U.S. patent application number 13/357962 was filed with the patent office on 2012-06-07 for uses of il-174 antagonists for inhibiting a th2 immune response.
This patent application is currently assigned to Schering Corporation. Invention is credited to Stephen D. Hurst, Donna M. Rennick, Sandra M. Zurawski.
Application Number | 20120141411 13/357962 |
Document ID | / |
Family ID | 22733587 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120141411 |
Kind Code |
A1 |
Hurst; Stephen D. ; et
al. |
June 7, 2012 |
Uses of IL-174 Antagonists for Inhibiting A Th2 Immune Response
Abstract
Agonists or antagonists of cytokine designated IL-174, and
various methods of their use are provided. In particular, the
methods make use of facts that many activities of the IL-174
cytokine are described.
Inventors: |
Hurst; Stephen D.; (Palo
Alto, CA) ; Zurawski; Sandra M.; (Midlothian, TX)
; Rennick; Donna M.; (Los Altos, CA) |
Assignee: |
Schering Corporation
Kenilworth
NJ
|
Family ID: |
22733587 |
Appl. No.: |
13/357962 |
Filed: |
January 25, 2012 |
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13090483 |
Apr 20, 2011 |
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13357962 |
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12614003 |
Nov 6, 2009 |
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13090483 |
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11957994 |
Dec 17, 2007 |
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12614003 |
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10705183 |
Nov 10, 2003 |
7357924 |
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11957994 |
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09836385 |
Apr 17, 2001 |
6676939 |
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10705183 |
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Current U.S.
Class: |
424/85.2 ;
424/145.1; 424/158.1 |
Current CPC
Class: |
A61P 1/18 20180101; A61P
31/10 20180101; A61P 35/00 20180101; C07K 16/244 20130101; A61P
17/02 20180101; A61P 33/10 20180101; A61P 29/00 20180101; A61P 3/10
20180101; A61P 7/00 20180101; A61P 31/00 20180101; A61P 37/08
20180101; A61P 1/04 20180101; A61P 11/00 20180101; A61P 1/16
20180101; A61P 7/06 20180101; A61K 39/3955 20130101; A61P 37/06
20180101; A61K 38/20 20130101; A61P 3/00 20180101; A61P 37/02
20180101; A61P 19/00 20180101; A61P 17/00 20180101; A61P 11/06
20180101; Y02A 50/423 20180101; A61K 2039/505 20130101; A61K
2039/57 20130101; A61P 19/02 20180101; A61P 37/00 20180101; A61P
31/14 20180101; A61P 43/00 20180101; A61P 33/12 20180101; A61P
25/00 20180101; A61P 37/04 20180101; A61P 1/00 20180101; A61K 45/06
20130101; A61P 17/06 20180101; A61P 31/12 20180101; A61P 33/00
20180101; A61K 38/20 20130101; A61K 2300/00 20130101; A61K 39/3955
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/85.2 ;
424/145.1; 424/158.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 37/00 20060101 A61P037/00; A61P 29/00 20060101
A61P029/00; A61P 1/00 20060101 A61P001/00; A61P 37/04 20060101
A61P037/04; A61P 7/00 20060101 A61P007/00; A61P 37/06 20060101
A61P037/06; A61P 25/00 20060101 A61P025/00; A61P 3/10 20060101
A61P003/10; A61P 17/06 20060101 A61P017/06; A61P 31/10 20060101
A61P031/10; A61P 33/00 20060101 A61P033/00; A61P 33/12 20060101
A61P033/12; A61P 33/10 20060101 A61P033/10; A61P 31/12 20060101
A61P031/12; A61P 37/08 20060101 A61P037/08; A61P 17/00 20060101
A61P017/00; A61P 11/00 20060101 A61P011/00; A61P 1/18 20060101
A61P001/18; A61P 1/16 20060101 A61P001/16; A61P 35/00 20060101
A61P035/00; A61P 11/06 20060101 A61P011/06; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of: a) directing a mammalian immune response towards a
Th2 type response, said method comprising administering an IL-174
agonist to immune cells of the mammal; b) stimulating an mammalian
innate immune response, said method comprising administering an
IL-174 agonist to immune cells of the mammal; c) augmenting a
mammalian inflammatory response from epithelial or fibroblast
cells, said method comprising further administering an IL-174
agonist to said mammal; d) inducing gut cell growth, said method
comprising administering an IL-174 agonist to said cell; e)
promoting mammalian extra medulary hematopoiesis, said method
comprising administering an IL-174 agonist to said mammal; f)
directing a mammalian immune response away from a Th2 type
response, said method comprising administering an IL-174 antagonist
to immune cells of the mammal; g) preventing mammalian inflammation
or granuloma formation, comprising administering an IL-174
antagonist to immune system cells; or h) augmenting antibody
response in serum and fecal material, said method comprising
administering an IL-174 agonist to immune cells of the mammal.
2. The method of claim 1: a) directing a mammalian immune response
towards a Th2 type response, said method comprising administering
an IL-174 agonist to immune cells of the mammal; b) stimulating an
mammalian innate immune response, said method comprising
administering an IL-174 agonist to immune cells of the mammal; c)
augmenting a mammalian inflammatory response from epithelial or
fibroblast cells, said method comprising further administering an
IL-174 agonist to said mammal; d) inducing gut cell growth, said
method comprising administering an IL-174 agonist to said cell; or
e) promoting mammalian extra medulary hematopoiesis, said method
comprising administering an IL-174 agonist to said mammal.
3. The method of claim 2: d) inducing gut cell growth, said method
comprising administering an IL-174 agonist to said cell; e)
promoting mammalian extra medulary hematopoiesis, said method
comprising administering an IL-174 agonist to said mammal.
4. The method of claim 1: f) directing a mammalian immune response
away from a Th2 type response, said method comprising administering
an IL-174 antagonist to immune cells of the mammal; g) preventing
mammalian inflammation or granuloma formation, comprising
administering an IL-174 antagonist to immune system cells; or h)
augmenting antibody responses in serum and fecal material, said
method comprising administering an IL-174 agonist to immune cells
of the mammal.
5. The method of claim 2 administering an agonist, wherein said
administering: a) induces cytokine production by a fibroblast,
epithelial, or endothelial cell; b) downregulates an inflammatory
response which accompanies an infection; c) stimulates growth of an
epithelial cell; or d) induces growth of gut epithelial,
fibroblast, or goblet cells.
6. The method of claim 2 administering an agonist, wherein said
mammal exhibits, or has experienced conditions to stimulate: a) an
autoimmune condition; b) an infectious disease immune response; c)
a wound healing response; or d) a Th1 mediated condition.
7. The method of claim 6, wherein: a) said autoimmune condition is
selected from: i) multiple sclerosis; ii) systemic lupus
erythematosis; iii) rheumatoid arthritis; iv) diabetes; or v)
psoriasis; b) said infectious response is symptomatic of i) an
Aspergillis infection; ii) a fungal infection, including
Candidaisis, Blastomycosis, or Aspergillosis; iii) a parasitic
infection, including Schistosomiasis, fluke worm, Helminth, or
Filariasis; or iv) a viral infection, including hepatitis; or c)
said Th1 mediated condition is an inflammatory condition, including
Crohn's disease, ulcerative colitis, pancreatitis, hepatitis, or
eosinophilic gastritis.
8. The method of claim 7 treating an infectious response, further
comprising administering another therapeutic entity to treat said
infection.
9. A method of claim 2, stimulating an mammalian innate immune
response.
10. The method of claim 4 administering an antagonist, wherein said
antagonist is a monoclonal antibody against IL-174.
11. The method of claim 4 administering an antagonist, wherein: a)
said administering blocks eosinophil attraction, tissue remodeling,
or fibrosis; or b) said mammal exhibits, or has experienced
conditions to stimulate: i) an allergic condition; ii) an
inflammatory condition; or iii) a Th2 mediated condition.
12. The method of claim 11, wherein: a) said eosinophils are
attracted to the lung, liver or intestine b) said fibrosis is
pancreatic duct or peribiliary fibrosis; c) said antagonist
suppresses production of IL-4, IL-5, and/or IL-13; d) said
antagonist decreases eotaxin, CCR4, and/or CCR4 expression in BAL;
e) symptoms of said allergic condition are in the lung; f) said
allergic condition is a systemic anaphylactic response, skin
hypersensitivity response, or a food allergy; or g) said
inflammatory or Th2 mediated condition is a dermatitis or asthmatic
inflammation.
13. The method of claim 11, wherein said mammal exhibits, or has
experienced conditions to stimulate: a) an allergic condition; b)
an inflammatory condition; or c) a Th2 mediated condition.
14. A composition comprising: a) an IL-174 agonist and: i) an
antimicrobial, including an antibiotic, antiviral, or antifungal
compound; or ii) a chemotherapy agent; or b) an IL-174 antagonist
and: i) an allergy medicament; ii) an asthma medicament; iii) a
dermatitis medicament; iv) a fibrosis medicament; or v) an
eosinophilic gastritis medicament.
Description
[0001] The present application is a continuation of Ser. No.
10/705,183, filed Nov. 10, 2003 which is a divisional of Ser. No.
09/836,385, filed Apr. 17, 2001, now U.S. Pat. No. 6,676,939, which
claims benefit of U.S. provisional patent application No.
60/198,488, filed Apr. 18, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to compositions related to
proteins which function in controlling physiology, development, and
differentiation of mammalian cells, e.g., cells of a mammalian
immune system. In particular, it provides nucleic acids, proteins,
antibodies, and mimetics which regulate cellular physiology,
development, differentiation, or function of various cell types,
including hematopoietic cells.
BACKGROUND OF THE INVENTION
[0003] The immune system of vertebrates consists of a number of
organs and several different cell types. Two major cell types
include the myeloid and lymphoid lineages. Among the lymphoid cell
lineage are B cells, which were originally characterized as
differentiating in fetal liver or adult bone marrow, and T cells,
which were originally characterized as differentiating in the
thymus. See, e.g., Paul (ed. 1998) Fundamental Immunology (4th ed.)
Raven Press, New York.
[0004] In many aspects of the development of an immune response or
cellular differentiation, soluble proteins known as cytokines play
a critical role in regulating cellular interactions. These
cytokines apparently mediate cellular activities in many ways. They
have been shown, in many cases, to modulate proliferation, growth,
and differentiation of hematopoietic stem cells into the vast
number of progenitors composing the lineages responsible for an
immune response.
[0005] However, the cellular molecules which are expressed by
different developmental stages of cells in these maturation
pathways are still incompletely identified. Moreover, the roles and
mechanisms of action of signaling molecules which induce, sustain,
or modulate the various physiological, developmental, or
proliferative states of these cells is poorly understood. Clearly,
the immune system and its response to various stresses had
relevance to medicine, e.g., infectious diseases, cancer related
responses and treatment, allergic and transplantation rejection
responses. See, e.g., Thorn, et al. Harrison's Principles of
Internal Medicine McGraw/Hill, New York.
[0006] Medical science relies, in large degree, on appropriate
recruitment or suppression of the immune system in effecting cures
for insufficient or improper physiological responses to
environmental factors. However, the lack of understanding of how
the immune system is regulated or differentiates has blocked the
ability to advantageously modulate the normal defensive mechanisms
to biological challenges. Medical conditions characterized by
abnormal or inappropriate regulation of the development or
physiology of relevant cells thus remain unmanageable. The
discovery and characterization of specific cytokines will
contribute to the development of therapies for a broad range of
degenerative or other conditions which affect the immune system,
hematopoietic cells, as well as other cell types. The present
invention provides solutions to some of these and many other
problems.
SUMMARY OF THE INVENTION
[0007] The present invention is based, in part, upon biological
activities of the cytokine known as IL-174. In particular, a number
of assays indicate that this cytokine has functions in regulating
establishment of Th2 type immune responses, innate immunity,
inflammatory responses, mucosal and fibroblast growth, certain
hematopoietic activities, and granuloma formation.
[0008] The present invention provides methods: directing a
mammalian immune response towards a Th2 type response, the method
comprising administering an IL-174 agonist to immune cells of the
mammal; stimulating an mammalian innate immune response, the method
comprising administering an IL-174 agonist to immune cells of the
mammal; augmenting a mammalian inflammatory response from
epithelial or fibroblast cells, the method comprising further
administering an IL-174 agonist to the mammal; inducing gut cell
growth, the method comprising administering an IL-174 agonist to
the cell; promoting mammalian extra medulary hematopoiesis, the
method comprising administering an IL-174 agonist to the mammal; or
augmenting antibody responses in serum and fecal material,
comprising administering an IL-174 agonist to the cell.
[0009] In various embodiments, the method involves administering an
agonist, wherein the administering: induces cytokine production by
a hematopoietic, fibroblast, epithelial, or endothelial cell;
downregulates an inflammatory response which accompanies an
infection; stimulates growth of an epithelial cell; or induces
growth of gut epithelial, fibroblast, or goblet cells. In some
embodiments, the method involves administering an agonist, wherein
the mammal exhibits, or has experienced conditions to stimulate: an
autoimmune condition; an infectious disease immune response; a
wound healing response; or a Th1 mediated condition. In further
embodiments, the autoimmune condition is selected from: multiple
sclerosis, systemic lupus erythematosis, rheumatoid arthritis,
diabetes, or psoriasis; the infectious response is symptomatic of
an Aspergillis infection, a fungal infection (including
Candidaisis, Blastomycosis, or Aspergillosis), a parasitic
infection (including Schistosomiasis, fluke worm, Helminth, or
Filariasis); or a viral infection (including hepatitis); or the Th1
mediated condition is an inflammatory condition (including Crohn's
disease, ulcerative colitis, pancreatitis, or hepatitis).
Additionally, the invention provides methods treating an infectious
response, farther comprising administering another therapeutic
entity to treat the infection.
[0010] The invention also provides other methods, e.g., directing a
mammalian immune response away from a Th2 type response, the method
comprising administering an IL-174 antagonist to immune cells of
the mammal; or preventing mammalian inflammation or granuloma
formation, comprising administering an IL-174 antagonist to immune
system cells. Often, the antagonist is a monoclonal or polyclonal
antibody against IL-174. The method may involve administering an
antagonist, wherein: the administering blocks eosinophil
attraction, tissue remodeling, or fibrosis; or the mammal exhibits,
or has experienced conditions to stimulate: an allergic condition;
an inflammatory condition; or a Th2 mediated condition. In further
embodiments, the eosinophils are attracted to the lung (i.e.,
asthma), liver or intestine (i.e., eosinophilic gastritis); the
fibrosis is pancreatic duct or peribiliary fibrosis; the antagonist
suppresses production of IL-4, IL-5, and/or IL-13; the antagonist
decreases eotaxin, CCR4, and/or CCR4 expression in BAL; symptoms of
the allergic condition are in the lung; the allergic condition is a
systemic anaphylactic response, skin hypersensitivity response, or
a food allergy; or the inflammatory or Th2 mediated condition is a
dermatitis or asthmatic inflammation. In yet other embodiments, the
mammal exhibits, or has experienced conditions to stimulate: an
allergic condition; an inflammatory condition; or a Th2 mediated
condition.
[0011] The invention also provides a composition comprising: an
IL-174 agonist and: an antimicrobial (including an antibiotic,
antiviral, or antifungal compound) or a chemotherapy agent; or an
IL-174 antagonist and: an allergy medicament, an asthma medicament,
a dermatitis medicament, a fibrosis medicament, or eosinophilic
gastritis medicament.
DETAILED DESCRIPTION OF THE INVENTION
[0012] Outline
[0013] I. General
[0014] II. Cytokine Agonists and Antagonists [0015] A. IL-174 and
Variants [0016] B. Antibodies [0017] C. Other Molecules
[0018] III. Immunoassays
[0019] IV. Uses
I. General
[0020] The invention is based, in part, on the surprising discovery
that the cytokine designated IL-174 has roles in various aspects of
immune responses. The IL-174 is one of a family of genes encoding
proteins which exhibit structural features characteristic of
cytokines, particularly related to the cytokine designated CTLA-8
(also referred to as IL-17).
[0021] Rat, mouse, human forms and a viral homolog of CTLA-8 have
been described and their sequences available from GenBank. See
Rouvier, et al, (1993) J. Immunol, 150:5445-5456; Yao, et al.
(1995) Immunity 3:811-821; Yao, et al. (1995) J. Immunol.
155:5483-5486; and Kennedy, et al. (1996) J. Interferon and
Cvtokine Res. 16:611-617. The CTLA-8 has activities implicated in
arthritis, kidney graft rejection, tumorigenicity, virus-host
interactions, and innate immunity; and appears to exhibit certain
regulatory functions similar to IL-6. See PubMed (search for
IL-17); Chabaud, et al. (1998) J. Immunol, 63:139-148; Amin, et al.
(1998) Curr. Opin. Rheumatol. 10:263-268; Van Kooten, et al. (1998)
J. Am. Soc. Nephrol, 9:1526-1534; Fossiez, et al. (1998) Int. Rev.
Immunol. 16:541-551; Knappe, et al. (1998) J. Virol. 72:5797-5801;
Seow (1998) Vet. Immuno. Immunopathol. 63:139-48; and Teunissen, et
al. (1998) J. Invest. Dermatol. 111:645-649. A report on the
signaling through the NF.kappa.B transcription factor implicates a
signal pathway which is used in innate immunity. Shalom-Barak, et
al. (1998) J. Biol. Chem. 273:27467-27473.
[0022] The IL-174 cDNA sequences exhibit various features which are
characteristic of mRNAs encoding cytokines, growth factors, and
oncogenes. The IL-17 is the first member of this newly recognized
family of cytokines related to TGF-.beta., and a number of members
of this family designated "IL-170" have been identified. The fold
for this family is predicted to be that of the TGF-.beta. family of
cytokines. The TGF-.beta. family of cytokines, and the IL-170
family share the common feature of a cystine knot motif,
characterized by a particular spacing of cysteine residues. See,
e.g., Sun and Davies (1995) Ann. Rev, Biophys. Biomolec. Struct.
24:269-291; McDonald, et al. (1993) Cell 73:421-424; and Isaacs
(1995) Curr. Op. Struct. Biol. 5:391-395. In particular, the
structures suggest a number of conserved cysteines. The disulfide
linkages should be cysteines 2 with 5; and 3 with 6; and 1 with 4.
Functional significance of the fold similarity suggests formation
of dimers for the IL-170 family. As a consequence, IL-170 dimers
would bring together two cell surface receptors, through which
signal transduction will occur.
[0023] These new proteins are designated CTLA-8 related, or
generally IL-170, proteins. The natural proteins should be capable
of mediating various physiological responses which would lead to
biological or physiological responses in target cells, e.g., those
responses characteristic of cytokine signaling.
[0024] Purified CTLA-8, when cultured with synoviocytes, is able to
induce the secretion of IL-6 from these cells. This induction is
reversed upon the addition of a neutralizing antibody raised
against human CTLA-8. Endothelial, epithelial, fibroblast and
carcinoma cells also exhibit responses to treatment with CTLA-8.
This data suggests that CTLA-8 may be implicated in inflammatory
fibrosis, e.g., psoriasis, sclerodermia, lung fibrosis, or
cirrhosis. CTLA-8 may also cause proliferation of carcinomas or
other cancer cells inasmuch as IL-6 often acts as a growth factor
for such cells. As such, the newly discovered other related family
members are likely to have similar or related biological
activities.
[0025] The cytokine IL-174, from mouse and human, has been
described earlier in PCT/US00/00006, which is incorporated herein
by reference for all purposes. The nucleic acid sequence and the
corresponding amino acid sequence encoding human IL-174 are set
forth as SEQ ID Nos. 1 and 2 respectively. Important predicted
structural motifs include, e.g., cAMP PK sites at 21-24, 53-56, and
95-98; Ca phosphorylation sites at 15-17, 16-18, and 45-47;
myristoly sites at 12-16, 115-119, and 118-122; N-glycosyl site at
104-107; phosphorylation sites at 21, 23, 43, 53, 56, 95, 98, and
131; PKC phosphorylation sites at 41.43 and 119-121; and tyrosine
kinase site at 95-102, The mouse nucleic acid and corresponding
amino acid sequence encoding IL-174 are set forth as SEQ ID Nos. 3
and 4 respectively. Important predicted motifs include, e.g., cAMP
PK sites at 29-32 and 61-64; Ca phosphorylation sites at 18-20,
53-55, and 67-69; myristoly site at 123-127; N-glycosylation site
at 112-114; and phosphorylation sites at 29, 31, 51, 53, 61, 64,
139, and 141; and PKC phosphorylation sites at 2-4, 49-51, and
127-129.
II. Cytokine Agonists and Antagonists
[0026] Mammalian IL-174 cytokines were described previously in
PCT/US00/00006. Various agonists and antagonists of the natural
ligands can be produced.
A. IL-174 and Variants
[0027] IL-174 agonists will exhibit some or all of the signaling
functions of the cytokine. Various mammalian IL-174 sequences may
be evaluated to determine what residues are conserved across
species, suggesting what residues may be changed without dramatic
effects on biological activity. Alternatively, conservative
substitutions are likely to retain biological activity, thus
leading to variant forms of the cytokine which will retain agonist
activity. Standard methods for screening mutant or variant IL-174
polypeptides will determine what sequences will be useful
therapeutic agonists.
[0028] In addition, certain nucleic acid expression methods may be
applied. Various promoters may be operably linked to the gene,
thereby allowing for regulated expression.
[0029] Alternatively, antagonist activity may be tested for. Tests
for ability to antagonize cytokine activity can be developed using
assays as described below. Various ligand homologs can be created
which retain receptor binding capacity, but lacking signaling
capability can be prepared. Small molecules may also be screened
for ability to antagonize IL-174 function. See generally Gilman, et
al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of
Therapeutics, 8th Ed,, Pergarnon Press; Remington's Pharmaceutical
Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Pa., each
of which is incorporated herein by reference.
B. Antibodies
[0030] The present invention provides for the use of an antibody or
binding composition which specifically binds to IL-174, preferably
mammalian, e.g., primate, human, cat, dog, rat, or mouse, and
neutralizes the ability of the cytokine to mediate its signal.
Antibodies can be raised to various IL-174 proteins, including
individual, polymorphic, allelic, strain, or species variants, and
fragments thereof, both in their naturally occurring (full-length)
forms or in their recombinant forms. Additionally, antibodies can
be raised to IL-174 polypeptides in both their native (or active)
forms or in their inactive, e.g., denatured, forms, which may
neutralize ligand capacity to mediate its signal. Antibodies may
block the interaction of the ligand with its receptor.
[0031] A number of immunogens may be selected to produce antibodies
specifically reactive, or selective for binding, with IL-174
proteins. Recombinant protein is a preferred immunogen for the
production of monoclonal or polyclonal antibodies. Naturally
occurring protein, from appropriate sources, e.g., primate, rodent,
etc., may also be used either in pure or impure form. Synthetic
peptides, made using the IL-174 protein sequences described herein,
may also be used as an immunogen for the production of antibodies
to IL-174 proteins. Recombinant protein can be expressed and
purified in eukaryotic or prokaryotic cells as described, e.g., in
Coligan, et al. (eds. 1995 and periodic supplements) Current
Protocols in Protein Science John Wiley & Sons, New York, N.Y.;
and Ausubel, et al (eds. 1987 and periodic supplements) Current
Protocols in Molecular Biology, Greene/Wiley, New York, N.Y.
Naturally folded or denatured material can be used, as appropriate,
for producing antibodies. Either monoclonal or polyclonal
antibodies may be generated, e.g., for subsequent use in
immunoassays to measure the protein, or for immunopurification
methods.
[0032] Methods of producing polyclonal antibodies are well known to
those of skill in the art. Typically, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized with the mixture. The animal's immune response to the
immunogen preparation is monitored by taking test bleeds and
determining the titer of reactivity to the IL-174 protein or
peptide of interest. For example, when appropriately high titers of
antibody to the immunogen are obtained, usually after repeated
immunizations, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be performed, if desired.
See, e.g., Harlow and Lane Antibodies, A Laboratory Manual CSH
Press, NY; or Coligan (ed.) Current Protocols in Immunology.
Immunization can also be performed through other methods, e.g., DNA
vector immunization. See, e.g., Wang, et al. (1997) Virology
228:278-284.
[0033] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Typically, following
immunizations, spleen cells from the animal are immortalized,
commonly by fusion with a myeloma cell. See, Kohler and Milstein
(1976) Eur. J. immunol. 6:511-519. Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods known in the art. See,
e.g., Doyle, et al. (eds. 1994 and periodic supplements) Cell and
Tissue Culture: Laboratory Procedures, Wiley, New York , N.Y.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity
for the antigen, and yield of the monoclonal antibodies produced by
such cells may be enhanced by various techniques, including
injection into the peritoneal cavity of a vertebrate host.
Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according, e.g., to the general
protocol outlined by Huse, et al. (1989) Science 246:1275-1281.
[0034] Antibodies or binding compositions, including binding
fragments and single chain versions, against predetermined
fragments of IL-174 polypeptides can be raised by immunization of
animals with conjugates of the fragments with carrier proteins as
described above. Monoclonal antibodies are prepared from cells
secreting the desired antibody.
[0035] These antibodies can be screened for binding to normal or
defective IL-174 protein. 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 10 .mu.M, more typically
at least about 3 .mu.M, preferably at least about 10 and more
preferably at least about 3 .mu.M or better.
[0036] In some instances, it is desirable to prepare monoclonal
antibodies (mAbs) 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.)
[0037] Academic Press, New York, N.Y.; and particularly in Kohler
and Milstein (1975) Nature 256:495-497, which discusses one method
of generating monoclonal antibodies. Summarized briefly, this
method involves injecting an animal with an immunogen. The animal
is then sacrificed and cells taken from its spleen, which are then
fused with myeloma cells. The result is a hybrid cell or
"hybridoma" that is capable of reproducing in vitro. The population
of hybridomas is then screened to isolate individual clones, each
of which secrete a single antibody species to the immunogen. In
this manner, the individual antibody species obtained are the
products of immortalized and cloned single B cells from the immune
animal generated in response to a specific site recognized on the
immunogenic substance.
[0038] Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, e.g., 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 341:544546. 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; and Queen, et al. (1989) Proc. Nat'l Acad. Sci. USA
86:10029-10033; or made in transgenic mice, see Mendez, et al.
(1997) Nature Genetics 15:146-156; Abgenix and Medarex
technologies,
[0039] Antibody binding compounds, including binding fragments, of
this invention can have significant diagnostic or therapeutic
value. They can be useful as non-neutralizing binding compounds and
can be coupled to toxins or radionuclides so that when the binding
compound binds to the antigen, a cell expressing it, e.g., on its
surface, is killed. Further, these binding compounds can be
conjugated to drugs or other therapeutic agents, either directly or
indirectly by means of a linker, and may effect drug targeting.
C. Other Molecules
[0040] Antibodies are merely one form of specific binding
compositions. Other binding compositions, which will often have
similar uses, include molecules that bind with specificity to
IL-174, e.g., in a protein-binding partner fashion, an
antibody-antigen interaction, or in a natural physiologically
relevant protein-protein interaction, either covalent or
non-covalent, e.g., proteins which specifically associate with
IL-174 protein. The molecule may be a polymer, or chemical reagent.
A functional analog may be a protein with structural modifications,
or may be a structurally unrelated molecule, e.g., which has a
molecular shape which interacts with the appropriate binding
determinants.
[0041] Drug screening using antibodies or IL-174 or fragments
thereof can be performed to identify compounds having binding
affinity to 1L-174, or can block or simulate the natural
interaction with ligand. Subsequent biological assays can then be
utilized to determine if the compound has intrinsic blocking
activity and is therefore an antagonist. Likewise, a compound
having intrinsic stimulating activity can signal to the cells via
the IL-174 pathway and is thus an agonist in that it simulates the
activity of a ligand. Mutein antagonists may be developed which
maintain receptor binding but lack signaling.
[0042] Structural studies of the ligands will lead to design of new
variants, particularly analogs exhibiting agonist or antagonist
properties on the receptor. This can be combined with previously
described screening methods to isolate muteins exhibiting desired
spectra of activities.
[0043] As receptor specific binding molecules are provided, also
included are small molecules identified by screening procedures. In
particular, it is well known in the art how to screen for small
molecules which interfere, e.g., with ligand binding to the
receptor, often by specific binding to the receptor and blocking of
binding by natural ligand. See, e.g., meetings on High Throughput
Screening, International Business Communications, Southborough,
Mass. 01772-1749. Such molecules may compete with natural ligands,
and selectively bind to the IL-174.
III. Immunoassays
[0044] Immunoassays are valuable in diagnosing a disease or
disorder associated with IL-174 imbalance or pathology. Qualitative
or quantitative measurement of a particular protein can be
performed by a variety of immunoassay methods. For a review of
immunological and immunoassay procedures in general, see Stites and
Terr (eds.) (1991) Basic and Clinical Immunology (7th ed.).
Moreover, the immunoassays of the present invention can be
performed in many configurations, which are reviewed extensively in
Maggio (ed. 1980) Enzyme Immunoassay CRC Press, Boca Raton,
Florida; Tijan (1985) "Practice and Theory of Enzyme Immunoassays,"
Laboratory Techniques in Biochemist and Molecular Biology, Elsevier
Science Publishers B.V., Amsterdam; and Harlow and Lane Antibodies:
A Laboratory Manual, supra. See also Chan (ed. 1987) Immunoassay: A
Practical Guide Academic Press, Orlando, Fla.; Price and Newman
(eds. 1991) Principles and Practice of Immunoassays Stockton Press,
N.Y.; and Ngo (ed. 1988) Non-isotopic Immunoassays Plenum Press,
NY.
[0045] Immunoassays for measurement of IL-174 proteins or peptides
can be performed by a variety of methods known to those skilled in
the art. In brief, immunoassays to measure the protein can be
either competitive or noncompetitive binding assays. In competitive
binding assays, the sample to be analyzed competes with a labeled
analyte for specific binding sites on a capture agent bound to a
solid surface. Preferably the capture agent is an antibody
specifically reactive with IL-174 proteins produced as described
above. The concentration of labeled analyte bound to the capture
agent is inversely proportional to the amount of free analyte
present in the sample.
[0046] In a competitive binding immunoassay, the IL-174 protein
present in the sample competes with labeled protein for binding to
a specific binding agent, e.g., an antibody specifically reactive
with IL-174 protein. The binding agent may be bound to a solid
surface to effect separation of bound labeled protein from the
unbound labeled protein. Alternatively, the competitive binding
assay may be conducted in liquid phase and a variety of techniques
known in the art may be used to separate the bound labeled protein
from the unbound labeled protein. Following separation, the amount
of bound labeled protein is determined. The amount of protein
present in the sample is inversely proportional to the amount of
labeled protein binding.
[0047] Alternatively, a homogeneous immunoassay may be performed in
which a separation step is not needed. In these immunoassays, the
label on the protein is altered by the binding of the protein to
its specific binding agent. This alteration in the labeled protein
results in a decrease or increase in the signal emitted by label,
so that measurement of the label at the end of the immunoassay
allows for detection or quantitation of the protein.
[0048] IL-174 proteins may also be determined by a variety of
noncompetitive immunoassay methods. For example, a two-site, solid
phase sandwich immunoassay may be used. In this type of assay, a
binding agent for the protein, for example an antibody, is attached
to a solid support. A second protein binding agent, which may also
be an antibody, and which binds the protein at a different site, is
labeled. After binding at both sites on the protein has occurred,
the unbound labeled binding agent is removed and the amount of
labeled binding agent bound to the solid phase is measured. The
amount of labeled binding agent bound is directly proportional to
the amount of protein in the sample.
[0049] Western blot analysis can be used to determine the presence
of IL-174 proteins in a sample. Electrophoresis is carried out,
e.g., on a tissue sample suspected of containing the protein.
Following electrophoresis to separate the proteins, and transfer of
the proteins to a suitable solid support, e.g., a nitrocellulose
filter, the solid support is incubated with an antibody reactive
with the protein. This antibody may be labeled, or alternatively
may be detected by subsequent incubation with a second labeled
antibody that binds the primary antibody.
[0050] The immunoassay formats described above may employ labeled
assay components. The label may be coupled directly or indirectly
to the desired component of the assay according to methods well
known in the art. A wide variety of labels and methods may be used.
Traditionally, a radioactive label incorporating .sup.3H,
.sup.125I, .sup.35s, .sup.14C, or .sup.32P was used.
Non-radioactive labels include ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on sensitivity
required, ease of conjugation with the compound, stability
requirements, and available instrumentation. For a review of
various labeling or signal producing systems which may be used, see
U.S. Pat. No. 4,391,904.
[0051] Antibodies reactive with a particular protein can also be
measured by a variety of immunoassay methods. Thus modifications of
the above procedures may be used to determine the amounts or
affinities of various IL-174 antibodies or antibody
preparations.
[0052] For a review of immunological and immunoassay procedures
applicable to the measurement of antibodies by immunoassay
techniques, see Stites and Terr (eds.) Basic and Clinical
Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra;
and Harlow and Lane Antibodies, A Laboratory Manual, supra.
[0053] Screens to evaluate the binding and activity of mAbs and
binding compositions encompass a variety of methods. Binding can be
assayed by detectably labeling the antibody or binding composition
as described above. Cells responsive to IL-174 can be used to assay
antibody or binding composition.
[0054] Evaluation of antibodies can be performed in other animals,
e.g., humans using various methods. For example, blood samples are
withdrawn from patients suffering from an indicated disease or
disorder before and after treatment, e.g., with a candidate
mAb.
IV. Uses
[0055] IL-174 now has demonstrated effects on various cells, which
effects may be indirect, as well as direct. A statistically
significant change in the numbers or effects on cells will
typically be at least about 10%, preferably 20%, 30%, 50%, 70%,
90%, or more. Effects of greater than 100%, e.g., 130%, 150%, 2X,
3X, 5X, etc., will often be desired. The effects may be specific in
reducing symptoms or signs of the indicated conditions.
[0056] The present invention is useful in the treatment of medical
conditions or diseases associated with immunological conditions
described. See, e.g., Frank, et al. (eds. 1995)
[0057] Samter's Immunologic Diseases, 5th Ed., vols, I-II, Little,
Brown and Co., Boston, Mass.
[0058] The agonists or antagonists described may be combined with
other treatments of the medical conditions described herein, e.g.,
another cytokine involved in Th1/Th2 balance (such as IL-1.gamma.
and/or IL-12 to Th1; or IL-4 to Th2), an antibiotic, immune
suppressive therapeutic, immune adjuvant, analgesic,
anti-inflammatory drug, growth factor, vasodilator, or
vasoconstrictor.
[0059] Preferred combination therapies include IL-174 agonists with
various therapeutics which promote Th2 type immune responses (e.g.,
IL-4, IL-1.gamma. antagonists, IL-12 antagonists), inflammatory
agents, such as topical, transdermal, or systemic irritants,
various growth factors, and hematopoietic factors. Antagonists of
IL-174, e.g., antibodies or ligand muteins, can be combined with
therapeutics which promote Th1 type immune responses (e.g.,
IL-1.gamma. and/or IL-12, IL-4 antagonists), or anti-inflammatories
(e.g., steroids, glucocortical steroids).
[0060] Standard immunological techniques are described, e.g,, in
Hertzenberg, et al. (eds. 1996) Weir's Handbook of Immunology vols.
1-4, Blackwell Science; Coligan (1991) Current Protocols in
Immunology Wiley/Greene, N.Y.; and Methods in Enzymology volumes
70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163.
These will allow use of the reagents for purifying cell
subpopulations, etc.
[0061] To prepare pharmaceutical or sterile compositions including
IL-174, antagonists, or combinations, the reagent is admixed with a
pharmaceutically acceptable carrier or excipient which is
preferably inert. Preparation of such pharmaceutical compositions
is known in the art, see, e.g., Remington's Pharmaceutical Sciences
and U.S. Pharmacopeia; National Formulary, Mack Publishing Company,
Easton, Pa. (1984).
[0062] Agonists, e.g., natural ligand, or antagonists, e.g.,
antibodies or binding compositions, are normally administered
parenterally, preferably intravenously. Since such protein or
peptide antagonists may be immunogenic they are preferably
administered slowly, either by a conventional IV administration set
or from a subcutaneous depot, e.g. as taught by Tomasi, et al.,
U.S. Pat. No. 4,732,863.
[0063] When administered parenterally the therapeutics will often
be formulated in a unit dosage injectable form (solution,
suspension, emulsion) in association with a pharmaceutically
acceptable parenteral vehicle. Such vehicles are typically
inherently nontoxic and nontherapeutic. Agonists, which typically
will be smaller biologics, will typically be administered in
smaller doses than antibody biologics. The antagonist may be
administered in aqueous vehicles such as water, saline or buffered
vehicles with or without various additives and/or diluting agents.
Alternatively, a suspension, such as a zinc suspension, can be
prepared to include the peptide. Such a suspension can be useful
for subcutaneous (SQ), intradermal (ID), or intramuscular (IM)
injection. The proportion of therapeutic entity and additive can be
varied over a broad range so long as both are present in effective
amounts. The therapeutic is preferably formulated in purified form
substantially free of aggregates, other proteins, endotoxins, and
the like, at concentrations of about 5 to 30 mg/ml, preferably 10
to 20 mg/ml. Preferably, the endotoxin levels are less than 2.5
EU/ml. See, e.g., Avis, et al. (eds. 1993) Pharmaceutical Dosage
Forms: Parenteral Medications 2d ed., Dekker, N.Y.; Lieberman, et
al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets 2d ed.,
Dekker, N.Y.; Lieberman, et at (eds. 1990) Pharmaceutical Dosage
Forms: Disperse Systems Dekker, N.Y.; Fodor, et al. (1991) Science
251:767-773; Coligan (ed.) Current Protocols in Immunology; Hood,
et al. Immunology Benjamin/Cummings; Paul (ed. 1997) Fundamental
Immunology 4th ed., Academic Press; Parce, et al. (1989) Science
246:243-247; Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA
87:4007-4011; and Blundell and Johnson (1976) Protein
Crystallography, Academic Press, New York.
[0064] Selecting an administration regimen for a therapeutic
agonist or antagonist depends on several factors, including the
serum or tissue turnover rate of the therapeutic, the
immunogenicity of the therapeutic, or the accessibility of the
target cells. Preferably, an administration regimen maximizes the
amount of therapeutic delivered to the patient consistent with an
acceptable level of side effects. Accordingly, the amount of
therapeutic delivered depends in part on the particular agonist or
antagonist and the severity of the condition being treated.
Guidance in selecting appropriate doses of antibodies is found in
the literature on therapeutic uses, e.g. Bach et al., chapter 22,
in Ferrone, et at. (eds. 1985), Handbook of Monoclonal Antibodies
Noges Publications, Park Ridge, N.J.; and Russell, pgs. 303-357,
and Smith et at, pgs. 365-389, in Haber, et al. (eds. 1977)
Antibodies in Human Diagnosis and Therapy Raven Press, New York,
N.Y.
[0065] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known in the art to
affect treatment or predicted to affect treatment. Generally, the
dose begins with an amount somewhat less than the optimum dose and
it is increased by small increments thereafter until the desired or
optimum effect is achieved relative to any negative side effects.
Preferably, an antibody or binding composition thereof that will be
used is derived from the same species as the animal targeted for
treatment, thereby minimizing a humoral response to the
reagent.
[0066] The total weekly dose ranges for antibodies or fragments
thereof; which specifically bind to IL-174, range generally from
about 1 ng, more generally from about 10 ng, typically from about
100 ng; more typically from about 1 .mu.g, more typically from
about 10 .mu.g, preferably from about 100 .mu.g, and more
preferably from about 1 mg per kilogram body weight. Although
higher amounts may be more efficacious, the lower doses typically
will have fewer adverse effects. Generally the range will be less
than 100 mg, preferably less than about 50 mg, and more preferably
less than about 25 mg per kilogram body weight.
[0067] The weekly dose ranges for antagonists, e.g., antibody,
binding fragments, range from about 10 .mu.g, preferably at least
about. 50 ug, and more preferably at least about 100 .mu.g per
kilogram of body weight. Generally, the range will be less than
about 1000 .mu.g, preferably less than about 500 .mu.g, and more
preferably less than about 100 .mu.g per kilogram of body weight.
Dosages are on a schedule which effects the desired treatment and
can be periodic over shorter or longer term. In general, ranges
will be from at least about 10 .mu.g to about 50 mg, preferably
about 100 .mu.g to about 10 mg per kilogram body weight.
[0068] Agonists, or other antagonists of the ligands, e.g.,
muteins, are also contemplated. Hourly dose ranges for cytokine or
muteins range from at least about 10 .mu.g, generally at least
about 50 .mu.g, typically at least about 100 .mu.g, and preferably
at least 500 .mu.g per hour. Generally the dosage will be less than
about 100 mg, typically less than about 30 mg, preferably less than
about 10 mg, and more preferably less than about 6 mg per hour.
General ranges will be from at least about 1 .mu.g to about 1000
.mu.g, preferably about 10 .mu.g to about 500 .mu.g per hour.
[0069] The present invention also provides for administration of
IL-174 antibodies or binding compositions in combination with known
therapies, e.g., therapeutics which alleviate the symptoms
associated with excessive inflammatory responses, such as steroids,
particularly glucocorticoids. Daily dosages for glucocorticoids
will range from at least about 1 mg, generally at least about 2 mg,
and preferably at least about S mg per day. Generally, the dosage
will be less than about 100 mg, typically less than about 50 mg,
preferably less than about 20 mg, and more preferably at least
about 10 mg per day. In general, the ranges will be from at least
about 1 mg to about 100 mg, preferably from about 2 mg to 50 mg per
day.
[0070] The phrase "effective amount" means an amount sufficient to
effect a desired response, or to ameliorate a symptom or sign of
the indicated condition. Typical mammalian hosts will include mice,
rats, cats, dogs, and primates, including humans. An effective
amount for a particular patient may vary depending on factors such
as the condition being treated, the overall health of the patient,
the method, route, and dose of administration and the severity of
side affects. Preferably, the effect will result in a change in
quantitation of at least about 10%, preferably at least 20%, 30%,
50%, 70%, or even 90% or more. When in combination, an effective
amount is in ratio to a combination of components and the effect is
not limited to individual components alone.
[0071] An effective amount of therapeutic will modulate the
symptoms typically by at least about 10%; usually by at least about
20%; preferably at least about 30%; or more preferably at least
about 50%. Alternatively, modulation of movement will mean that the
movement or trafficking of various cell types is affected. Such
will result in, e.g., statistically significant and quantifiable
changes in the numbers of cells being affected. This may be an
increase or decrease in the numbers of target cells being attracted
within a time period OT target area.
[0072] The present invention provides reagents which will find use
in therapeutic applications as described elsewhere herein, e.g., in
the general description for treating disorders associated with the
indicated conditions. See, e.g., 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, NY;
Gilman, et al. (eds. 1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press;
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Pa.; Langer (1990) Science 249:1527-1533;
and Merck Index, Merck & Co., Rahway, N.J.
[0073] Antibodies to IL-174 proteins may be used for the
identification or sorting of cell populations expressing IL-174
protein. Methods to sort such populations are well known in the
art, see, e.g., 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.
Populations of cells expressing the IL-174 receptor can also be
purified using magnetic beads as described, e.g., in Bieva, et al.
(1989) Exp. Hematol. 17:914-920; Hernebtub, et al. (1990) Bioconj.
Chem. 1:411-418; Vaccaro (1990) Am. Biotechnol. Lab. 3:30.
[0074] Moreover, antisense nucleic acids maybe used. For example,
antisense constructs specific to nucleic acids encoding, e.g., the
ligand, may function in a manner like ligand antagonists, and
antisense constructs specific to those encoding the receptor may
function like receptor antagonists. See, e.g., Stepkowski, et al,
(1998) Transplantation 66:699-707; and ISIS Pharmaceuticals
technology. Thus, it may be possible to block the signaling through
these pathways with antisense nucleic acids.
[0075] Using the assay methods described above, the antibodies or
binding compositions are useful in diagnosing disease states which
result in the indicated disorders. Antibodies raised against an
IL-174 protein 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. Combinations of these signals may be also pursued.
[0076] The broad scope of this invention is best understood with
reference to the following examples, which are not intended to
limit the inventions to the specific embodiments.
EXAMPLES
I. General Methods
[0077] The cytokine IL-174, from mouse and human, has been
described earlier in PCT/US00/00006, which is incorporated herein
by reference for all purposes.
[0078] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d
ed.), vols. 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene/Wiley,
New York; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to
Methods and Applicatioqs Academic Press, N.Y. 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" in Methods in Enzymology, vol. 182, and other
volumes in this series; manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif.; and Coligan, et al. (eds.) (1995 and
periodic supplements) Current Protocols in Protein Science, John
Wiley & Sons, New York, N.Y. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) OIA express: The High Level Expression
& Protein Purification System QIAGEN, Inc., Chatsworth,
Calif.
[0079] 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) 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.
[0080] 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.
II. Antibody Production
[0081] Appropriate mammals are immunized with appropriate amounts
of IL-174 gene transfected cells, e.g., intraperitoneally every 2
weeks for 8 weeks. Typically, rodents are used, though other
species should accommodate production of selective and specific
antibodies. The final immunization is given intravenously (IV)
through the tail vein.
[0082] Generic polyclonal antibodies may be collected.
Alternatively, monoclonal antibodies can be produced. For example,
four days after the IV injection, the spleen is removed and fused
to SP2/0 and NS1 cells. HAT resistant hybridomas are selected,
e.g., using a protocol designed by Stem Cell Technologies
(Vancouver, BC). After 10 days of HAT selection, resistant foci are
transferred to 96 well plates and expanded for 3 days. Antibody
containing supernatants are analyzed, e.g., by FACS for binding to
NIH3T3/surface IL-174 transfectants. Many different IL-174 mAbs are
typically produced. Those antibodies may be isolated and modified,
e.g., by labeling or other means as is standard in the art. See,
e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH
Press; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed.) Academic Press, New York, N.Y. Methods to conjugate
magnetic reagents, toxic entities, labels, attach the antibodies to
solid substrates, to sterile filter, etc., are known in the
art.
III. IL-174 Antagonists
[0083] Various antagonists of IL-174 are made available. For
example, antibodies against the cytokine itself may block the
binding of ligand to its receptor, thereby serving as a direct
receptor antagonist, Other antagonists may function by blocking the
binding of ligand to receptor, e.g., by binding to the ligand in a
way to preclude the possibility of binding to the receptor. Other
antagonists, e.g., mutein antagonists, may bind to the receptor
without signaling, thereby blocking a true agonist from binding.
Many of these may serve to block the signal transmitted to target
cells, specifically IL-174 responsive cells.
[0084] Information on the criticality of particular residues is
determined using standard procedures and analysis. Standard
mutagenesis analysis is performed, e.g., by generating many
different variants at determined positions, e.g., at the positions
identified above, and evaluating biological activities of the
variants. This may be performed to the extent of determining
positions which modify activity, or to focus on specific positions
to determine the residues which can be substituted to either
retain, block, or modulate biological activity.
[0085] Alternatively, analysis of natural variants can indicate
what positions tolerate natural mutations. This may result from
populational analysis of variation among individuals, or across
strains or species. Samples from selected individuals are analyzed,
e.g., by PCR analysis and sequencing, This allows evaluation of
population polymorphisms.
IV. Adenoviral infection
[0086] Adenovirus constructs were prepared. Mice were infected with
adenovirus-IL-74 via the intranasal route or the intravenous route.
See, e.g., Hitt, et al. (1997) Adv. Pharmacol, 40:137-195. The
doses of adenovirus-IL-74 given ranged from 5.times.10.sup.9 to
5.times.10.sup.10 particles. Mice were evaluated 7, 14, 21, and 35
days post infection using standard histologic, immunologic, and
hematologic methods
V. Histologic Analysis
[0087] Tissues from mice were fixed in formalin, routinely
processed, sectioned, and stained with hematoxylin and eosin for
microscopic examination. The organs examined included the lung,
heart, liver, kidney, spleen, bone marrow, and gastrointestinal
tract. See, e.g., Kerr (1999) Atlas of functional Histology;
Sternberg (ed. 1998) Histology for Pathologists; and Stevens and
Lowe (1996) Human Histology.
VI. Hematopoietic Colony Forming Assays
[0088] Hematopoietic progenitors in the spleens of mice were
enumerated in routine colony-forming assays. Spleen cells were
depleted of erythrocytes by hypotonic lysis.
[0089] Spleen cells were incubated with unmodified rat monoclonal
antibodies specific for CD2 (RM2.2; see Yagata, et al. (1989) Proc.
Nat'l Acad, Sci. USA 86:645-649), CD8 (53.6.7; see Ledbetter, et
al. Immunol. Rev. 47:63-90), B220 (RA3-6B2; see Coffman, et al.
(1981) Nature 289:681-683), Mac1 (M1/70; see Springer, et al.
(1979) Eur. J. Inununol. 9:301-306), GRI (RB6-8C5; see Julia, et
al. (1988) Eur. J. Immunol. 19:1819-1826), and erythrocytes
(Ter-119; see Spangrude, et al. (1990) Exp. Hemat. 18:920-926). The
lineage positive cells depleted by negative selection using goat
anti-rat coated BioMagR. beads (PerSeptive Biosystems, Framingham,
Mass.) in two successive rounds of treatment. The remaining lineage
negative spleen cells were seeded in 35 mm culture dishes
containing 1 ml modified Iscove's medium (JRH, Kansas City, Kans.),
20% fetal calf serum (FCS) (JRH) 50 mM 2-mercaptoethanol, and 0.8%
(wt/vol) methylcellulose. All cultures were supplemented with
saturating concentrations of various growth factors. Cultures were
incubated at 37.degree. C. in a humidified atmosphere flushed with
5% CO2. After 10 days of incubation, colonies were analyzed for
number and size. Cell morphologies were determined after colonies
were applied to glass slides and stained with Wright's Giemsa for
microscopic examination. See Metcalf (1984) The Hemopoietic Colony
Stimulating Factors, Elsevier, Amsterdam.
VII. Blood Cell Analysis
[0090] Blood from mice was collected in Isotonic Buffered Diluent
(BioChem
[0091] ImmunoSystems, Allentown, Pa.) and hemoglobin, hematocrit,
white blood cell, red blood cell, and platelet counts were
determined by an automatic cell counter (Serono 9010, Serono-Baker
Diagnostics, Inc., Allentown, Pa.). Blood smears were stained with
Wright's Giemsa and differential counts were performed with the aid
of a microscope. See, e.g., Dainiak (1990) The Biology of
Hematopoiesis, Wiley-Liss Inc., New York; Testa and Molineux (1993)
Haemopoiesis, Oxford University Press, New York.
VIII. Determination of Antibody Isotypes
[0092] Sera from mice were serially diluted in isotonic buffer in
microtiter wells coated with rat anti-mouse heavy-chain
class-specific antibodies (anti-.alpha., -.gamma.1, -.gamma.2a, and
-.epsilon.). A biotinylated secondary antibody (specific for
heavy-chain classes) was added and then reacted with
streptavidin-HRP (Jackson ImmunoResearch Laboratories Inc., West
Grove, Pa.). The colored product produced by this TMB peroxidase
substrate system (Kirkegaard & Perry Laboratories,
Gaithersburg, Md.) is detected under a long-wavelength UV lamp (450
nanometers). See, e.g., Coligan, et al. (eds. 1991 and supplements)
Current Protocols in Immunology, Greene/Wiley.
IX. Bronchio-Alveolar Lavage Fluid Analysis
[0093] Bronchioalveolar lavage (BAL) fluid was collected from
sacrificed mice by inserting a needle into the trachea and
dispensing 1 ml of DMEM media. 200 .mu.l of the recovered sample
was examined for cell composition by cytospin followed by
Wright-Giemsa stain. Differential counts were performed with the
aid of a microscope.
X. Quantification of Gene Expression by Real-Time RT-PCR
[0094] Sample tissues were excised from mice following treatment
and were snap-frozen in liquid nitrogen. Total RNA was extracted
using RNAstat60 (Molecular Research Center) according to the
manufacturer's directions and were stored at -80.degree. C. in
nuclease-free. For cDNA synthesis RNA was incubated with 10 units
of DNase I (Boehringer Mannheim) in the presence of RNasin
(Promega) for 30 minutes at 37.degree. C. The samples were then
heat inactivated at 95.degree. C. for 10 minutes, chilled, and
reverse transcribed with Superscript II reverse transcriptase
(Gibco/BRL) with random hexamers according to the manufacturer's
protocol. Primers were either obtained from Perkin Elmer or
generated with Primer Express software (Perkin Elmer) and were
synthesized in the DNAX primer core facility. Whenever possible,
primer pairs were designed to span intron/exon borders. Samples
were then subjected to 40 cycles of amplification of 95.degree. C.
for 15 seconds followed by 60.degree. C. for 1 minute using an ABI
GeneAmp 5700 sequence detection system and SYBR green buffer
according to the manufacturer (Perkin Elmer). PCR amplification of
the housekeeping gene ubiquitin was performed for each sample to
control for sample loading and to allow normalization between
samples according to the manufacturer's instructions (Perkin
Elmer). Both water and genomic DNA controls were included to insure
specificity. Each data point was examined for integrity by analysis
of the amplification plot and disassociation curves.
[0095] All citations herein are incorporated herein by reference to
the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0096] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited by the terms of the appended claims,
along with the full scope of equivalents to which such claims are
entitled; and the invention is not to be limited by the specific
embodiments that have been presented herein by way of example.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 4 <210> SEQ ID NO 1 <211> LENGTH: 504 <212>
TYPE: DNA <213> ORGANISM: primate <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (19)..(501)
<221> NAME/KEY: mat_peptide <222> LOCATION: (67)..(501)
<400> SEQUENCE: 1 tgagtgtgca gtgccagc atg tac cag gtg gtt gca
ttc ttg gca atg gtc 51 Met Tyr Gln Val Val Ala Phe Leu Ala Met Val
-15 -10 atg gga acc cac acc tac agc cac tgg ccc agc tgc tgc ccc agc
aaa 99 Met Gly Thr His Thr Tyr Ser His Trp Pro Ser Cys Cys Pro Ser
Lys -5 -1 1 5 10 ggg cag gac acc tct gag gag ctg ctg agg tgg agc
act gtg cct gtg 147 Gly Gln Asp Thr Ser Glu Glu Leu Leu Arg Trp Ser
Thr Val Pro Val 15 20 25 cct ccc cta gag cct gct agg ccc aac cgc
cac cca gag tcc tgt agg 195 Pro Pro Leu Glu Pro Ala Arg Pro Asn Arg
His Pro Glu Ser Cys Arg 30 35 40 gcc agt gaa gat gga ccc ctc aac
agc agg gcc atc tcc ccc tgg aga 243 Ala Ser Glu Asp Gly Pro Leu Asn
Ser Arg Ala Ile Ser Pro Trp Arg 45 50 55 tat gag ttg gac aga gac
ttg aac cgg ctc ccc cag gac ctg tac cac 291 Tyr Glu Leu Asp Arg Asp
Leu Asn Arg Leu Pro Gln Asp Leu Tyr His 60 65 70 75 gcc cgt tgc ctg
tgc ccg cac tgc gtc agc cta cag aca ggc tcc cac 339 Ala Arg Cys Leu
Cys Pro His Cys Val Ser Leu Gln Thr Gly Ser His 80 85 90 atg gac
ccc cgg ggc aac tcg gag ctg ctc tac cac aac cag act gtc 387 Met Asp
Pro Arg Gly Asn Ser Glu Leu Leu Tyr His Asn Gln Thr Val 95 100 105
ttc tac cgg cgg cca tgc cat ggc gag aag ggc acc cac aag ggc tac 435
Phe Tyr Arg Arg Pro Cys His Gly Glu Lys Gly Thr His Lys Gly Tyr 110
115 120 tgc ctg gag cgc agg ctg tac cgt gtt tcc tta gct tgt gtg tgt
gtg 483 Cys Leu Glu Arg Arg Leu Tyr Arg Val Ser Leu Ala Cys Val Cys
Val 125 130 135 cgg ccc cgt gtg atg ggc tag 504 Arg Pro Arg Val Met
Gly 140 145 <210> SEQ ID NO 2 <211> LENGTH: 161
<212> TYPE: PRT <213> ORGANISM: primate <400>
SEQUENCE: 2 Met Tyr Gln Val Val Ala Phe Leu Ala Met Val Met Gly Thr
His Thr -15 -10 -5 -1 Tyr Ser His Trp Pro Ser Cys Cys Pro Ser Lys
Gly Gln Asp Thr Ser 1 5 10 15 Glu Glu Leu Leu Arg Trp Ser Thr Val
Pro Val Pro Pro Leu Glu Pro 20 25 30 Ala Arg Pro Asn Arg His Pro
Glu Ser Cys Arg Ala Ser Glu Asp Gly 35 40 45 Pro Leu Asn Ser Arg
Ala Ile Ser Pro Trp Arg Tyr Glu Leu Asp Arg 50 55 60 Asp Leu Asn
Arg Leu Pro Gln Asp Leu Tyr His Ala Arg Cys Leu Cys 65 70 75 80 Pro
His Cys Val Ser Leu Gln Thr Gly Ser His Met Asp Pro Arg Gly 85 90
95 Asn Ser Glu Leu Leu Tyr His Asn Gln Thr Val Phe Tyr Arg Arg Pro
100 105 110 Cys His Gly Glu Lys Gly Thr His Lys Gly Tyr Cys Leu Glu
Arg Arg 115 120 125 Leu Tyr Arg Val Ser Leu Ala Cys Val Cys Val Arg
Pro Arg Val Met 130 135 140 Gly 145 <210> SEQ ID NO 3
<211> LENGTH: 985 <212> TYPE: DNA <213> ORGANISM:
rodent <220> FEATURE: <221> NAME/KEY: CDS <222>
LOCATION: (1)..(507) <221> NAME/KEY: mat_peptide <222>
LOCATION: (49)..(507) <400> SEQUENCE: 3 atg tac cag gct gtt
gca ttc ttg gca atg atc gtg gga acc cac acc 48 Met Tyr Gln Ala Val
Ala Phe Leu Ala Met Ile Val Gly Thr His Thr -15 -10 -5 -1 gtc agc
ttg cgg atc cag gag ggc tgc agt cac ttg ccc agc tgc tgc 96 Val Ser
Leu Arg Ile Gln Glu Gly Cys Ser His Leu Pro Ser Cys Cys 1 5 10 15
ccc agc aaa gag caa gaa ccc ccg gag gag tgg ctg aag tgg agc tct 144
Pro Ser Lys Glu Gln Glu Pro Pro Glu Glu Trp Leu Lys Trp Ser Ser 20
25 30 gca tct gtg tcc ccc cca gag cct ctg agc cac acc cac cac gca
gaa 192 Ala Ser Val Ser Pro Pro Glu Pro Leu Ser His Thr His His Ala
Glu 35 40 45 tcc tgc agg gcc agc aag gat ggc ccc ctc aac agc agg
gcc atc tct 240 Ser Cys Arg Ala Ser Lys Asp Gly Pro Leu Asn Ser Arg
Ala Ile Ser 50 55 60 cct tgg agc tat gag ttg gac agg gac ttg aat
cgg gtc ccc cag gac 288 Pro Trp Ser Tyr Glu Leu Asp Arg Asp Leu Asn
Arg Val Pro Gln Asp 65 70 75 80 ctg tac cac gct cga tgc ctg tgc cca
cac tgc gtc agc cta cag aca 336 Leu Tyr His Ala Arg Cys Leu Cys Pro
His Cys Val Ser Leu Gln Thr 85 90 95 ggc tcc cac atg gac ccg ctg
ggc aac tcc gtc cca ctt tac cac aac 384 Gly Ser His Met Asp Pro Leu
Gly Asn Ser Val Pro Leu Tyr His Asn 100 105 110 cag acg gtc ttc tac
cgg cgg cca tgc cat ggt gag gaa ggt acc cat 432 Gln Thr Val Phe Tyr
Arg Arg Pro Cys His Gly Glu Glu Gly Thr His 115 120 125 cgc cgc tac
tgc ttg gag cgc agg ctc tac cga gtc tcc ttg gct tgt 480 Arg Arg Tyr
Cys Leu Glu Arg Arg Leu Tyr Arg Val Ser Leu Ala Cys 130 135 140 gtg
tgt gtg cgg ccc cgg gtc atg gct tagtcatgct caccacctgc 527 Val Cys
Val Arg Pro Arg Val Met Ala 145 150 ctgaggctga tgcccggttg
ggagagaggg ccaggtgtac aatcaccttg ccaatgcggg 587 ccgggttcaa
gccctccaaa gccctacctg aagcagcagg ctcccgggac aagatggagg 647
acttggggag aaactctgac ttttgcactt tttggaagca cttttgggaa ggagcaggtt
707 ccgcttgtgc tgctagagga tgctgttgtg gcatttctac tcaggaacgg
actccaaagg 767 cctgctgacc ctggaagcca tactcctggc tcctttcccc
tgaatccccc aactcctggc 827 acaggcactt tctccacctc tccccctttg
ccttttgttg tgtttgtttg tgcatgccaa 887 ctctgcgtgc agccaggtgt
aattgccttg aaggatggtt ctgaggtgaa agctgttatc 947 gaaagtgaag
agatttatcc aaataaacat ctgtgttt 985 <210> SEQ ID NO 4
<211> LENGTH: 169 <212> TYPE: PRT <213> ORGANISM:
rodent <400> SEQUENCE: 4 Met Tyr Gln Ala Val Ala Phe Leu Ala
Met Ile Val Gly Thr His Thr -15 -10 -5 -1 Val Ser Leu Arg Ile Gln
Glu Gly Cys Ser His Leu Pro Ser Cys Cys 1 5 10 15 Pro Ser Lys Glu
Gln Glu Pro Pro Glu Glu Trp Leu Lys Trp Ser Ser 20 25 30 Ala Ser
Val Ser Pro Pro Glu Pro Leu Ser His Thr His His Ala Glu 35 40 45
Ser Cys Arg Ala Ser Lys Asp Gly Pro Leu Asn Ser Arg Ala Ile Ser 50
55 60 Pro Trp Ser Tyr Glu Leu Asp Arg Asp Leu Asn Arg Val Pro Gln
Asp 65 70 75 80 Leu Tyr His Ala Arg Cys Leu Cys Pro His Cys Val Ser
Leu Gln Thr 85 90 95 Gly Ser His Met Asp Pro Leu Gly Asn Ser Val
Pro Leu Tyr His Asn 100 105 110 Gln Thr Val Phe Tyr Arg Arg Pro Cys
His Gly Glu Glu Gly Thr His 115 120 125 Arg Arg Tyr Cys Leu Glu Arg
Arg Leu Tyr Arg Val Ser Leu Ala Cys 130 135 140 Val Cys Val Arg Pro
Arg Val Met Ala 145 150
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 4 <210>
SEQ ID NO 1 <211> LENGTH: 504 <212> TYPE: DNA
<213> ORGANISM: primate <220> FEATURE: <221>
NAME/KEY: CDS <222> LOCATION: (19)..(501) <221>
NAME/KEY: mat_peptide <222> LOCATION: (67)..(501) <400>
SEQUENCE: 1 tgagtgtgca gtgccagc atg tac cag gtg gtt gca ttc ttg gca
atg gtc 51 Met Tyr Gln Val Val Ala Phe Leu Ala Met Val -15 -10 atg
gga acc cac acc tac agc cac tgg ccc agc tgc tgc ccc agc aaa 99 Met
Gly Thr His Thr Tyr Ser His Trp Pro Ser Cys Cys Pro Ser Lys -5 -1 1
5 10 ggg cag gac acc tct gag gag ctg ctg agg tgg agc act gtg cct
gtg 147 Gly Gln Asp Thr Ser Glu Glu Leu Leu Arg Trp Ser Thr Val Pro
Val 15 20 25 cct ccc cta gag cct gct agg ccc aac cgc cac cca gag
tcc tgt agg 195 Pro Pro Leu Glu Pro Ala Arg Pro Asn Arg His Pro Glu
Ser Cys Arg 30 35 40 gcc agt gaa gat gga ccc ctc aac agc agg gcc
atc tcc ccc tgg aga 243 Ala Ser Glu Asp Gly Pro Leu Asn Ser Arg Ala
Ile Ser Pro Trp Arg 45 50 55 tat gag ttg gac aga gac ttg aac cgg
ctc ccc cag gac ctg tac cac 291 Tyr Glu Leu Asp Arg Asp Leu Asn Arg
Leu Pro Gln Asp Leu Tyr His 60 65 70 75 gcc cgt tgc ctg tgc ccg cac
tgc gtc agc cta cag aca ggc tcc cac 339 Ala Arg Cys Leu Cys Pro His
Cys Val Ser Leu Gln Thr Gly Ser His 80 85 90 atg gac ccc cgg ggc
aac tcg gag ctg ctc tac cac aac cag act gtc 387 Met Asp Pro Arg Gly
Asn Ser Glu Leu Leu Tyr His Asn Gln Thr Val 95 100 105 ttc tac cgg
cgg cca tgc cat ggc gag aag ggc acc cac aag ggc tac 435 Phe Tyr Arg
Arg Pro Cys His Gly Glu Lys Gly Thr His Lys Gly Tyr 110 115 120 tgc
ctg gag cgc agg ctg tac cgt gtt tcc tta gct tgt gtg tgt gtg 483 Cys
Leu Glu Arg Arg Leu Tyr Arg Val Ser Leu Ala Cys Val Cys Val 125 130
135 cgg ccc cgt gtg atg ggc tag 504 Arg Pro Arg Val Met Gly 140 145
<210> SEQ ID NO 2 <211> LENGTH: 161 <212> TYPE:
PRT <213> ORGANISM: primate <400> SEQUENCE: 2 Met Tyr
Gln Val Val Ala Phe Leu Ala Met Val Met Gly Thr His Thr -15 -10 -5
-1 Tyr Ser His Trp Pro Ser Cys Cys Pro Ser Lys Gly Gln Asp Thr Ser
1 5 10 15 Glu Glu Leu Leu Arg Trp Ser Thr Val Pro Val Pro Pro Leu
Glu Pro 20 25 30 Ala Arg Pro Asn Arg His Pro Glu Ser Cys Arg Ala
Ser Glu Asp Gly 35 40 45 Pro Leu Asn Ser Arg Ala Ile Ser Pro Trp
Arg Tyr Glu Leu Asp Arg 50 55 60 Asp Leu Asn Arg Leu Pro Gln Asp
Leu Tyr His Ala Arg Cys Leu Cys 65 70 75 80 Pro His Cys Val Ser Leu
Gln Thr Gly Ser His Met Asp Pro Arg Gly 85 90 95 Asn Ser Glu Leu
Leu Tyr His Asn Gln Thr Val Phe Tyr Arg Arg Pro 100 105 110 Cys His
Gly Glu Lys Gly Thr His Lys Gly Tyr Cys Leu Glu Arg Arg 115 120 125
Leu Tyr Arg Val Ser Leu Ala Cys Val Cys Val Arg Pro Arg Val Met 130
135 140 Gly 145 <210> SEQ ID NO 3 <211> LENGTH: 985
<212> TYPE: DNA <213> ORGANISM: rodent <220>
FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(507)
<221> NAME/KEY: mat_peptide <222> LOCATION: (49)..(507)
<400> SEQUENCE: 3 atg tac cag gct gtt gca ttc ttg gca atg atc
gtg gga acc cac acc 48 Met Tyr Gln Ala Val Ala Phe Leu Ala Met Ile
Val Gly Thr His Thr -15 -10 -5 -1 gtc agc ttg cgg atc cag gag ggc
tgc agt cac ttg ccc agc tgc tgc 96 Val Ser Leu Arg Ile Gln Glu Gly
Cys Ser His Leu Pro Ser Cys Cys 1 5 10 15 ccc agc aaa gag caa gaa
ccc ccg gag gag tgg ctg aag tgg agc tct 144 Pro Ser Lys Glu Gln Glu
Pro Pro Glu Glu Trp Leu Lys Trp Ser Ser 20 25 30 gca tct gtg tcc
ccc cca gag cct ctg agc cac acc cac cac gca gaa 192 Ala Ser Val Ser
Pro Pro Glu Pro Leu Ser His Thr His His Ala Glu 35 40 45 tcc tgc
agg gcc agc aag gat ggc ccc ctc aac agc agg gcc atc tct 240 Ser Cys
Arg Ala Ser Lys Asp Gly Pro Leu Asn Ser Arg Ala Ile Ser 50 55 60
cct tgg agc tat gag ttg gac agg gac ttg aat cgg gtc ccc cag gac 288
Pro Trp Ser Tyr Glu Leu Asp Arg Asp Leu Asn Arg Val Pro Gln Asp 65
70 75 80 ctg tac cac gct cga tgc ctg tgc cca cac tgc gtc agc cta
cag aca 336 Leu Tyr His Ala Arg Cys Leu Cys Pro His Cys Val Ser Leu
Gln Thr 85 90 95 ggc tcc cac atg gac ccg ctg ggc aac tcc gtc cca
ctt tac cac aac 384 Gly Ser His Met Asp Pro Leu Gly Asn Ser Val Pro
Leu Tyr His Asn 100 105 110 cag acg gtc ttc tac cgg cgg cca tgc cat
ggt gag gaa ggt acc cat 432 Gln Thr Val Phe Tyr Arg Arg Pro Cys His
Gly Glu Glu Gly Thr His 115 120 125 cgc cgc tac tgc ttg gag cgc agg
ctc tac cga gtc tcc ttg gct tgt 480 Arg Arg Tyr Cys Leu Glu Arg Arg
Leu Tyr Arg Val Ser Leu Ala Cys 130 135 140 gtg tgt gtg cgg ccc cgg
gtc atg gct tagtcatgct caccacctgc 527 Val Cys Val Arg Pro Arg Val
Met Ala 145 150 ctgaggctga tgcccggttg ggagagaggg ccaggtgtac
aatcaccttg ccaatgcggg 587 ccgggttcaa gccctccaaa gccctacctg
aagcagcagg ctcccgggac aagatggagg 647 acttggggag aaactctgac
ttttgcactt tttggaagca cttttgggaa ggagcaggtt 707 ccgcttgtgc
tgctagagga tgctgttgtg gcatttctac tcaggaacgg actccaaagg 767
cctgctgacc ctggaagcca tactcctggc tcctttcccc tgaatccccc aactcctggc
827 acaggcactt tctccacctc tccccctttg ccttttgttg tgtttgtttg
tgcatgccaa 887 ctctgcgtgc agccaggtgt aattgccttg aaggatggtt
ctgaggtgaa agctgttatc 947 gaaagtgaag agatttatcc aaataaacat ctgtgttt
985 <210> SEQ ID NO 4 <211> LENGTH: 169 <212>
TYPE: PRT <213> ORGANISM: rodent <400> SEQUENCE: 4 Met
Tyr Gln Ala Val Ala Phe Leu Ala Met Ile Val Gly Thr His Thr -15 -10
-5 -1 Val Ser Leu Arg Ile Gln Glu Gly Cys Ser His Leu Pro Ser Cys
Cys 1 5 10 15 Pro Ser Lys Glu Gln Glu Pro Pro Glu Glu Trp Leu Lys
Trp Ser Ser 20 25 30 Ala Ser Val Ser Pro Pro Glu Pro Leu Ser His
Thr His His Ala Glu 35 40 45 Ser Cys Arg Ala Ser Lys Asp Gly Pro
Leu Asn Ser Arg Ala Ile Ser 50 55 60 Pro Trp Ser Tyr Glu Leu Asp
Arg Asp Leu Asn Arg Val Pro Gln Asp 65 70 75 80 Leu Tyr His Ala Arg
Cys Leu Cys Pro His Cys Val Ser Leu Gln Thr 85 90 95 Gly Ser His
Met Asp Pro Leu Gly Asn Ser Val Pro Leu Tyr His Asn 100 105 110 Gln
Thr Val Phe Tyr Arg Arg Pro Cys His Gly Glu Glu Gly Thr His 115 120
125 Arg Arg Tyr Cys Leu Glu Arg Arg Leu Tyr Arg Val Ser Leu Ala Cys
130 135 140 Val Cys Val Arg Pro Arg Val Met Ala 145 150
* * * * *