1989)
Nature 342:920-923) suggested a prospective therapeutic use as a
molecule that could speed repair of damaged or severed motor
neurons (Sendtner, et al. (1990) Nature 345:440-441; and Curtis, et
al. (1993) Nature 365:253-256) or prevent nerve degeneration
(Sendtner, et al. (1992) Nature 358:502-504; Emerich, et al. (1997)
Nature 386:395-399; and Gravel, et al. (1997) Nature Med.
3:765-770). However, CNTF is oddly a protein without a secretory
signal peptide, and does not appear to escape the cell (Stockli, et
al., ibid); furthermore, engineered (Masu, et al. (1993) Nature
365:27-32) or naturally occurring (Takahashi, et al. (1994) Nature
Genet. 7:79-84) disruptions of the CNTF gene are not deleterious.
By contrast, gene disruptions of the primary receptor for CNTF
(CNTF-R.alpha.) prove lethal shortly after birth. DeChiara, et al.
(1995) Cell 83:313-322). Together, these observations point to the
existence of a second ligand for CNTF-R.alpha. that is
physiologically responsible for the in vitro observed, or in vivo
desired, actions of CNTF. This work demonstrates that the composite
cytokine IL-B60/CLF-1 is likely this long sought-after factor that
is both developmentally critical, it is secreted from target organs
and directs their innervation by motor neurons, as well as
therapeutically promising, since nerve transection results in a
fast and long lasting induction of both IL-B60 and CLF-1,
indicating a role for the complex in regeneration. In support of
this model, gene disruption of CLF-1 (Alexander, et al. (1999)
Curr. Biol. 9:605-608) is quite similar in phenotype to the
CNTF-R.alpha. knock-out.
[0097] In an intriguing twist, while IL-B60 has a signal peptide,
its secretion remains critically dependent on complexing with
CLF-1, as described. Once secreted, IL-B60 signals via a tripartite
receptor system that is otherwise identical to that of CNTF,
consisting of the two ubiquitously expressed signal-transducing
components gp130 and LIF-R, and the specificity-determining
receptor, CNTF-R.alpha.. Strikingly, the role of CLF-1 seems to be
restricted to that of a chaperone, since it is discarded from the
signaling complex after delivering IL-B60 to CNTF-R.alpha.; indeed,
the requirement for CLF-1 can be sidestepped by fusing IL-B60
directly to a soluble form of the CNTF-R.alpha. chain. All three
protein chains involved in this novel system, IL-B60, CLF-1, and
CNTF-R.alpha., represent the most highly conserved sequences in the
hematopoietic cytokine/receptor superfamily, indicating an
evolutionarily critical interaction. Furthermore, the conditional
use of a hematopoietic receptor as a secretion factor and escort
presents a novel paradigm for cytokine activity.
[0098] To summarize, this work sheds light on the entwined
biological function of two orphan molecules, IL-B60 and CLF-1, by
describing their novel engagement of the CNTF receptor complex. In
doing so, we present a strong argument that it is IL-B60/CLF-1
cytokine that serves as a key physiological factor in motor neuron
development and regeneration.
[0099] II. Purified IL-B60 or Complex
[0100] Human IL-B60 amino acid sequence, is shown, in one
embodiment within SEQ ID NO: 2. Other naturally occurring nucleic
acids which encode the protein can be isolated by standard
procedures using the provided sequence, e.g., PCR techniques, or by
hybridization. These amino acid sequences, provided amino to
carboxy, are important in providing sequence information for the
cytokine allowing for distinguishing the protein antigen from other
proteins and exemplifying numerous variants. Moreover, the peptide
sequences allow preparation of peptides to generate antibodies to
recognize such segments, and nucleotide sequences allow preparation
of oligonucleotide probes, both of which are strategies for
detection or isolation, e.g., cloning, of genes encoding such
sequences.
[0101] As used herein, the term "human soluble IL-B60" shall
encompass, when used in a protein context, a protein having amino
acid sequence corresponding to a soluble polypeptide from SEQ ID
NO: 2. Significant fragments thereof will often retain similar
functions, e.g., antigenicity. Preferred embodiments comprise 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 may be recited, longer lengths, of various
sizes, may be appropriate, e.g., one of length 7, and two of length
12. Similar features apply to polynucleotides.
[0102] Binding components, e.g., antibodies, typically bind to an
IL-B60 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. Counterpart proteins
will be found in mammalian species other than human, e.g., other
primates, ungulates, or rodents. Non-mammalian species should also
possess structurally or functionally related genes and proteins,
e.g., birds or amphibians.
[0103] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least about
12 amino acids, typically at least about 16 amino acids, preferably
at least about 20 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 35, 40,
45, 50, etc. Such fragments may have ends which begin and/or end at
virtually all positions, e.g., beginning at residues 1, 2, 3, etc.,
and ending at, e.g., 150, 149, 148, etc., in all practical
combinations. Particularly interesting peptides have ends
corresponding to structural domain boundaries, e.g., helices A, B,
C, and/or D. See Table 1.
[0104] The term "binding composition" refers to molecules that bind
with specificity to IL-B60, e.g., in an antibody-antigen
interaction. The specificity may be more or less inclusive, e.g.,
specific to a particular embodiment, or to groups of related
embodiments, e.g., primate, rodent, etc. Depletion or absorptions
can provide desired selectivities. Also provided are compounds,
e.g., proteins, which specifically associate with IL-B60, including
in a natural physiologically relevant protein-protein interaction,
either covalent or non-covalent. The molecule may be a polymer, or
chemical reagent. A functional analog may be a protein with
structural modifications, or it may be a molecule which has a
molecular shape which interacts with the appropriate binding
determinants. The compounds may serve as agonists or antagonists of
a receptor binding interaction, see, e.g., Goodman, et al. (eds.)
Goodman & Gilman's: The Pharmacological Bases of Therapeutics
(current ed.) Pergamon Press.
[0105] Substantially pure, e.g., in a protein context, typically
means that the protein is free from other contaminating proteins,
nucleic acids, or other biologicals derived from the original
source organism. Purity may be assayed by standard methods,
typically by weight, and will ordinarily be at least about 40%
pure, generally at least about 50% pure, often at least about 60%
pure, typically at least about 80% pure, preferably at least about
90% pure, and in most preferred embodiments, at least about 95%
pure. Carriers or excipients will often be added.
[0106] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. For
diagnostic purposes, the temperature will usually be about room
temperature or warmer, but less than the denaturation temperature
of components in the assay. For therapeutic purposes, the
temperature will usually be body temperature, typically about
37.degree. C. for humans and mice, though under certain situations
the temperature may be raised or lowered in situ or in vitro.
[0107] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents.
[0108] The solvent and electrolytes will usually be a biologically
compatible buffer, of a type used for preservation of biological
activities, and will usually approximate a physiological aqueous
solvent. Usually the solvent will have a neutral pH, typically
between about 5 and 10, and preferably about 7.5. On some
occasions, one or more detergents will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammoni- o]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein. In other
instances, a harsh detergent may be used to effect significant
denaturation.
[0109] An IL-B60 polypeptide that specifically binds to or that is
specifically immunoreactive with an antibody, e.g., such as a
polyclonal antibody, generated against a defined immunogen, e.g.,
such as an immunogen consisting of an amino acid sequence of SEQ ID
NO: 2 or fragments thereof or a polypeptide generated from the
nucleic acid of SEQ ID NO: 1 is typically determined in an
immunoassay. Included within the metes and bounds of the present
invention are those nucleic acid sequences described herein,
including functional variants, that encode polypeptides that
selectively bind to polyclonal antibodies generated against the
prototypical IL-B60 polypeptide as structurally and functionally
defined herein. The immunoassay typically uses a polyclonal
antiserum which was raised, e.g., to a protein of SEQ ID NO: 2.
This antiserum is selected, or depleted, to have low
crossreactivity against appropriate other closely related family
members, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption or depletion prior
to use in the immunoassay. Appropriate selective serum preparations
can be isolated, and characterized.
[0110] 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 protein of SEQ ID NO: 2 using a
standard adjuvant, such as Freund's adjuvant, and a standard mouse
immunization protocol (see Harlow and Lane). Alternatively, a
substantially full length synthetic peptide derived from the
sequences disclosed herein can be used as 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 closely related family members, e.g., LIF,
CT-1, CNTF, DIL-40, or other members of the IL-6 family, using a
competitive binding immunoassay such as the one described in Harlow
and Lane, supra, at pages 570-573. Preferably at least two
IL-6/IL-12 family members are used in this determination in
conjunction with the target. These long chain cytokine family
members can be produced as recombinant proteins and isolated using
standard molecular biology and protein chemistry techniques as
described herein. Thus, antibody preparations can be identified or
produced having desired selectivity or specificity for subsets of
IL-B60 family members. Alternatively, antibodies may be prepared
which bind to the complex comprising the IL-B60 with the CLF-1.
[0111] 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 protein of SEQ ID NO: 2. 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.
[0112] 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. 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, e.g., SEQ ID NO: 2 that is
required, then the second protein is said to specifically bind to
an antibody generated to the immunogen.
[0113] III. Physical Variants
[0114] This invention also encompasses proteins or peptides having
substantial amino acid sequence identity with the amino acid
sequence of the IL-B60 antigen. The variants include species,
polymorphic, or allelic variants.
[0115] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See also 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 IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
Sequence identity 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. The conservation may apply to biological
features, functional features, or structural features. Homologous
amino acid sequences are typically intended to include natural
polymorphic or allelic and interspecies variations of a protein
sequence. Typical homologous proteins or peptides will have from
25-100% identity (if gaps can be introduced), to 50-100% identity
(if conservative substitutions are included) with the amino acid
sequence of the IL-B60. Identity measures will be at least about
35%, generally at least about 40%, often at least about 50%,
typically at least about 60%, usually at least about 70%,
preferably at least about 80%, and more preferably at least about
90%.
[0116] The isolated IL-B60 DNA can be readily modified by
nucleotide substitutions, nucleotide deletions, nucleotide
insertions, and inversions of short nucleotide stretches. These
modifications result in novel DNA sequences which encode these
antigens, their derivatives, or proteins having similar
physiological, immunogenic, antigenic, or other functional
activity. These modified sequences can be used to produce mutant
antigens or to enhance expression. Enhanced expression may involve
gene amplification, increased transcription, increased translation,
and other mechanisms. "Mutant IL-B60" encompasses a polypeptide
otherwise falling within the sequence identity definition of the
IL-B60 as set forth above, but having an amino acid sequence which
differs from that of IL-B60 as normally found in nature, whether by
way of deletion, substitution, or insertion. This generally
includes proteins having significant identity with a protein having
sequence of SEQ ID NO: 2, and as sharing various biological
activities, e.g., antigenic or immunogenic, with those sequences,
and in preferred embodiments contain most of the natural full
length disclosed sequences. Full length sequences will typically be
preferred, though truncated versions will also be useful, likewise,
genes or proteins found from natural sources are typically most
desired. Similar concepts apply to different IL-B60 proteins,
particularly those found in various warm blooded animals, e.g.,
mammals and birds. These descriptions are generally meant to
encompass all IL-B60 proteins, not limited to the particular
primate embodiments specifically discussed.
[0117] IL-B60 mutagenesis can also be conducted by making amino
acid insertions or deletions. Substitutions, deletions, insertions,
or any 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 mutants can then be screened for the desired activity.
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 or polymerase chain reaction (PCR) techniques.
See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987 and
Supplements); and Kunkel, et al. (1987) Methods in Enzymol.
154:367-382. Preferred embodiments include, e.g., 1-fold, 2-fold,
3-fold, 5-fold, 7-fold, etc., preferably conservative substitutions
at the nucleotide or amino acid levels. Preferably the
substitutions will be away from the conserved cysteines, and often
will be in the regions away from the helical structural domains.
Such variants may be useful to produce specific antibodies, and
often will share many or all biological properties. See Table 2.
Recognition of the cytokine structure provides important insight
into the structure and positions of residues which may be modified
to effect desired changes in receptor interaction. Also, the
interaction of the IL-B60 with the CLF-1 protein requires
complementary structural features in the interacting surface.
[0118] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
A similar concept applies to heterologous nucleic acid
sequences.
[0119] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
target-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.
[0120] 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, e.g., PCR techniques.
[0121] Structural analysis can be applied to this gene, in
comparison to the IL-6 family of cytokines. The family includes,
e.g., IL-6, IL-11, IL-12, G-CSF, LIF, OSM, CNTF, and Ob. Alignment
of the human and mouse IL-B60 sequences with other members of the
IL-6 family should allow definition of structural features. In
particular, .beta.-sheet and .alpha.-helix residues can be
determined using, e.g., RASMOL program, see 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. See, also, Wilkins, et al.
(eds. 1997) Proteome Research: New Frontiers in Functional Genomics
Springer-Verlag, NY. Preferred residues for substitutions include
the surface exposed residues which would be predicted to interact
with receptor. Other residues which should conserve function will
be conservative substitutions, particularly at position far from
the surface exposed residues.
[0122] IV. Functional Variants
[0123] The blocking of physiological response to IL-B60s may result
from the competitive inhibition of binding of the ligand to its
receptor.
[0124] In vitro assays of the present invention will often use
isolated protein, soluble fragments comprising receptor binding
segments of these proteins, or fragments attached to solid phase
substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations
and modifications, or cytokine mutations and modifications, e.g.,
IL-B60 analogs.
[0125] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
cytokine, or receptor binding fragments compete with a test
compound.
[0126] "Derivatives" of IL-B60 antigens include amino acid sequence
mutants from naturally occurring forms, glycosylation variants, and
covalent or aggregate conjugates with other chemical moieties.
Covalent derivatives can be prepared by linkage of functionalities
to groups which are found in IL-B60 amino acid side chains or at
the N- or C-termini, e.g., by standard means. See, e.g., Lundblad
and Noyes (1988) Chemical Reagents for Protein Modification, vols.
1-2, CRC Press, Inc., Boca Raton, Fla.; Hugli (ed. 1989) Techniques
in Protein Chemistry, Academic Press, San Diego, Calif.; and Wong
(1991) Chemistry of Protein Conjugation and Cross Linking, CRC
Press, Boca Raton, Fla.
[0127] 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. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also
embraced are versions of the peptides with the same primary amino
acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0128] Fusion polypeptides between IL-B60s and other homologous or
heterologous proteins are also provided. Many cytokine receptors or
other surface proteins are multimeric, e.g., homodimeric entities,
and a repeat construct may have various advantages, including
lessened susceptibility to proteolytic cleavage. Typical examples
are fusions of a reporter polypeptide, e.g., luciferase, with a
segment or domain of a protein, e.g., a receptor-binding segment,
so that the presence or location of the fused ligand may be easily
determined. See, e.g., Dull, et al., U.S. Pat. No. 4,859,609. Other
gene fusion partners include bacterial .beta.-galactosidase, trpE,
Protein A, .beta.-lactamase, alpha amylase, alcohol dehydrogenase,
yeast alpha mating factor, and detection or purification tags such
as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al.
(1988) Science 241:812-816.
[0129] Fusion peptides 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, e.g., in Sambrook, et al. (1989) Molecular
Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring
Harbor Laboratory; and Ausubel, et al. (eds. 1993) Current
Protocols in Molecular Biology, Greene and Wiley, NY. Techniques
for synthesis of polypeptides are described, e.g., in Merrifield
(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science
232: 341-347; Atherton, et al. (1989) Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford; and Grant
(1992) Synthetic Peptides: A User's Guide, W. H. Freeman, NY.
Refolding methods may be applicable to synthetic proteins.
[0130] This invention also contemplates the use of derivatives of
IL-B60 proteins other than variations in amino acid sequence or
glycosylation. Such derivatives may involve covalent or aggregative
association with chemical moieties or protein carriers. Covalent or
aggregative derivatives will be useful as immunogens, as reagents
in immunoassays, or in purification methods such as for affinity
purification of binding partners, e.g., other antigens. An IL-B60
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 anti-IL-B60 antibodies or an alternative binding
composition. The IL-B60 proteins can also be labeled with a
detectable group, e.g., for use in diagnostic assays. Purification
of IL-B60 may be effected by an immobilized antibody or
complementary binding partner, e.g., binding portion of a
receptor.
[0131] A solubilized IL-B60 or fragment of this invention can be
used as an immunogen for the production of antisera or antibodies
specific for binding. Purified antigen can be used to screen
monoclonal antibodies or antigen-binding fragments, encompassing
antigen binding fragments of natural antibodies, e.g., Fab, Fab',
F(ab).sub.2, etc. Purified IL-B60 antigens can also be used as a
reagent to detect antibodies generated in response to the presence
of elevated levels of the cytokine, which may be diagnostic of an
abnormal or specific physiological or disease condition. This
invention contemplates antibodies raised against amino acid
sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, or
fragments of proteins containing it. In particular, this invention
contemplates antibodies having binding affinity to or being raised
against specific domains, e.g., helices A, B, C, or D of the
IL-B60, or the Ig domains of the CLF-1.
[0132] The present invention contemplates the isolation of
additional closely related species variants. Southern and Northern
blot analysis will establish that similar genetic entities exist in
other mammals. It is likely that IL-B60s are widespread in species
variants, e.g., rodents, lagomorphs, carnivores, artiodactyla,
perissodactyla, and primates.
[0133] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the molecules will be greatly accelerated
by the isolation and characterization of additional distinct
species or polymorphic variants of them. In particular, the present
invention provides useful probes for identifying additional
homologous genetic entities in different species.
[0134] The isolated genes will allow transformation of cells
lacking expression of an IL-B60, e.g., either species types or
cells which lack corresponding proteins and exhibit negative
background activity. This should allow analysis of the function of
IL-B60 in comparison to untransformed control cells.
[0135] Dissection of critical structural elements which effect the
various physiological functions mediated through these antigens is
possible using standard techniques of modern molecular biology,
particularly in comparing members of the related class. See, e.g.,
the homolog-scanning mutagenesis technique described in Cunningham,
et al. (1989) Science 243:1339-1336; and approaches used in O'Dowd,
et al. (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter, et
al. (1990) EMBO J. 9:4381-4390.
[0136] Intracellular functions would probably involve receptor
signaling. However, protein internalization may occur under certain
circumstances, and interaction between intracellular components and
cytokine may occur. Specific segments of interaction of IL-B60 with
interacting components may be identified by mutagenesis or direct
biochemical means, e.g., cross-linking or affinity methods.
Structural analysis by crystallographic or other physical methods
will also be applicable. Further investigation of the mechanism of
signal transduction will include study of associated components
which may be isolatable by affinity methods or by genetic means,
e.g., complementation analysis of mutants.
[0137] Further study of the expression and control of IL-B60 will
be pursued. The controlling elements associated with the antigens
should exhibit differential physiological, developmental, tissue
specific, or other expression patterns. Upstream or downstream
genetic regions, e.g., control elements, are of interest.
[0138] Structural studies of the IL-B60 antigens will lead to
design of new antigens, particularly analogs exhibiting agonist or
antagonist properties on the molecule. This can be combined with
previously described screening methods to isolate antigens
exhibiting desired spectra of activities.
[0139] V. Antibodies
[0140] Antibodies can be raised to various epitopes of the IL-B60
proteins, including species, polymorphic, or allelic variants, and
fragments thereof, both in their naturally occurring forms and in
their recombinant forms. Additionally, antibodies can be raised to
IL-B60s in either their active forms or in their inactive forms,
including native or denatured versions. Anti-idiotypic antibodies
are also contemplated.
[0141] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens 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 IL-B60s, or screened
for agonistic or antagonistic activity, e.g., mediated through a
receptor. Antibodies may be agonistic or antagonistic, e.g., by
sterically blocking binding to a receptor. 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.
[0142] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they can be screened for ability to bind to the antigens without
inhibiting binding to a receptor. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying IL-B60 protein or its receptors.
See, e.g., Chan (ed. 1987) Immunology: A Practical Guide, Academic
Press, Orlando, Fla.; Price and Newman (eds. 1991) Principles and
Practice of Immunoassay, Stockton Press, N.Y.; and Ngo (ed. 1988)
Nonisotopic Immunoassay, Plenum Press, N.Y. Cross absorptions or
other tests will identify antibodies which exhibit various spectra
of specificities, e.g., unique or shared species specificities.
[0143] Further, the antibodies, including antigen binding
fragments, of this invention can be potent antagonists that bind to
the antigen and inhibit functional binding, e.g., to a receptor
which may elicit a biological response. They also can be useful as
non-neutralizing antibodies and can be coupled to toxins or
radionuclides so that when the antibody binds to antigen, a cell
expressing it, e.g., on its surface, is killed. Further, these
antibodies can be conjugated to drugs or other therapeutic agents,
either directly or indirectly by means of a linker, and may effect
drug targeting.
[0144] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its 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; Williams, et al. (1967) Methods in
Immunology and Immunochemistry, vol. 1, Academic Press, New York;
and Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH
Press, NY, for descriptions of methods of preparing polyclonal
antisera.
[0145] 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.
[0146] 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. 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
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; Moore, et al., U.S. Pat. No. 4,642,334; and Queen, et
al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
[0147] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support. See,
e.g., Wilchek et al. (1984) Meth. Enzymol. 104:3-55. Conversely,
protein can be used for depletion or cross absorptions to prepare
selectively specific binding compositions.
[0148] Antibodies raised against each IL-B60 will also be useful to
raise anti-idiotypic antibodies. These will be useful in detecting
or diagnosing various immunological conditions related to
expression of the respective antigens.
[0149] VI. Nucleic Acids
[0150] The described peptide sequences and the related reagents are
useful in detecting, isolating, or identifying a DNA clone encoding
IL-B60, e.g., from a natural source. Typically, it will be useful
in isolating a gene from a mammal, and similar procedures will be
applied to isolate genes from other species, e.g., warm blooded
animals, such as birds and mammals. Cross hybridization will allow
isolation of IL-B60 from the same, e.g., polymorphic variants, or
other species. A number of different approaches will be available
to successfully isolate a suitable nucleic acid clone.
[0151] The purified protein or polypeptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold
Spring Harbor Press.
[0152] For example, a specific binding composition could be used
for screening of an expression library made from a cell line which
expresses an IL-B60. Screening of intracellular expression can be
performed by various staining or immunofluorescence procedures.
Binding compositions could be used to affinity purify or sort out
cells expressing a surface fusion protein.
[0153] The peptide segments can also be used to predict appropriate
oligonucleotides to screen a library. The genetic code can be used
to select appropriate oligonucleotides useful as probes for
screening. See, e.g., SEQ ID NO: 1. In combination with polymerase
chain reaction (PCR) techniques, synthetic oligonucleotides will be
useful in selecting correct clones from a library. Complementary
sequences will also be used as probes, primers, or antisense
strands. Various fragments should be particularly useful, e.g.,
coupled with anchored vector or poly-A complementary PCR techniques
or with complementary DNA of other peptides.
[0154] This invention contemplates use of isolated DNA or fragments
to encode a biologically active corresponding IL-B60 polypeptide,
particularly lacking the portion coding the untranslated portions
of the described sequence. In addition, this invention covers
isolated or recombinant DNA which encodes a biologically active
protein or polypeptide and which is capable of hybridizing under
appropriate conditions with the DNA sequences described herein.
Said biologically active protein or polypeptide can be an intact
antigen, or fragment, and have an amino acid sequence disclosed in,
e.g., SEQ ID NO: 2, particularly a mature, secreted polypeptide.
Further, this invention covers the use of isolated or recombinant
DNA, or fragments thereof, which encode proteins which exhibit high
identity to a secreted IL-B60. The isolated DNA can have the
respective regulatory sequences in the 5' and 3' flanks, e.g.,
promoters, enhancers, poly-A addition signals, and others.
Alternatively, expression may be effected by operably linking a
coding segment to a heterologous promoter, e.g., by inserting a
promoter upstream from an endogenous gene.
[0155] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and/or 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 and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. Generally, the nucleic acid will be
in a vector or fragment less than about 50 kb, usually less than
about 30 kb, typically less than about 10 kb, and preferably less
than about 6 kb.
[0156] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0157] A "recombinant" nucleic acid is 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 selection or
production. 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. Thus, e.g.,
products made by transforming cells with any unnaturally occurring
vector is encompassed, as are nucleic acids comprising sequence
derived using any synthetic oligonucleotide process. Such 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 sequence recognition site.
[0158] Alternatively, it 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. 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. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
or polymorphic variants.
[0159] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least about 22 nucleotides, ordinarily at least about 29
nucleotides, more often at least about 35 nucleotides, typically at
least about 41 nucleotides, usually at least about 47 nucleotides,
preferably at least about 55 nucleotides, and in particularly
preferred embodiments will be at least about 60 or more
nucleotides, e.g., 67, 73, 81, 89, 95, etc.
[0160] A DNA which codes for an IL-B60 protein will be particularly
useful to identify genes, mRNA, and cDNA species which code for
related or similar proteins, as well as DNAs which code for
homologous proteins from different species. There will be homologs
in other species, including primates, rodents, canines, felines,
and birds. Various IL-B60 proteins should be homologous and are
encompassed herein. However, even proteins that have a more distant
evolutionary relationship to the antigen can readily be isolated
under appropriate conditions using these sequences if they are
sufficiently homologous. Primate IL-B60 proteins are of particular
interest.
[0161] Recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp.
1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991)
Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson
(ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical
Oncology 10:180-199.
[0162] Substantial homology, e.g., 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 50% of the nucleotides, generally at
least about 58%, ordinarily at least about 65%, often at least
about 71%, typically at least about 77%, usually at least about
85%, preferably at least about 95 to 98% or more, and in particular
embodiments, as high as about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence of IL-B60, e.g., in SEQ
ID NO: 1. Typically, selective hybridization will occur when there
is at least about 55% identity over a stretch of at least about 30
nucleotides, preferably at least about 75% over a stretch of about
25 nucleotides, and most preferably at least about 90% over about
20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213.
The length of identity comparison, as described, may be over longer
stretches, and in certain embodiments will be over a stretch of at
least about 17 nucleotides, usually at least about 28 nucleotides,
typically at least about 40 nucleotides, and preferably at least
about 75 to 100 or more nucleotides.
[0163] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., usually in excess of about 37.degree. C.,
typically in excess of about 55.degree. C., preferably in excess of
about 70.degree. C. Stringent salt conditions will ordinarily be
less than about 1000 mM, usually less than about 400 mM, typically
less than about 250 mM, preferably less than about 150 mM,
including about 100, 50, or even 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. Hybridization under stringent conditions should give a
background of at least 2-fold over background, preferably at least
3-5 or more.
[0164] 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.
[0165] 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 Needleman 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).
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] IL-B60 from other mammalian species can be cloned and
isolated by cross-species hybridization of closely related species.
Homology may be relatively low between distantly related species,
and thus hybridization of relatively closely related species is
advisable. Alternatively, preparation of an antibody preparation
which exhibits less species specificity may be useful in expression
cloning approaches.
[0171] VII. Making IL-B60 or Complex; Mimetics
[0172] DNA which encodes the IL-B60 or fragments thereof can be
obtained by chemical synthesis, screening cDNA libraries, or
screening genomic libraries prepared from a wide variety of cell
lines or tissue samples. See, e.g., Okayama and Berg (1982) Mol.
Cell. Biol. 2:161-170; Gubler and Hoffman (1983) Gene 25:263-269;
and Glover (ed. 1984) DNA Cloning: A Practical Approach, IRL Press,
Oxford. Alternatively, the sequences provided herein provide useful
PCR primers or allow synthetic or other preparation of suitable
genes encoding an IL-B60; including naturally occurring
embodiments.
[0173] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length IL-B60 or fragments which can in
turn, e.g., be used to generate polyclonal or monoclonal
antibodies; for binding studies; for construction and expression of
modified molecules; and for structure/function studies.
[0174] 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. 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, Mass.
[0175] For purposes of this invention, DNA 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. See, e.g., Rodriguez, et al., Chapter 10,
pp. 205-236; Balbas and Bolivar (1990) Methods in Enzymology
185:14-37; and Ausubel, et al. (1993) Current Protocols in
Molecular Biology, Greene and Wiley, NY.
[0176] Representative examples of suitable expression vectors
include pCDNA1; pCD, see Okayama, et al. (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
See, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-199.
[0177] It will often be desired to express an IL-B60 polypeptide in
a system which provides a specific or defined glycosylation
pattern. See, e.g., Luckow and Summers (1988) Bio/Technology
6:47-55; and Kaufman (1990) Meth. Enzymol. 185:487-511.
[0178] The IL-B60, or a fragment thereof, may be engineered to be
phosphatidyl inositol (PI) linked to a cell membrane, but can be
removed from membranes by treatment with a phosphatidyl inositol
cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C. This
releases the antigen in a biologically active form, and allows
purification by standard procedures of protein chemistry. See,
e.g., Low (1989) Biochim. Biophys. Acta 988:427-454; Tse, et al.
(1985) Science 230:1003-1008; and Brunner, et al. (1991) J. Cell
Biol. 114:1275-1283.
[0179] Now that the IL-B60 has been characterized, 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; Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.
1991) Techniques in Protein Chemistry II, Academic Press, San
Diego, Calif.
[0180] VIII. Uses
[0181] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
IL-B60 mediated conditions, or below in the description of kits for
diagnosis. The gene may be useful in forensic sciences, e.g., to
distinguish rodent from human, or as a marker to distinguish
between different cells exhibiting differential expression or
modification patterns. The provided compositions are useful
reagents for, e.g., in vitro assays, scientific research, and the
synthesis or manufacture of nucleic acids, polypeptides, or
antibodies.
[0182] This invention also provides reagents with significant
commercial and/or therapeutic potential. The IL-B60 (naturally
occurring or recombinant), fragments thereof, and antibodies
thereto, along with compounds identified as having binding affinity
to IL-B60, should be useful as reagents for teaching techniques of
molecular biology, immunology, or physiology. Appropriate kits may
be prepared with the reagents, e.g., in practical laboratory
exercises in production or use of proteins, antibodies, cloning
methods, histology, etc.
[0183] The reagents will also be useful in the treatment of
conditions associated with abnormal physiology or development,
including inflammatory conditions. They may be useful in vitro
tests for presence or absence of interacting components, which may
correlate with success of particular treatment strategies. In
particular, modulation of physiology of various, e.g.,
hematopoietic or lymphoid, cells will be achieved by appropriate
methods for treatment using the compositions provided herein. See,
e.g., Thomson (1994; ed.) The Cytokine Handbook (2d ed.) Academic
Press, San Diego; Metcalf and Nicola (1995) The Hematopoietic
Colony Stimulating Factors Cambridge University Press; and Aggarwal
and Gutterman (1991) Human Cytokines Blackwell Pub.
[0184] For example, a disease or disorder associated with abnormal
expression or abnormal signaling by an IL-B60 should be a likely
target for an agonist or antagonist. The new cytokine should play a
role in regulation or development of hematopoietic cells, e.g.,
lymphoid cells, which affect immunological responses, e.g.,
inflammation and/or autoimmune disorders. Alternatively, it may
affect vascular physiology or development, or neuronal effects.
[0185] In particular, the cytokine should mediate, in various
contexts, cytokine synthesis by the cells, proliferation, etc.
Antagonists of IL-B60, such as mutein variants of a naturally
occurring form of IL-B60 or blocking antibodies, may provide a
selective and powerful way to block immune responses, e.g., in
situations as inflammatory or autoimmune responses. See also
Samter, et al. (eds.) Immunological Diseases vols. 1 and 2, Little,
Brown and Co.
[0186] In addition, certain combination compositions would be
useful, e.g., with other modulators of inflammation. Such other
molecules may include steroids, other versions of IL-6 and/or
G-CSF, including species variants, or viral homologs, and their
respective antagonists.
[0187] Various abnormal conditions are known in each of the cell
types shown to produce IL-B60 mRNA by Northern blot analysis. See
Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &
Co., Rahway, N.J.; Thorn, et al. Harrison's Principles of Internal
Medicine, McGraw-Hill, N.Y.; and Weatherall, et al. (eds.) Oxford
Textbook of Medicine, Oxford University Press, Oxford. Many other
medical conditions and diseases involve activation by macrophages
or monocytes, and many of these will be responsive to treatment by
an agonist or antagonist provided herein. See, e.g., Stites and
Terr (eds.; 1991) Basic and Clinical Immunology Appleton and Lange,
Norwalk, Conn.; and Samter, et al. (eds.) Immunological Diseases
Little, Brown and Co. These problems should be susceptible to
prevention or treatment using compositions provided herein. The
pancreatic islet localization suggests a possible relevance to
diabetes.
[0188] IL-B60, antagonists, antibodies, etc., can be purified and
then administered to a patient, veterinary or human. These reagents
can be combined for therapeutic use with additional active or inert
ingredients, e.g., in conventional pharmaceutically acceptable
carriers or diluents, e.g., immunogenic adjuvants, along with
physiologically innocuous stabilizers, excipients, or
preservatives. These combinations can be sterile 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,
including forms which are not complement binding.
[0189] Drug screening using IL-B60 or fragments thereof can be
performed to identify compounds having binding affinity to or other
relevant biological effects on IL-B60 functions, 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 cytokine. Likewise, a compound
having intrinsic stimulating activity can activate the signal
pathway and is thus an agonist in that it simulates the activity of
IL-B60. This invention further contemplates the therapeutic use of
blocking antibodies to IL-B60 as antagonists and of stimulatory
antibodies as agonists. This approach should be particularly useful
with other IL-B60 species variants.
[0190] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, 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. 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. 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 a slow release apparatus will often be utilized
for continuous or long term administration. See, e.g., Langer
(1990) Science 249:1527-1533.
[0191] IL-B60, fragments thereof, and antibodies to it 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 typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
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, topical, or parenteral (including
subcutaneous, intramuscular, intravenous and intradermal)
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any 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, New
York; Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms:
Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)
Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.
The therapy of this invention may be combined with or used in
association with other agents, e.g., other cytokines, including
IL-6 or G-CSF, or their respective antagonists.
[0192] Both naturally occurring and recombinant forms of the
IL-B60s of this invention are particularly useful in kits and assay
methods which are capable of screening compounds for binding
activity to the proteins. Several methods of automating assays have
been developed in recent years so as to permit screening of tens of
thousands of compounds in a short period. See, e.g., Fodor, et al.
(1991) Science 251:767-773, which describes means for testing of
binding affinity by a plurality of defined polymers synthesized on
a solid substrate. The development of suitable assays can be
greatly facilitated by the availability of large amounts of
purified, soluble IL-B60 as provided by this invention.
[0193] Other methods can be used to determine the critical residues
in IL-B60-IL-B60 receptor interactions. Mutational analysis can be
performed, e.g., see Somoza, et al. (1993) J. Exptl. Med.
178:549-558, to determine specific residues critical in the
interaction and/or signaling. PHD (Rost and Sander (1994) Proteins
19:55-72) and DSC (King and Sternberg (1996) Protein Sci.
5:2298-2310) can provide secondary structure predictions of
.alpha.-helix (H), .beta.-strand (E), or coil (L). Helices A and D
are most important in receptor interaction, with the D helix the
more important region. See Table 2.
[0194] For example, antagonists can normally be found once the
antigen has been structurally defined, e.g., by tertiary structure
data. Testing of potential interacting analogs is now possible upon
the development of highly automated assay methods using a purified
IL-B60. In particular, new agonists and antagonists will be
discovered by using screening techniques described herein. Of
particular importance are compounds found to have a combined
binding affinity for a spectrum of IL-B60 molecules, e.g.,
compounds which can serve as antagonists for species variants of
IL-B60.
[0195] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing an IL-B60. Cells may be
isolated which express an IL-B60 in isolation from other molecules.
Such cells, either in viable or fixed form, can be used for
standard binding partner 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.
[0196] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to an IL-B60 and is described in detail
in Geysen, European Patent Application 84/03564, published on Sep.
13, 1984. First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g., plastic pins
or some other appropriate surface, see Fodor, et al. (1991). Then
all the pins are reacted with solubilized, unpurified or
solubilized, purified IL-B60, and washed. The next step involves
detecting bound IL-B60.
[0197] Rational drug design may also be based upon structural
studies of the molecular shapes of the IL-B60 and other effectors
or analogs. Effectors may be other proteins which mediate other
functions in response to binding, or other proteins which normally
interact with IL-B60, e.g., a 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, as modeled,
e.g., against other cytokine-receptor models. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson (1976) Protein Crystallography, Academic
Press, New York.
[0198] IX. Kits
[0199] This invention also contemplates use of IL-B60 proteins,
fragments thereof, peptides, and their fusion products in a variety
of diagnostic kits and methods for detecting the presence of
another IL-B60 or binding partner. Typically the kit will have a
compartment containing either a defined IL-B60 peptide or gene
segment or a reagent which recognizes one or the other, e.g.,
IL-B60 fragments or antibodies.
[0200] A kit for determining the binding affinity of a test
compound to an IL-B60 would typically comprise a test compound; a
labeled compound, for example a binding partner or antibody having
known binding affinity for IL-B60; a source of IL-B60 (naturally
occurring or recombinant); and a means for separating bound from
free labeled compound, such as a solid phase for immobilizing the
molecule. Once compounds are screened, those having suitable
binding affinity to the antigen can be evaluated in suitable
biological assays, as are well known in the art, to determine
whether they act as agonists or antagonists to the IL-B60 signaling
pathway. The availability of recombinant IL-B60 polypeptides also
provide well defined standards for calibrating such assays.
[0201] A preferred kit for determining the concentration of, e.g.,
an IL-B60 in a sample would typically comprise a labeled compound,
e.g., binding partner or antibody, having known binding affinity
for the antigen, a source of cytokine (naturally occurring or
recombinant) and a means for separating the bound from free labeled
compound, e.g., a solid phase for immobilizing the IL-B60.
Compartments containing reagents, and instructions, will normally
be provided.
[0202] Antibodies, including antigen binding fragments, specific
for the IL-B60 or fragments are useful in diagnostic applications
to detect the presence of elevated levels of IL-B60 and/or its
fragments. Such diagnostic assays can employ lysates, live cells,
fixed cells, immunofluorescence, cell cultures, body fluids, and
further can involve the detection of antigens related to the
antigen in serum, or the like. Diagnostic assays may be homogeneous
(without a separation step between free reagent and antigen-binding
partner 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.
See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525;
Harlow and Lane (1980) Antibodies: A Laboratory Manual, CSH Press,
N.Y.; and Coligan, et al. (eds. 1993) Current Protocols in
Immunology, Greene and Wiley, N.Y.
[0203] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against an IL-B60, as such may be diagnostic
of various abnormal states. For example, overproduction of IL-B60
may result in production of various immunological reactions which
may be diagnostic of abnormal physiological states, particularly in
proliferative cell conditions such as cancer or abnormal activation
or differentiation. Moreover, the distribution pattern available
provides information that the cytokine is expressed in pancreatic
islets, suggesting the possibility that the cytokine may be
involved in function of that organ, e.g., in a diabetes relevant
medical condition.
[0204] 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
binding partner, or labeled IL-B60 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.
Desirably, the reagents are provided as a dry lyophilized powder,
where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay.
[0205] Many of the aforementioned constituents of the drug
screening and 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 any of these assays, the binding partner, test compound, IL-B60,
or antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels
such as .sup.125I, enzymes 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. Possibilities for
indirect labeling include biotinylation of one constituent followed
by binding to avidin coupled to one of the above label groups.
[0206] There are also numerous methods of separating the bound from
the free IL-B60, or alternatively the bound from the free test
compound. The IL-B60 can be immobilized on various matrixes
followed by washing. Suitable matrixes include plastic such as an
ELISA plate, filters, and beads. See, e.g., Coligan, et al. (eds.
1993) Current Protocols in Immunology, Vol. 1, Chapter 2, Greene
and Wiley, NY. Other suitable separation techniques include,
without limitation, the fluorescein antibody magnetizable particle
method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461,
and the double antibody magnetic particle separation as described
in U.S. Pat. No. 4,659,678.
[0207] Methods for linking proteins or their fragments to the
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.
[0208] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an IL-B60. These sequences can be used as probes for detecting
levels of the IL-B60 message in samples from patients suspected of
having an abnormal condition, e.g., inflammatory or autoimmune.
Since the cytokine may be a marker or mediator for activation, it
may be useful to determine the numbers of activated cells to
determine, e.g., when additional therapy may be called for, e.g.,
in a preventative fashion before the effects become and progress to
significance. 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. See, e.g., Langer-Safer, et al. (1982) Proc. Nat'l.
Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967; and
Wilchek et al. (1988) Anal. Biochem. 171:1-32.
[0209] Diagnostic kits which also test for the qualitative or
quantitative expression of other molecules 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. Other kits may be used to evaluate other cell
subsets.
[0210] X. Isolating a IL-B60 Receptor
[0211] Having isolated a ligand of a specific ligand-receptor
interaction, methods exist for isolating the receptor. See,
Gearing, et al. (1989) EMBO J. 8:3667-3676. For example, means to
label the IL-B60 cytokine without interfering with the binding to
its receptor can be determined. For example, an affinity label can
be fused to either the amino- or carboxyl-terminus of the ligand.
Such label may be a FLAG epitope tag, or, e.g., an Ig or Fc domain.
An expression library can be screened for specific binding of the
cytokine, e.g., by cell sorting, or other screening to detect
subpopulations which express such a binding component. See, e.g.,
Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271; and
Liu, et al. (1994) J. Immunol. 152:1821-29. Alternatively, a
panning method may be used. See, e.g., Seed and Aruffo (1987) Proc.
Nat'l Acad. Sci. USA 84:3365-3369.
[0212] Protein cross-linking techniques with label can be applied
to isolate binding partners of the IL-B60 cytokine. This would
allow identification of proteins which specifically interact with
the cytokine, e.g., in a ligand-receptor like manner.
[0213] Early experiments will be performed to determine whether the
known IL-6 or G-CSF receptor components are involved in response(s)
to IL-B60. It is also quite possible that these functional receptor
complexes may share many or all components with an IL-B60 receptor
complex, either a specific receptor subunit or an accessory
receptor subunit.
[0214] 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 only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
EXAMPLES
[0215] I. General Methods
[0216] Many of the standard methods below are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, NY; 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.; Ausubel, et al. (1987
and Supplements) Current Protocols in Molecular Biology
Wiley/Greene, NY; Innis, et al. (eds. 1990) PCR Protocols: A Guide
to Methods and Applications Academic Press, NY; Bonifacino, et al.
Current Protocols in Cell Biology Wiley, NY; and Doyle, et al. Cell
and Tissue Culture: Laboratory Protocols Wiley, NY. 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); Deutscher (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan, et al. (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New York,
N.Y.; Matsudaira (ed. 1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif.; 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 (epitope tags), e.g., to a
FLAG sequence or an equivalent which can be fused, e.g., via a
protease-removable sequence. See, e.g., Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate Absorbent"
in Setlow (ed.) Genetic Engineering, Principle and Methods
12:87-98, Plenum Press, NY; and Crowe, et al. (1992) QIAexpress:
The High Level Expression & Protein Purification System
QUIAGEN, Inc., Chatsworth, Calif.
[0217] Computer sequence analysis is performed, e.g., using
available software programs, including those from the University of
Wisconsin Genetics Computer Group (GCG), Madison, Wis., the NCBI at
NIH, and GenBank, NCBI, EMBO, and other sources of public sequence.
Other analysis sources include, e.g., RASMOL program, see 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; and DSC,
see King and Sternberg (1996) Protein Sci. 5:2298-2310. See, also,
Wilkins, et al. (eds. 1997) Proteome Research: New Frontiers in
Functional Genomics Springer-Verlag, NY; Salzberg, et al. (eds.
1998) Computational Methods in Molecular Biology Elsevier, N.Y.;
and Birren, et al. (eds. 1997) Genome Analysis: A Laboratory Manual
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
[0218] Standard immunological techniques are described, e.g., in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan (1991 and updates)
Current Protocols in Immunology Wiley/Greene, NY; and Methods in
Enzymology vols. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150,
162, and 163. Cytokine assays are described, e.g., in Thomson (ed.
1994) The Cytokine Handbook (2d ed.) Academic Press, San Diego;
Metcalf and Nicola (1995) The Hematopoietic Colony Stimulating
Factors Cambridge University Press; and Aggarwal and Gutterman
(1991) Human Cytokines Blackwell Pub.
[0219] Assays for vascular biological activities are well known in
the art. They will cover angiogenic and angiostatic activities in
tumor, or other tissues, e.g., arterial smooth muscle proliferation
(see, e.g., Koyoma, et al. (1996) Cell 87:1069-1078), monocyte
adhesion to vascular epithelium (see McEvoy, et al. (1997) J. Exp.
Med. 185:2069-2077), etc. See also Ross (1993) Nature 362:801-809;
Rekhter and Gordon (1995) Am. J. Pathol. 147:668-677; Thyberg, et
al. (1990) Atherosclerosis 10:966-990; and Gumbiner (1996) Cell
84:345-357.
[0220] Assays for neural cell biological activities are described,
e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neurosciences Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of developmental systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Biology CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
[0221] FACS analyses are described in Melamed, et al. (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson,
et al. (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
[0222] II. Cloning of Human IL-B60
[0223] The sequence of the gene is provided in Table 1. The
sequence is derived from a genomic human sequence. These sequences
allow preparation of PCR primers, or probes, to determine cellular
distribution of the gene. The sequences allow isolation of genomic
DNA which encode the message.
[0224] Using the probe or PCR primers, various tissues or cell
types are probed to determine cellular distribution. PCR products
are cloned using, e.g., a TA cloning kit (Invitrogen). The
resulting cDNA plasmids are sequenced from both termini on an
automated sequencer (Applied Biosystems).
[0225] III. Cellular Expression of IL-B60
[0226] An appropriate probe or primers specific for cDNA encoding
primate IL-B60 are prepared. Typically, the probe is labeled, e.g.,
by random priming. The expression is probably in the cell types
described, and perhaps also in pancreatic islets.
[0227] The presence of a leader sequence led to the expectation of
finding IL-B60 secreted when expressed in mammalian cells.
Transfection of 293T cells with a tagged form of hIL-B60
(hIL-B60-Etag) did not result in efficient secretion of IL-B60.
Instead, IL-B60 could only be immunoprecipitated from the lysate of
the transfected cells. The possibility was investigated of IL-B60
being a composite factor like IL-12 (p35/p40) and thus needing a
partner for secretion. Among the non-signaling receptors of the
IL-6 family the recently described and, thus far, orphan, receptor
CLF-1 (NR6) also showed a high level of homology between human and
murine forms (>95% amino acid identity). Based on these
observations a hypothesis was generated that the IL-B60 and CLF-1
are partners.
[0228] Southern Analysis: DNA (5 .mu.g) from a primary amplified
cDNA library is 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.).
[0229] Samples for human mRNA isolation can include, e.g.,
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 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);
CD28-T cell clone; 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);
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); epithelial cells, unstimulated; epithelial cells,
IL-1.beta. activated; 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). Expression of IL-B60 transcript was very high in
elutriated monocytes, activated with LPS, IFN.gamma., anti-IL-10
for 4, 16 h pooled (M106); elutriated monocytes, activated with
LPS, IFN.gamma., anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102);
elutriated monocytes, activated LPS for 6 h (M109); and elutriated
monocytes, activated LPS for 1 h (M108).
[0230] Samples for mouse mRNA expression can include, e.g., resting
mouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to
estrogen receptor) transfected cells, control (C201); Mell4+ naive
T cells from spleen, resting (T209); Mell4+ naive T cells from
spleen, stimulated with IFN.gamma., IL-12, and anti IL-4 to
polarize to TH1 cells, exposed to IFN.gamma. and IL-4 for 6, 12, 24
h, pooled (T210); Mell4+ naive T cells from spleen, stimulated with
IL-4 and anti IFN.gamma. to polarize to Th2 cells, exposed to IL-4
and anti IFN.gamma. for 6, 13, 24 h, pooled (T211); T cells, TH1
polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7
days with IFN-.gamma. and anti IL-4; T200); T cells, TH2 polarized
(Mell4 bright, CD4+ cells from spleen, polarized for 7 days with
IL-4 and anti-IFN-.gamma.; T201); T cells, highly TH1 polarized
3.times. from transgenic Balb/C (see Openshaw, et al. (1995) J.
Exp. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 24 h
pooled; T202); T cells, highly TH2 polarized 3.times. from
transgenic Balb/C (activated with anti-CD3 for 2, 6, 24 h pooled
(T203); T cells, highly TH1 polarized 3.times. from transgenic C57
bl/6 (activated with anti-CD3 for 2, 6, 24 h pooled; T212); T
cells, highly TH2 polarized 3.times. from transgenic C57 bl/6
(activated with anti-CD3 for 2, 6, 24 h pooled; T213); T cells,
highly TH1 polarized (naive CD4+ T-cells from transgenic Balb/C,
polarized 3.times. with IFN.gamma., IL-12, and anti-IL-4;
stimulated with IGIF, IL-12, and anti IL-4 for 6, 12, 24 h,
pooled); 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); unstimulated B cell line CH12
(B201); unstimulated mature B cell leukemia cell line A20 (B200);
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); unstimulated bone marrow derived dendritic cells
depleted with anti B220, anti CD3, and anti Class II, cultured in
GM-CSF and IL-4 (D202); bone marrow derived dendritic cells
depleted with anti B220, anti CD3, and anti Class II, cultured in
GM-CSF and IL-4, stimulated with anti CD40 for 1, 5 d, pooled
(D203); monocyte cell line RAW 264.7 activated with LPS 4 h (M200);
bone-marrow macrophages derived with GM and M-CSF (M201);
bone-marrow macrophages derived with GM-CSF, stimulated with LPS,
IFN.gamma., and IL-10 for 24 h (M205); bone-marrow macrophages
derived with GM-CSF, stimulated with LPS, IFN.gamma., and anti
IL-10 for 24 h (M206); peritoneal macrophages (M207); 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);
unstimulated mast cell lines MC-9 and MCP-12 (M208); immortalized
endothelial cell line derived from brain microvascular endothelial
cells, unstimulated (E200); immortalized endothelial cell line
derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. (E201); immortalized endothelial cell
line derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. (E202); immortalized endothelial cell
line derived from brain microvascular endothelial cells, stimulated
overnight with TNF.alpha. and IL-10 (E203); total aorta from wt C57
bl/6 mouse; total aorta from 5 month ApoE KO mouse (X207); total
aorta from 12 month ApoE KO mouse (X207); wt thymus (O214); total
thymus, rag-1 (O208); total kidney, rag-1 (O209); total kidney, NZ
B/W mouse; and total heart, rag-1 (O202).
[0231] The human IL-B60 was found expressed in T cells; the Th0
clone Mot72 (activated); activated PBL; monocytes; dendritic cells;
fetal lung, and heavy smoker lung samples.
[0232] The CLF-1 was found expressed in dendritic cells;
splenocytes; Th1 cells; fetal lung; and lung samples. This
distribution is consistent with the complex being important in
immune function, e.g., dendritic and immune cells, and in lung
physiology.
[0233] Since CLF-1 is necessary for IL-B60 secretion in vitro,
various human and mouse cDNA libraries were screened for
co-expression of both mRNAs. Highest expression for both was found
in adult human splenocytes, T cells, activated monocytes and
dendritic cells and in fetal lung, and uterus. In mouse libraries,
co-expression was strongest in adult lung.
[0234] IV. Chromosome Mapping of IL-B60
[0235] An isolated cDNA encoding the IL-B60 is used. Chromosome
mapping is a standard technique. See, e.g., BIOS Laboratories (New
Haven, Conn.) and methods for using a mouse somatic cell hybrid
panel with PCR.
[0236] The human IL-B60 gene has been localized to human chromosome
11.
[0237] V. Purification of IL-B60 Protein or Complexes
[0238] Multiple transfected cell lines are screened for one which
expresses the cytokine at a high level compared with other cells.
Alternatively, a recombinant construct with both subunits can be
made. Various cell lines are screened and selected for their
favorable properties in handling. Natural IL-B60 can be isolated
from natural sources, or by expression from a transformed cell
using an appropriate expression vector. Purification of the
expressed protein or complex is achieved by standard procedures, or
may be combined with engineered means for effective purification at
high efficiency from cell lysates or supernatants. FLAG or
His.sub.6 segments can be used for such purification features.
Alternatively, affinity chromatography may be used with specific
antibodies, see below.
[0239] Protein is produced in coli, insect cell, or mammalian
expression systems, as desired.
[0240] VI. Isolation of Homologous IL-B60 Genes
[0241] The IL-B60 cDNA, or other species counterpart sequence, can
be used as a hybridization probe to screen a library from a desired
source, e.g., a primate cell cDNA library. Many different species
can be screened both for stringency necessary for easy
hybridization, and for presence using a probe. Appropriate
hybridization conditions will be used to select for clones
exhibiting specificity of cross hybridization.
[0242] Screening by hybridization using degenerate probes based
upon the peptide sequences will also allow isolation of appropriate
clones. Alternatively, use of appropriate primers for PCR screening
will yield enrichment of appropriate nucleic acid clones.
[0243] Similar methods are applicable to isolate either species,
polymorphic, or allelic variants. Species variants are isolated
using cross-species hybridization techniques based upon isolation
of a full length isolate or fragment from one species as a
probe.
[0244] Alternatively, antibodies raised against human IL-B60 will
be used to screen for cells which express cross-reactive proteins
from an appropriate, e.g., cDNA library. The purified protein or
defined peptides are useful for generating antibodies by standard
methods, as described above. Synthetic peptides or purified protein
are presented to an immune system to generate monoclonal or
polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols
in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies:
A Laboratory Manual Cold Spring Harbor Press. The resulting
antibodies are used for screening, purification, or diagnosis, as
described.
[0245] VII. Antibodies Specific for IL-B60 or Complexes
[0246] Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Polyclonal serum, or hybridomas may be
prepared. 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. Immunoselection,
immunodepletion, and related techniques are available to prepare
selective reagents, as desired, e.g., for the IL-B60 alone, or the
complex between the two subunits.
[0247] VIII. IL-B60 and CLF-1 Coprecipitate
[0248] A CLF-1-FLAG construct was prepared in an expression vector.
An IL-B60Etag (epitope tagged) construct was also prepared.
Transient transfection into COS cells either with the IL-B60Etag
construct alone, the CLF-1-FLAG construct alone, or both together.
Cells were labeled with .sup.35S methionine. The supernatants and
cells were collected.
[0249] Upon co-transfection of cells with IL-B60-Etag and soluble
receptor CLF-1-Flag, secretion of both ligand and soluble receptor
was greatly enhanced. Both could be immunoprecipitated with
antibodies against either the ligand (anti Etag) or the receptor
(anti Flag), indicating that IL-B60 and CLF-1 form a soluble
cytokine/receptor complex similar to IL-12 (p35/p40). See Gubler,
et al. (1991) Proc. Nat'l Acad. Sci. USA 88:4143-4147; Wolf, et al.
(1991) J. Immunol. 146:3074-3081. Thus, coexpression with a correct
partner will result in a dramatic increase in the secretion of the
gene products. Coexpression of IL-B60 with other soluble receptors
including Ebi3 (Devergne, et al. (1996) J. Virol. 70:1143-1153),
IL-12 p40, and sCNTFR (Davis, et al. (1991) Science 253.:59-63) did
not result in efficient secretion of the ligand.
[0250] The supernatants were immunoprecipitated with either
anti-FLAG M2 (precipitates CLF-1) or anti-Etag Ab (precipitates
IL-B60). In IL-B60Etag transfectants alone, the level of expression
in the supernatant detected using the antiEtag antibodies was very
low. In contrast, in the double transfectants, the IL-B60Etag and a
second labeled band were immunoprecipitated. The second band
corresponds to the CLF-1. Thus, the Etag antibody
immunoprecipitates both proteins, e.g., they form a complex. In the
single transfectant CLF-1FLAG, a little bit of CLF-1FLAG protein is
immunoprecipitated with the anti-FLAG M2 Ab. This result is
consistent with the other soluble receptors, e.g., for p40
component of IL-12. However, in the double transfectants not only
is more CLF-1 seen, but now also IL-B60. The immunoprecipitation
works in both directions.
[0251] IX. IL-B60 Binds to the CNTFR
[0252] To identify the signaling receptors for IL-B60/CLF-1
conditioned medium from hIL-B60 and mCLF-1 cotransfected 293T cells
was added to BA/F3 cells stably transfected with human gp130 alone
or hgp130 in combination with the hIL-6R, hOSMR, hLIFR, or hLIFR
and hCNTFR, respectively. Only BA/F3 cells expressing gp130, LIFR,
and CNTFR showed a proliferative response upon stimulation with
IL-B60/CLF-1. To analyze the possibility of a signaling complex
consisting of CNTFR/gp130 or CNTFR/LIFR only, two soluble fusion
proteins were designed connecting either the CNTFR or CLF-1 to
IL-B60 via a flexible linker. Similar so-called hyper-cytokines
have been shown to be 100-1000.times. more active on cells than
cytokine and soluble receptor added separately. See Fischer, et al.
(1997) Nature Biotechnol. 15:142-145. Hyper-CNTFR-IL-B60 was able
to induce proliferation of BAF3/gp130/LIFR cells but not of
BAF3/gp130 cells, showing that the LIFR is a component of the
signaling complex. Stimulation of cells with hyper-CLF-1-IL-B60 did
not result in proliferation of any cell line. This indicated that
although necessary for IL-B60 secretion, CLF-1 is not a subunit of
the active signaling receptor complex.
[0253] Involvement of gp130 in the active receptor complex was
shown with a neutralizing antibody against gp130 which completely
blocked this response. Furthermore, analysis of signal transducers
in lysates from BA/F3 cells expressing gp130, LIFR, and CNTFR
showed that STAT3 is only phosphorylated after stimulation with
either co-expressed IL-B60 and CLF-1 or with the CNTFR-IL-B60
fusion protein but not with the CLF-1-IL-B60 fusion.
[0254] X. Evaluation of Breadth of Biological Functions
[0255] Biological activities of IL-B60 or complex are tested based,
e.g., on the sequence and structural homology between IL-B60 and
IL-6 and G-CSF. Initially, assays that had shown biological
activities of IL-6 or G-CSF are examined.
[0256] A. Regulation of IL-B60 and CLF-1 After Sciatic Nerve
Injury
[0257] IL-B60 and CLF-1 expression in the mouse spinal cord was
analyzed in unilateral transection of the sciatic nerve followed by
separation of proximal and distal nerve stumps, thus preventing
regeneration. At various time points, tissue from the transection
area was collected and analyzed by quantitative PCR for expression
of IL-B60 and CLF-1. Transection of the sciatic nerve resulted in
fast and long lasting upregulation of ligand and receptor. After 6
hrs IL-B60 and CLF-1 were upregulated. Expression was still
elevated 20 days after transection when compared to non-lesioned or
sham-lesioned nerves. In regenerating axons (crushed nerves) both
IL-B60 and CLF-1 are downregulated after 12 h, but whereas IL-B60
expression almost reaches levels of non-lesioned nerves after 20
days, CLF-1 levels peak after 20 days. This might point to an
additional function of CLF-1, possibly in remyelination, which
starts after two weeks. Transection of the sciatic nerve in mice
lacking GM-CSF and a macrophage response in nerve shows that IL-B60
expression after 4 days is not altered compared to normal mice.
However, CLF-1 levels in those mice are heterogeneous, with a range
from no alteration to an almost 4 fold increase of expression
compared to normal littermates.
[0258] B. Effects on Proliferation of Cells
[0259] The effect on proliferation of various cell types are
evaluated with various concentrations of cytokine. A dose response
analysis is performed, in combinations with the related cytokines
IL-6, G-CSF, etc. A cytosensor machine may be used, which detects
cell metabolism and growth (Molecular Devices, Sunnyvale,
Calif.).
[0260] C. Effects on the Expression of Cell Surface Molecules on
Human Monocytes
[0261] Monocytes are purified by negative selection from peripheral
blood mononuclear cells of normal healthy donors. Briefly,
3.times.10.sup.8 ficoll banded mononuclear cells are incubated on
ice with a cocktail of monoclonal antibodies (Becton-Dickinson;
Mountain View, Calif.) consisting, e.g., of 200 .mu.l of .alpha.CD2
(Leu-5A), 200 .mu.l of .alpha.CD3 (Leu-4), 100 .mu.l of .alpha.CD8
(Leu 2a), 100 .mu.l of .alpha.CD19 (Leu-12), 100 .mu.l of
.alpha.CD20 (Leu-16), 100 .mu.l of .alpha.CD56 (Leu-19), 100 .mu.l
of .alpha.CD67 (IOM 67; Immunotech, Westbrook, Me.), and
anti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody
bound cells are washed and then incubated with sheep anti-mouse IgG
coupled magnetic beads (Dynal, Oslo, Norway) at a bead to cell
ratio of 20:1. Antibody bound cells are separated from monocytes by
application of a magnetic field. Subsequently, human monocytes are
cultured in Yssel's medium (Gemini Bioproducts, Calabasas, Calif.)
containing 1% human AB serum in the absence or presence of IL-B60,
IL-6, G-CSF or combinations.
[0262] Analyses of the expression of cell surface molecules can be
performed by direct immunofluorescence. For example,
2.times.10.sup.5 purified human monocytes are incubated in
phosphate buffered saline (PBS) containing 1% human serum on ice
for 20 minutes. Cells are pelleted at 200.times.g. Cells are
resuspended in 20 ml PE or FITC labeled mAb. Following an
additional 20 minute incubation on ice, cells are washed in PBS
containing 1% human serum followed by two washes in PBS alone.
Cells are fixed in PBS containing 1% paraformaldehyde and analyzed
on FACScan flow cytometer (Becton Dickinson; Mountain View,
Calif.). Exemplary mAbs are used, e.g.: CD11b (anti-mac1), CD11c (a
gp150/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BB1/B7), HLA-DR
(L243) from Becton-Dickinson and CD86 (FUN 1; Pharmingen), CD64
(32.2; Medarex), CD40 (mAb89; Schering-Plough France).
[0263] D. Effects of IL-B60 or Complex on Cytokine Production by
Human Monocytes
[0264] Human monocytes are isolated as described and cultured in
Yssel's medium (Gemini Bioproducts, Calabasas, Calif.) containing
1% human AB serum in the absence or presence of IL-B60 ({fraction
(1/100)} dilution baculovirus expressed material). In addition,
monocytes are stimulated with LPS (E. coli 0127:B8 Difco) in the
absence or presence of IL-B60 and the concentration of cytokines
(IL-1.beta., IL-6, TNF.alpha., GM-CSF, and IL-10) in the cell
culture supernatant determined by ELISA.
[0265] For intracytoplasmic staining for cytokines, monocytes are
cultured (1 million/ml) in Yssel's medium in the absence or
presence of IL-B60 and LPS (E. coli 0127:B8 Difco) and 10 mg/ml
Brefeldin A (Epicentre technologies Madison Wis.) for 12 hrs. Cells
are washed in PBS and incubated in 2% formaldehyde/PBS solution for
20 minutes at RT. Subsequently cells are washed, resuspended in
permeabilization buffer (0.5% saponin (Sigma) in PBS/BSA
(0.5%)/Azide (1 mM)) and incubated for 20 minutes at RT. Cells
(2.times.10.sup.5) are centrifuged and resuspended in 20 ml
directly conjugated anti-cytokine mAbs diluted 1:10 in
permeabilization buffer for 20 minutes at RT. The following
antibodies can be used: IL-1.alpha.-PE (364-3B3-14); IL-6-PE
(MQ2-13A5); TNF.alpha.-PE (MAb11); GM-CSF-PE (BVD2-21C11); and
IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.). Subsequently,
cells are washed twice in permeabilization buffer and once in
PBS/BSA/Azide and analyzed on FACScan flow cytometer (Becton
Dickinson; Mountain View, Calif.).
[0266] E. Effects of IL-B60 on Proliferation of Human Peripheral
Blood Mononuclear Cells (PBMC)
[0267] Total PBMC are isolated from buffy coats of normal healthy
donors by centrifugation through ficoll-hypaque as described
(Boyum, et al.). PBMC are cultured in 200 .mu.l Yssel's medium
(Gemini Bioproducts, Calabasas, Calif.) containing 1% human AB
serum in 96 well plates (Falcon, Becton-Dickinson, NJ) in the
absence or presence of IL-B60. Cells are cultured in medium alone
or in combination with 100 U/ml IL-2 (R&D Systems) for 120
hours. 3H-Thymidine (0.1 mCi) is added during the last six hours of
culture and 3H-Thymidine incorporation determined by liquid
scintillation counting.
[0268] The native, recombinant, and fusion proteins would be tested
for agonist and antagonist activity in many other biological assay
systems, e.g., on T-cells, B-cells, NK, macrophages, dendritic
cells, hematopoietic progenitors, etc. Because of the IL-6 and
G-CSF structural relationship, assays related to those activities
should be analyzed
[0269] IL-B60 is evaluated for agonist or antagonist activity on
transfected cells expressing IL-6 or G-CSF receptor and controls.
See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90,
11267-11271; Ho, et al. (1995) Mol. Cell. Biol. 15:5043-5053; and
Liu, et al. (1994). J. Immunol. 152:1821-1829.
[0270] IL-B60 is evaluated for effect in macrophage/dendritic cell
activation and antigen presentation assays, T cell cytokine
production and proliferation in response to antigen or allogeneic
stimulus. See, e.g., de Waal Malefyt et al. (1991) J. Exp. Med.
174:1209-1220; de Waal Malefyt et al. (199.1) J. Exp. Med.
174:915-924; Fiorentino, et al. (.1991) J. Immunol. 147, 3815-3822;
Fiorentino, et al. (1991) J. Immunol. 146:3444-3451; and Groux, et
al. (1996) J. Exp. Med. 184:19-29.
[0271] IL-B60 will also be evaluated for effects on NK cell
stimulation. Assays may be based, e.g., on Hsu, et al. (1992)
Internat. Immunol. 4:563-569; and Schwarz, et al. (1994) J.
Immunother. 16:95-104.
[0272] B cell growth and differentiation effects will be analyzed,
e.g., by the methodology described, e.g., in Defrance, et al.
(1992). J. Exp. Med. 175:671-682; Rousset, et al. (1992) Proc.
Nat'l Acad. Sci. USA 89:1890-1893; including IgG2 and IgA2 switch
factor assays. Note that, unlike COS7 supernatants, NIH3T3 and COP
supernatants apparently do not interfere with human B cell
assays.
[0273] F. IL-B60 and CLF-1 Induce a Switch in Neurotransmitter
Properties
[0274] Cholineric sympathetic neurons innervate at least three
different targets: sweat glands, vasculature in skeletal muscle,
and periosteum. Mature innervation of sympathetic neurons begins at
the end of the first postnatal week and is characterized by the
appearance of cholineric properties. Cultures of sympathetic
neurons were analyzed for the induction of different
neuromodulators, which specify the cholinergic phenotype.
Cholecystokinin (CCK), vasoactive intestinal polypeptide (VIP),
substance P (SP) and somatostatin (SOM) are upregulated after
stimulation of neurons with conditioned medium from IL-B60/CLF-1
cotransfected cells or the CNTFR-IL-B60 fusion protein. Thus, the
complex exhibits significant developmental biology function, and
may be effective in inducing certain aspects of neural
development.
[0275] XI. Generation and Analysis of Genetically Altered
Animals
[0276] Transgenic mice can be generated by standard methods. Such
animals are useful to determine the effects of overexpression of
the gene, in specific tissues, or completely throughout the
organism. Such may provide interesting insight into development of
the animal or particular tissues in various stages. Moreover, the
effect on various responses to biological stress can be evaluated.
See, e.g., Hogan, et al. (1995) Manipulating the Mouse Embryo: A
Laboratory Manual (2d ed.) Cold Spring Harbor Laboratory Press.
[0277] Adenovirus techniques are available for expression of the
gene in various cells and organs. See, e.g., Hitt, et al. (1997)
Adv. Pharmacol. 40:137-195; and literature from Quantum
Biotechnologies, Montreal, Canada. Animals may be useful to
determine the effects of the gene on various developmental or
physiologically functional animal systems.
[0278] The genomic structure for the mouse IL-B60 has been
determined. A strategy for the production of IL-B60 knock-out mice
can be developed, and appropriate constructs made.
[0279] All references cited 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 in its entirety for all purposes.
[0280] 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 only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
Sequence CWU 1
1
13 1 1790 DNA primate CDS (162)..(806) mat_peptide (213)..(806) 1
ccgagcgaaa aaaacctgcg agtgggcctg gcggatggga ttattaaagc ttcgccggag
60 ccgcggctcg ccctcccact ccgccagcct ccgggagagg agccgcaccc
ggccggcccg 120 gccccagccc catggacctc cgagcagggg actcgtgggg g atg
tta gcg tgc ctg 176 Met Leu Ala Cys Leu -15 tgc acg gtg ctc tgg cac
ctc cct gca gtg cca gct ctc aat cgc aca 224 Cys Thr Val Leu Trp His
Leu Pro Ala Val Pro Ala Leu Asn Arg Thr -10 -5 -1 1 ggg gac cca ggg
cct ggc ccc tcc atc cag aaa acc tat gac ctc acc 272 Gly Asp Pro Gly
Pro Gly Pro Ser Ile Gln Lys Thr Tyr Asp Leu Thr 5 10 15 20 cgc tac
ctg gag cac caa ctc cgc agc ttg gct ggg acc tat ctg aac 320 Arg Tyr
Leu Glu His Gln Leu Arg Ser Leu Ala Gly Thr Tyr Leu Asn 25 30 35
tac ctg ggc ccc cct ttc aac gag cca gac ttc aac cct ccc cgc ctg 368
Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe Asn Pro Pro Arg Leu 40
45 50 ggg gca gag act ctg ccc agg gcc act gtt gac ttg gag gtg tgg
cga 416 Gly Ala Glu Thr Leu Pro Arg Ala Thr Val Asp Leu Glu Val Trp
Arg 55 60 65 agc ctc aat gac aaa ctg cgg ctg acc cag aac tac gag
gcc tac agc 464 Ser Leu Asn Asp Lys Leu Arg Leu Thr Gln Asn Tyr Glu
Ala Tyr Ser 70 75 80 cac ctt ctg tgt tac ttg cgt ggc ctc aac cgt
cag gct gcc act gct 512 His Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg
Gln Ala Ala Thr Ala 85 90 95 100 gag ctg cgc cgc agc ctg gcc cac
ttc tgc acc agc ctc cag ggc ctg 560 Glu Leu Arg Arg Ser Leu Ala His
Phe Cys Thr Ser Leu Gln Gly Leu 105 110 115 ctg ggc agc att gcg ggc
gtc atg gca gct ctg ggc tac cca ctg ccc 608 Leu Gly Ser Ile Ala Gly
Val Met Ala Ala Leu Gly Tyr Pro Leu Pro 120 125 130 cag ccg ctg cct
ggg act gaa ccc act tgg act cct ggc cct gcc cac 656 Gln Pro Leu Pro
Gly Thr Glu Pro Thr Trp Thr Pro Gly Pro Ala His 135 140 145 agt gac
ttc ctc cag aag atg gac gac ttc tgg ctg ctg aag gag ctg 704 Ser Asp
Phe Leu Gln Lys Met Asp Asp Phe Trp Leu Leu Lys Glu Leu 150 155 160
cag acc tgg ctg tgg cgc tcg gcc aag gac ttc aac cgg ctc aag aag 752
Gln Thr Trp Leu Trp Arg Ser Ala Lys Asp Phe Asn Arg Leu Lys Lys 165
170 175 180 aag atg cag cct cca gca gct gca gtc acc ctg cac ctg ggg
gct cat 800 Lys Met Gln Pro Pro Ala Ala Ala Val Thr Leu His Leu Gly
Ala His 185 190 195 ggc ttc tgacttctga ccttctcctc ttcgctcccc
cttcaaaccc tgctcccact 856 Gly Phe ttgtgagagc cagccctgta tgccaacacc
tgttgagcca ggagacagaa gctgtgagcc 916 tctggccctt tcctggaccg
gctgggcgtg tgatgcgatc agccctgtct cctccccacc 976 tcccaaaggt
ctaccgagct ggggaggagg tacagtaggc cctgtcctgt cctgtttcta 1036
caggaagtca tgctcgaggg agtgtgaagt ggttcaggtt ggtgcagagg cgctcatggc
1096 ctcctgcttc ttgcctacca cttggccagt gcccacccag cccctcaggt
ggcacatctg 1156 gagggcaggg gttgaggggc caccaccaca catgcctttc
tggggtgaag ccctttggct 1216 gccccactct ccttggatgg gtgttgctcc
cttatcccca aatcactcta tacatccaat 1276 tcaggaaaca aacatggtgg
caattctaca caaaaagaga tgagattaac agtgcagggt 1336 tggggtctgc
attggaggtg ccctataaac cagaagagaa aatactgaaa gcacaggggc 1396
agggacagac cagaccagac ccaggagtct ccaaagcaca gagtggcaaa caaaacccga
1456 gctgagcatc aggaccttgc ctcgaattgt cttccagtat tacggtgcct
cttctctgcc 1516 ccctttccca gggtatctgt gggttgccag gctggggagg
gcaaccatag ccacaccaca 1576 ggatttcctg aaagtttaca atgcagtagc
attttggggt gtagggtggc agctccccaa 1636 ggccctgccc cccagcccca
cccactcatg actctaagtg tgttgtatta atatttattt 1696 atttggagat
gttatttatt agatgatatt tattgcagaa tttctattct tgtattaaca 1756
aataaaatgc ttgccccaga acaaaaaaaa aaaa 1790 2 215 PRT primate 2 Met
Leu Ala Cys Leu Cys Thr Val Leu Trp His Leu Pro Ala Val Pro -15 -10
-5 Ala Leu Asn Arg Thr Gly Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys
-1 1 5 10 15 Thr Tyr Asp Leu Thr Arg Tyr Leu Glu His Gln Leu Arg
Ser Leu Ala 20 25 30 Gly Thr Tyr Leu Asn Tyr Leu Gly Pro Pro Phe
Asn Glu Pro Asp Phe 35 40 45 Asn Pro Pro Arg Leu Gly Ala Glu Thr
Leu Pro Arg Ala Thr Val Asp 50 55 60 Leu Glu Val Trp Arg Ser Leu
Asn Asp Lys Leu Arg Leu Thr Gln Asn 65 70 75 Tyr Glu Ala Tyr Ser
His Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg 80 85 90 95 Gln Ala Ala
Thr Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr 100 105 110 Ser
Leu Gln Gly Leu Leu Gly Ser Ile Ala Gly Val Met Ala Ala Leu 115 120
125 Gly Tyr Pro Leu Pro Gln Pro Leu Pro Gly Thr Glu Pro Thr Trp Thr
130 135 140 Pro Gly Pro Ala His Ser Asp Phe Leu Gln Lys Met Asp Asp
Phe Trp 145 150 155 Leu Leu Lys Glu Leu Gln Thr Trp Leu Trp Arg Ser
Ala Lys Asp Phe 160 165 170 175 Asn Arg Leu Lys Lys Lys Met Gln Pro
Pro Ala Ala Ala Val Thr Leu 180 185 190 His Leu Gly Ala His Gly Phe
195 3 648 DNA primate CDS (1)..(645) mat_peptide (52)..(645) 3 atg
tta gct tgc cta tgc acg gtg ctg tgg cac ctc cct gca gtg cca 48 Met
Leu Ala Cys Leu Cys Thr Val Leu Trp His Leu Pro Ala Val Pro -15 -10
-5 gct ctt aat cgc aca gga gat cca ggc cct ggc ccc tcc atc cag aaa
96 Ala Leu Asn Arg Thr Gly Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys
-1 1 5 10 15 acc tat gac ctc acc cgc tac ctg gag cat caa ctc cgc
agc tta gct 144 Thr Tyr Asp Leu Thr Arg Tyr Leu Glu His Gln Leu Arg
Ser Leu Ala 20 25 30 ggg acc tac ctg aac tac ctg ggg ccc cct ttc
aac gag cct gac ttc 192 Gly Thr Tyr Leu Asn Tyr Leu Gly Pro Pro Phe
Asn Glu Pro Asp Phe 35 40 45 aat cct cct cga ctg ggg gca gaa act
ctg ccc agg gcc acg gtc aac 240 Asn Pro Pro Arg Leu Gly Ala Glu Thr
Leu Pro Arg Ala Thr Val Asn 50 55 60 ttg gaa gtg tgg cga agc ctc
aat gac agg ctg cgg ctg acc cag aac 288 Leu Glu Val Trp Arg Ser Leu
Asn Asp Arg Leu Arg Leu Thr Gln Asn 65 70 75 tat gag gcg tac agt
cac ctc ctg tgt tac ttg cgt ggc ctc aac cgt 336 Tyr Glu Ala Tyr Ser
His Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg 80 85 90 95 cag gct gcc
aca gct gaa ctc cga cgt agc ctg gcc cac ttc tgt acc 384 Gln Ala Ala
Thr Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr 100 105 110 agc
ctc cag ggc ctg ctg ggc agc att gca ggt gtc atg gcg acg ctt 432 Ser
Leu Gln Gly Leu Leu Gly Ser Ile Ala Gly Val Met Ala Thr Leu 115 120
125 ggc tac cca ctg ccc cag cct ctg cca ggg act gag cca gcc tgg gcc
480 Gly Tyr Pro Leu Pro Gln Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala
130 135 140 cct ggc cct gcc cac agt gac ttc ctc cag aag atg gat gac
ttc tgg 528 Pro Gly Pro Ala His Ser Asp Phe Leu Gln Lys Met Asp Asp
Phe Trp 145 150 155 ctg ctg aag gag ctg cag acc tgg cta tgg cgt tca
gcc aag gac ttc 576 Leu Leu Lys Glu Leu Gln Thr Trp Leu Trp Arg Ser
Ala Lys Asp Phe 160 165 170 175 aac cgg ctt aag aag aag atg cag cct
cca gca gct tca gtc acc ctg 624 Asn Arg Leu Lys Lys Lys Met Gln Pro
Pro Ala Ala Ser Val Thr Leu 180 185 190 cac ttg gag gcc cat ggt ttc
tga 648 His Leu Glu Ala His Gly Phe 195 4 215 PRT primate 4 Met Leu
Ala Cys Leu Cys Thr Val Leu Trp His Leu Pro Ala Val Pro -15 -10 -5
Ala Leu Asn Arg Thr Gly Asp Pro Gly Pro Gly Pro Ser Ile Gln Lys -1
1 5 10 15 Thr Tyr Asp Leu Thr Arg Tyr Leu Glu His Gln Leu Arg Ser
Leu Ala 20 25 30 Gly Thr Tyr Leu Asn Tyr Leu Gly Pro Pro Phe Asn
Glu Pro Asp Phe 35 40 45 Asn Pro Pro Arg Leu Gly Ala Glu Thr Leu
Pro Arg Ala Thr Val Asn 50 55 60 Leu Glu Val Trp Arg Ser Leu Asn
Asp Arg Leu Arg Leu Thr Gln Asn 65 70 75 Tyr Glu Ala Tyr Ser His
Leu Leu Cys Tyr Leu Arg Gly Leu Asn Arg 80 85 90 95 Gln Ala Ala Thr
Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr 100 105 110 Ser Leu
Gln Gly Leu Leu Gly Ser Ile Ala Gly Val Met Ala Thr Leu 115 120 125
Gly Tyr Pro Leu Pro Gln Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala 130
135 140 Pro Gly Pro Ala His Ser Asp Phe Leu Gln Lys Met Asp Asp Phe
Trp 145 150 155 Leu Leu Lys Glu Leu Gln Thr Trp Leu Trp Arg Ser Ala
Lys Asp Phe 160 165 170 175 Asn Arg Leu Lys Lys Lys Met Gln Pro Pro
Ala Ala Ser Val Thr Leu 180 185 190 His Leu Glu Ala His Gly Phe 195
5 203 PRT rodent 5 Met Lys Val Leu Ala Ala Gly Ile Val Pro Leu Leu
Leu Leu Val Leu 1 5 10 15 His Trp Lys His Gly Ala Gly Ser Pro Leu
Pro Ile Thr Pro Val Asn 20 25 30 Ala Thr Cys Ala Ile Arg His Pro
Cys His Gly Asn Leu Met Asn Gln 35 40 45 Ile Lys Asn Gln Leu Ala
Gln Leu Asn Gly Ser Ala Asn Ala Leu Phe 50 55 60 Ile Ser Tyr Tyr
Thr Ala Gln Gly Glu Pro Phe Pro Asn Asn Val Glu 65 70 75 80 Lys Leu
Cys Ala Pro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn 85 90 95
Gly Thr Glu Lys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr 100
105 110 Leu Ser Ala Ser Leu Thr Asn Ile Thr Arg Asp Gln Lys Val Leu
Asn 115 120 125 Pro Thr Ala Val Ser Leu Gln Val Lys Leu Asn Ala Thr
Ile Asp Val 130 135 140 Met Arg Gly Leu Leu Ser Asn Val Leu Cys Arg
Leu Cys Asn Lys Tyr 145 150 155 160 Arg Val Gly His Val Asp Val Pro
Pro Val Pro Asp His Ser Asp Lys 165 170 175 Glu Ala Phe Gln Arg Lys
Lys Leu Gly Cys Gln Leu Leu Gly Thr Tyr 180 185 190 Lys Gln Val Ile
Ser Val Val Val Gln Ala Phe 195 200 6 202 PRT primate 6 Met Lys Val
Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp
Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn Ala 20 25
30 Thr Cys Ala Ile Arg His Pro Cys His Asn Asn Leu Met Asn Gln Ile
35 40 45 Arg Ser Gln Leu Ala Gln Leu Asn Gly Ser Ala Asn Ala Leu
Phe Ile 50 55 60 Leu Tyr Tyr Thr Ala Gln Gly Glu Pro Phe Pro Asn
Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro
Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu
Leu Tyr Arg Ile Val Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn
Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro 115 120 125 Ser Ala Leu Ser
Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu 130 135 140 Arg Gly
Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155
160 Val Gly His Val Asp Val Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp
165 170 175 Val Phe Gln Lys Lys Lys Leu Gly Cys Gln Leu Leu Gly Lys
Tyr Lys 180 185 190 Gln Ile Ile Ala Val Leu Ala Gln Ala Phe 195 200
7 201 PRT primate 7 Met Ser Arg Arg Glu Gly Ser Leu Glu Asp Pro Gln
Thr Asp Ser Ser 1 5 10 15 Val Ser Leu Leu Pro His Leu Glu Ala Lys
Ile Arg Gln Thr His Ser 20 25 30 Leu Ala His Leu Leu Thr Lys Tyr
Ala Glu Gln Leu Leu Gln Glu Tyr 35 40 45 Val Gln Leu Gln Gly Asp
Pro Phe Gly Leu Pro Ser Phe Ser Pro Pro 50 55 60 Arg Leu Pro Val
Ala Gly Leu Ser Ala Pro Ala Pro Ser His Ala Gly 65 70 75 80 Leu Pro
Val His Glu Arg Leu Arg Leu Asp Ala Ala Ala Leu Ala Ala 85 90 95
Leu Pro Pro Leu Leu Asp Ala Val Cys Arg Arg Gln Ala Glu Leu Asn 100
105 110 Pro Arg Ala Pro Arg Leu Leu Arg Arg Leu Glu Asp Ala Ala Arg
Gln 115 120 125 Ala Arg Ala Leu Gly Ala Ala Val Glu Ala Leu Leu Ala
Ala Leu Gly 130 135 140 Ala Ala Asn Arg Gly Pro Arg Ala Glu Pro Pro
Ala Ala Thr Ala Ser 145 150 155 160 Ala Ala Ser Ala Thr Gly Val Phe
Pro Ala Lys Val Leu Gly Leu Arg 165 170 175 Val Cys Gly Leu Tyr Arg
Glu Trp Leu Ser Arg Thr Glu Gly Asp Leu 180 185 190 Gly Gln Leu Leu
Pro Gly Gly Ser Ala 195 200 8 203 PRT rodent 8 Met Ser Gln Arg Glu
Gly Ser Leu Glu Asp His Gln Thr Asp Ser Ser 1 5 10 15 Ile Ser Phe
Leu Pro His Leu Glu Ala Lys Ile Arg Gln Thr His Asn 20 25 30 Leu
Ala Arg Leu Leu Thr Lys Tyr Ala Glu Gln Leu Leu Glu Glu Tyr 35 40
45 Val Gln Gln Gln Gly Glu Pro Phe Gly Leu Pro Gly Phe Ser Pro Pro
50 55 60 Arg Leu Pro Leu Ala Gly Leu Ser Gly Pro Ala Pro Ser His
Ala Gly 65 70 75 80 Leu Pro Val Ser Glu Arg Leu Arg Gln Asp Ala Ala
Ala Leu Ser Val 85 90 95 Leu Pro Ala Leu Leu Asp Ala Val Arg Arg
Arg Gln Ala Glu Leu Asn 100 105 110 Pro Arg Ala Pro Arg Leu Leu Arg
Ser Leu Glu Asp Ala Ala Arg Gln 115 120 125 Val Arg Ala Leu Gly Ala
Ala Val Glu Thr Val Leu Ala Ala Leu Gly 130 135 140 Ala Ala Ala Arg
Gly Pro Gly Pro Glu Pro Val Thr Val Ala Thr Leu 145 150 155 160 Phe
Thr Ala Asn Ser Thr Ala Gly Ile Phe Ser Ala Lys Val Leu Gly 165 170
175 Phe His Val Cys Gly Leu Tyr Gly Glu Trp Val Ser Arg Thr Glu Gly
180 185 190 Asp Leu Gly Gln Leu Val Pro Gly Gly Val Ala 195 200 9
200 PRT primate 9 Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His
Arg Arg Asp Leu 1 5 10 15 Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys
Ile Arg Ser Asp Leu Thr 20 25 30 Ala Leu Thr Glu Ser Tyr Val Lys
His Gln Gly Leu Asn Lys Asn Ile 35 40 45 Asn Leu Asp Ser Ala Asp
Gly Met Pro Val Ala Ser Thr Asp Gln Trp 50 55 60 Ser Glu Leu Thr
Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr 65 70 75 80 Arg Thr
Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val 85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu 100
105 110 Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met
Ile 115 120 125 Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly
Met Pro Ile 130 135 140 Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys
Leu Trp Gly Leu Lys 145 150 155 160 Val Leu Gln Glu Leu Ser Gln Trp
Thr Val Arg Ser Ile His Asp Leu 165 170 175 Arg Phe Ile Ser Ser His
Gln Thr Gly Ile Pro Ala Arg Gly Ser His 180 185 190 Tyr Ile Ala Asn
Asn Lys Lys Met 195 200 10 198 PRT rodent 10 Met Ala Phe Ala Glu
Gln Ser Pro Leu Thr Leu His Arg Arg Asp Leu 1 5 10 15 Cys Ser Arg
Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr 20 25 30 Ala
Leu Met Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile 35 40
45 Ser Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Arg Trp
50 55 60 Ser Glu Met Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln
Ala Tyr
65 70 75 80 Arg Thr Phe Gln Gly Met Leu Thr Lys Leu Leu Glu Asp Gln
Arg Val 85 90 95 His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala
Ile His Thr Leu 100 105 110 Thr Leu Gln Val Ser Ala Phe Ala Tyr Gln
Leu Glu Glu Leu Met Ala 115 120 125 Leu Leu Glu Gln Lys Val Pro Glu
Lys Glu Ala Asp Gly Met Pro Val 130 135 140 Thr Ile Gly Asp Gly Gly
Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys 145 150 155 160 Val Leu Gln
Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu 165 170 175 Arg
Val Ile Ser Ser His His Met Gly Ile Ser Ala His Glu Ser His 180 185
190 Tyr Gly Ala Lys Gln Met 195 11 208 PRT primate 11 Met Thr His
Leu Ser Leu Leu Gly Pro Leu Pro Cys Val Arg Thr Ser 1 5 10 15 Gln
Gln Leu Pro Glu Thr Gln Gln Val Thr Thr Pro Gly Lys Lys Pro 20 25
30 Val Ser Val Gly Arg Arg Glu Val Arg Val Pro Gly Thr Ala Leu Val
35 40 45 Pro Ser Leu Leu Ser Val Ser Val Leu Leu Gln Leu Gln Tyr
Gln Gly 50 55 60 Ser Pro Phe Ser Asp Pro Gly Phe Ser Ala Pro Glu
Leu Gln Leu Ser 65 70 75 80 Ser Leu Pro Pro Ala Thr Ala Phe Phe Lys
Thr Trp His Ala Leu Asp 85 90 95 Asp Gly Glu Arg Leu Ser Leu Ala
Gln Arg Ala Ile Asp Pro His Leu 100 105 110 Gln Leu Val Glu Asp Asp
Gln Ser Asp Leu Asn Pro Gly Ser Pro Ile 115 120 125 Leu Pro Ala Gln
Leu Gly Ala Ala Arg Leu Arg Ala Gln Gly Pro Leu 130 135 140 Gly Asn
Met Ala Ala Ile Met Thr Ala Leu Gly Leu Pro Ile Pro Pro 145 150 155
160 Glu Glu Asp Thr Pro Gly Leu Ala Ala Phe Gly Ala Ser Ala Phe Glu
165 170 175 Arg Lys Cys Arg Gly Tyr Val Val Thr Arg Glu Tyr Gly His
Trp Thr 180 185 190 Asp Arg Ala Val Arg Asp Leu Ala Leu Leu Lys Ala
Lys Tyr Ser Ala 195 200 205 12 410 PRT primate 12 Met Pro Ala Gly
Arg Arg Gly Pro Ala Ala Gln Ser Ala Arg Arg Pro 1 5 10 15 Pro Pro
Leu Leu Pro Leu Leu Leu Leu Leu Cys Val Leu Gly Ala Pro 20 25 30
Arg Ala Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro Gln Asp Pro 35
40 45 Thr Leu Leu Ile Gly Ser Ser Leu Leu Ala Thr Cys Ser Val His
Gly 50 55 60 Asp Pro Pro Gly Ala Thr Ala Glu Gly Leu Tyr Trp Thr
Leu Asn Gly 65 70 75 80 Arg Arg Leu Pro Pro Glu Leu Ser Arg Val Leu
Asn Ala Ser Thr Leu 85 90 95 Ala Leu Ala Leu Ala Asn Leu Asn Gly
Ser Arg Gln Arg Ser Gly Asp 100 105 110 Asn Leu Val Cys His Ala Arg
Asp Gly Ser Ile Leu Ala Gly Ser Cys 115 120 125 Leu Tyr Val Gly Leu
Pro Pro Glu Lys Pro Val Asn Ile Ser Cys Trp 130 135 140 Ser Lys Asn
Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala His 145 150 155 160
Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu Arg 165
170 175 Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr His Thr Val Gly
Pro 180 185 190 His Ser Cys His Ile Pro Lys Asp Leu Ala Leu Phe Thr
Pro Tyr Glu 195 200 205 Ile Trp Val Glu Ala Thr Asn Arg Leu Gly Ser
Ala Arg Ser Asp Val 210 215 220 Leu Thr Leu Asp Ile Leu Asp Val Val
Thr Thr Asp Pro Pro Pro Asp 225 230 235 240 Val His Val Ser Arg Val
Gly Gly Leu Glu Asp Gln Leu Ser Val Arg 245 250 255 Trp Val Ser Pro
Pro Ala Leu Lys Asp Phe Leu Phe Gln Ala Lys Tyr 260 265 270 Gln Ile
Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val Asp 275 280 285
Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly Leu Lys Pro Gly 290
295 300 Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe Gly Ile Tyr
Gly 305 310 315 320 Ser Lys Lys Ala Gly Ile Trp Ser Glu Trp Ser His
Pro Thr Ala Ala 325 330 335 Ser Thr Pro Arg Ser Glu Arg Pro Gly Pro
Gly Gly Gly Ala Cys Glu 340 345 350 Pro Arg Gly Gly Glu Pro Ser Ser
Gly Pro Val Arg Arg Glu Leu Lys 355 360 365 Gln Phe Leu Gly Trp Leu
Lys Lys His Ala Tyr Cys Ser Asn Leu Ser 370 375 380 Phe Arg Leu Tyr
Asp Gln Trp Arg Ala Trp Met Gln Lys Ser His Lys 385 390 395 400 Thr
Arg Asn Gln Val Leu Pro Asp Lys Leu 405 410 13 407 PRT rodent 13
Arg Pro Leu Ser Ser Leu Trp Ser Pro Leu Leu Leu Cys Val Leu Gly 1 5
10 15 Val Pro Arg Gly Gly Ser Gly Ala His Thr Ala Val Ile Ser Pro
Gln 20 25 30 Asp Pro Thr Leu Leu Ile Gly Ser Ser Leu Gln Ala Thr
Cys Ser Ile 35 40 45 His Gly Asp Thr Pro Gly Ala Thr Ala Glu Gly
Leu Tyr Trp Thr Leu 50 55 60 Asn Gly Arg Arg Leu Pro Ser Leu Ser
Arg Leu Leu Asn Thr Ser Thr 65 70 75 80 Leu Ala Leu Ala Leu Ala Asn
Leu Asn Gly Ser Arg Gln Gln Ser Gly 85 90 95 Asp Asn Leu Val Cys
His Ala Arg Asp Gly Ser Ile Leu Ala Gly Ser 100 105 110 Cys Leu Tyr
Val Gly Leu Pro Pro Glu Lys Pro Phe Asn Ile Ser Cys 115 120 125 Trp
Ser Arg Asn Met Lys Asp Leu Thr Cys Arg Trp Thr Pro Gly Ala 130 135
140 His Gly Glu Thr Phe Leu His Thr Asn Tyr Ser Leu Lys Tyr Lys Leu
145 150 155 160 Arg Trp Tyr Gly Gln Asp Asn Thr Cys Glu Glu Tyr His
Thr Val Gly 165 170 175 Pro His Ser Cys His Ile Pro Lys Asp Leu Ala
Leu Phe Thr Pro Tyr 180 185 190 Glu Ile Trp Val Glu Ala Thr Asn Arg
Leu Gly Ser Ala Arg Ser Asp 195 200 205 Val Leu Thr Leu Asp Val Leu
Asp Val Val Thr Thr Asp Pro Pro Pro 210 215 220 Asp Val His Val Ser
Arg Val Gly Gly Leu Glu Asp Gln Leu Ser Val 225 230 235 240 Arg Trp
Val Ser Pro Pro Ala Leu Lys Asp Phe Leu Phe Gln Ala Lys 245 250 255
Tyr Gln Ile Arg Tyr Arg Val Glu Asp Ser Val Asp Trp Lys Val Val 260
265 270 Asp Asp Val Ser Asn Gln Thr Ser Cys Arg Leu Ala Gly Leu Lys
Pro 275 280 285 Gly Thr Val Tyr Phe Val Gln Val Arg Cys Asn Pro Phe
Gly Ile Tyr 290 295 300 Gly Ser Lys Lys Ala Gly Ile Trp Ser Glu Trp
Ser His Pro Thr Ala 305 310 315 320 Ala Ser Thr Pro Arg Ser Glu Arg
Pro Gly Pro Gly Gly Gly Val Cys 325 330 335 Glu Pro Arg Gly Gly Glu
Pro Ser Ser Gly Pro Val Arg Arg Glu Leu 340 345 350 Lys Gln Phe Leu
Gly Trp Leu Lys Lys His Ala Tyr Cys Ser Asn Leu 355 360 365 Ser Phe
Arg Leu Tyr Asp Gln Trp Arg Ala Trp Met Gln Lys Ser His 370 375 380
Lys Thr Arg Asn Gln Asp Glu Gly Ile Leu Pro Ser Gly Arg Arg Gly 385
390 395 400 Ala Ala Arg Gly Pro Ala Gly 405
* * * * *