U.S. patent application number 11/538579 was filed with the patent office on 2007-03-01 for soluble zcytor14, anti-zcytor14 antibodies and binding partners and methods of using in inflammation.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Janine Bilsborough, Steven K. Burkhead, Zeren Gao, Stephen R. Jaspers, Rolf E. Kuestner, Steven D. Levin, Scott R. Presnell.
Application Number | 20070048257 11/538579 |
Document ID | / |
Family ID | 35407029 |
Filed Date | 2007-03-01 |
United States Patent
Application |
20070048257 |
Kind Code |
A1 |
Presnell; Scott R. ; et
al. |
March 1, 2007 |
SOLUBLE ZCYTOR14, ANTI-ZCYTOR14 ANTIBODIES AND BINDING PARTNERS AND
METHODS OF USING IN INFLAMMATION
Abstract
The present invention relates to blocking, inhibiting, reducing,
antagonizing or neutralizing the activity of IL-17F, IL-17A, or
both IL-17A and IL-17F polypeptide molecules. IL-17A and IL-17F are
cytokines that are involved in inflammatory processes and human
disease. ZcytoR14 is a common receptor for IL-17A and IL-17F. The
present invention includes soluble ZcytoR14, anti-ZcytoR14
antibodies and binding partners, as well as methods for
antagonizing IL-17F, IL-17A or both IL-17A and IL-17F using such
soluble receptors, antibodies and binding partners.
Inventors: |
Presnell; Scott R.; (Tacoma,
WA) ; Burkhead; Steven K.; (San Antonio, TX) ;
Levin; Steven D.; (Seattle, WA) ; Kuestner; Rolf
E.; (Bothell, WA) ; Gao; Zeren; (Redmond,
WA) ; Jaspers; Stephen R.; (Edmonds, WA) ;
Bilsborough; Janine; (Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
35407029 |
Appl. No.: |
11/538579 |
Filed: |
October 4, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11150533 |
Jun 10, 2005 |
|
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11538579 |
Oct 4, 2006 |
|
|
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60578805 |
Jun 10, 2004 |
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Current U.S.
Class: |
424/85.1 ;
514/1.4; 514/1.7; 514/12.2; 514/13.2; 514/16.6; 514/16.8; 514/18.7;
514/2.1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 19/02 20180101; A61K 2039/505 20130101; A61P 37/02 20180101;
C07K 2317/73 20130101; C07K 16/2866 20130101; A61P 31/04 20180101;
A61P 1/04 20180101; C07K 2317/76 20130101; A61P 29/00 20180101;
A61P 11/06 20180101; A61P 17/04 20180101; C07K 2319/30 20130101;
A61P 17/06 20180101; C07K 14/7155 20130101 |
Class at
Publication: |
424/085.1 ;
514/012 |
International
Class: |
A61K 38/19 20070101
A61K038/19; A61K 38/17 20070101 A61K038/17 |
Claims
1. A method of reducing IL-17A-induced or IL-17F-induced
inflammation comprising administering to a mammal with inflammation
a soluble receptor comprising a ZcytoR14 subunit and an IL-17RA
subunit in an amount sufficient to reduce inflammation.
2. A method of reducing IL-17A-induced and IL-17F-induced
inflammation comprising administering to a mammal with inflammation
a soluble receptor comprising a ZcytoR14 subunit and an IL-17RA
subunit in an amount sufficient to reduce inflammation.
3. A method of treating a mammal afflicted with an inflammatory
disease in which IL-17A or IL-17F plays a role, comprising:
administering a soluble receptor comprising a ZcytoR14 subunit and
an IL-17RA subunit to the mammal such that the inflammation is
reduced, wherein the inflammatory activity of either IL-17A (SEQ ID
NO:14) or IL-17F (SEQ ID NO:16) is reduced.
4. The method of claim 3, wherein the disease is asthma.
5. The method of claim 3, wherein the disease is a chronic
inflammatory disease.
6. The method of claim 5, wherein the disease is a chronic
inflammatory disease comprising inflammatory bowel disease,
ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis,
or psoriasis.
7. The method of claim 3, wherein the disease is an acute
inflammatory disease.
8. The method of claim 7, wherein the disease is an acute
inflammatory disease comprising endotoxemia, septicemia, toxic
shock syndrome or infectious disease.
9. A method of treating a mammal afflicted with an inflammatory
disease in which IL-17A and IL-17F plays a role, comprising:
administering a soluble receptor comprising a ZcytoR14 subunit and
an IL-17RA subunit to the mammal such that the inflammation is
reduced, wherein the inflammatory activity of both IL-17A (SEQ ID
NO:14) and IL-17F (SEQ ID NO:16) is reduced.
10. The method of claim 9, wherein the disease is asthma.
11. The method of claim 9, wherein the disease is a chronic
inflammatory disease.
12. The method of claim 11, wherein the disease is a chronic
inflammatory disease comprising inflammatory bowel disease,
ulcerative colitis, Crohn's disease, arthritis, atopic dermatitis,
or psoriasis.
13. The method of claim 9, wherein the disease is an acute
inflammatory disease.
14. The method of claim 13, wherein the disease is an acute
inflammatory disease comprising endotoxemia, septicemia, toxic
shock syndrome or infectious disease.
15. A method of treating a pathological condition in a subject
associated with IL-17A or IL-17F activity comprising administering
an effective amount of a soluble receptor comprising a ZcytoR14
subunit and an IL-17RA subunit, thereby treating said pathological
condition.
16. The method of claim 15, wherein said pathological condition is
asthma.
17. The method of claim 15, wherein said pathological condition is
a chronic inflammatory condition.
18. The method of claim 17, wherein said chronic inflammatory
condition comprising inflammatory bowel disease, ulcerative
colitis, Crohn's disease, arthritis, atopic dermatitis, or
psoriasis.
19. The method of claim 15, wherein said pathological condition is
an acute inflammatory condition.
20. The method of claim 19, wherein said acute inflammatory
condition comprises endotoxemia, septicemia, toxic shock syndrome,
or infectious disease.
21. A method of treating a pathological condition in a subject
associated with IL-17A and IL-17F activity comprising administering
an effective amount of a soluble receptor comprising a ZcytoR14
subunit and an IL-17RA subunit, thereby treating said pathological
condition.
22. The method of claim 21, wherein said pathological condition is
asthma.
23. The method of claim 21, wherein said pathological condition is
a chronic inflammatory condition.
24. The method of claim 23, wherein said chronic inflammatory
condition comprising inflammatory bowel disease, ulcerative
colitis, Crohn's disease, arthritis, atopic dermatitis, or
psoriasis.
25. The method of claim 21, wherein said pathological condition is
an acute inflammatory condition.
26. The method of claim 25, wherein said acute inflammatory
condition comprises endotoxemia, septicemia, toxic shock syndrome,
or infectious disease.
Description
REFERENCE TO RELATED INVENTIONS
[0001] This application a continuation of U.S. application Ser. No.
11/150,533, filed on Jun. 10, 2005, which claims the benefit of
U.S. Provisional Application Ser. No. 60/578,805, filed Jun. 10,
2004, both of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Cytokines are soluble, small proteins that mediate a variety
of biological effects, including the regulation of the growth and
differentiation of many cell types (see, for example, Arai et al.,
Ann. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol.
3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Proteins that
constitute the cytokine group include interleukins, interferons,
colony stimulating factors, tumor necrosis factors, and other
regulatory molecules. For example, human interleukin-17 is a
cytokine which stimulates the expression of interleukin-6,
intracellular adhesion molecule 1, interleukin-8, granulocyte
macrophage colony-stimulating factor, and prostaglandin E2
expression, and plays a role in the preferential maturation of
CD34+ hematopoietic precursors into neutrophils (Yao et al., J.
Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593
(1996)).
[0003] Receptors that bind cytokines are typically composed of one
or more integral membrane proteins that bind the cytokine with high
affinity and transduce this binding event to the cell through the
cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains.
[0004] The demonstrated in vivo activities of cytokines and their
receptors illustrate the clinical potential of, and need for, other
cytokines, cytokine receptors, cytokine agonists, and cytokine
antagonists. For example, demonstrated in vivo activities of the
pro-inflammatory cytokine family illustrates the enormous clinical
potential of, and need for antagonists of pro-inflammatory
molecules.
DETAILED DESCRIPTION OF THE INVENTION
[0005] The present invention addresses these needs by providing
antagonists to pro-inflammatory cytokines IL-17A and IL-17F.
Specifically, the pro-inflammatory cytokines IL-17A and IL-17F have
a high degree of sequence similarity, share many biological
properties, and are both produced by activated T cells. They have
both been implicated as factors that contribute to the progression
of various autoimmune and inflammatory diseases including
rheumatoid arthritis and asthma. In fact, reagents that negate
IL-17A function significantly ameliorate disease incidence and
severity in several mouse models of human disease. IL-17A mediates
its effects through interaction with its cognate receptor, the
IL-17 receptor (IL-17R), but the receptor for IL-17F has not yet
been identified. We now report that we have identified the
IL-17R-related molecule, ZcytoR14 as the receptor for IL-17F.
However, we have also noted that this receptor binds to both IL-17A
and IL-17F with a similar high affinity. IL-17R on the other hand,
binds IL-17A with high affinity, but binds IL-17F with very low
affinity. Consistent with this, we have shown that a soluble form
of IL-17R blocks IL-17A binding and signaling in cells expressing
either receptor, but does not interfere with binding or function of
IL-17F to ZcytoR14. In contrast, a soluble form of ZcytoR14
antagonizes both IL-17A and IL-17F, either singly or together, in
cells expressing either receptor. Since IL-17A intervention has
been proposed as an effective therapy for several auto-immune
diseases, using antagonists of the present invention, which may
block, inhibit, reduce, antagonize or neutralize the activity of
IL-17A, IL-17F, or both IL-17A and IL-17F, which include soluble
ZcytoR14 receptors and neutralizing anti-ZcytoR14 antibodies, will
have advantages over therapies that target only one of these two
cytokines. The invention further provides uses therefor in
inflammatory disease, as well as related compositions and
methods.
A) Overview
[0006] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0007] Though the genesis of these diseases often involves
multi-step pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0008] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, etc.
[0009] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0010] A central event in both humoral and cell mediated immune
responses is the activation and clonal expansion of helper T cells.
Helper T cell activation is initiated by the interaction of the T
cell receptor (TCR)-CD3 complex with an antigen-MHC on the surface
of an antigen presenting cell. This interaction mediates a cascade
of biochemical events that induce the resting helper T cell to
enter a cell cycle (the G0 to G1 transition) and results in the
expression of a high affinity receptor for IL-2 and sometimes IL-4.
The activated T cell progresses through the cycle proliferating and
differentiating into memory cells or effector cells.
[0011] In addition to the signals mediated through the TCR,
activation of T cells involves additional costimulation induced by
cytokines released by the antigen presenting cell or through
interactions with membrane bound molecules on the antigen
presenting cell and the T cell. The cytokines IL-1 and IL-6 have
been shown to provide a costimulatory signal. Also, the interaction
between the B7 molecule expressed on the surface of an antigen
presenting cell and CD28 and CTLA-4 molecules expressed on the T
cell surface effect T cell activation. Activated T cells express an
increased number of cellular adhesion molecules, such as ICAM-1,
integrins, VLA-4, LFA-1, CD56, etc.
[0012] T-cell proliferation in a mixed lymphocyte culture or mixed
lymphocyte reaction (MLR) is an established indication of the
ability of a compound to stimulate the immune system. In many
immune responses, inflammatory cells infiltrate the site of injury
or infection. The migrating cells may be neutrophilic,
eosinophilic, monocytic or lymphocytic as can be determined by
histologic examination of the affected tissues. Current Protocols
in Immunology, ed. John E. Coligan, 1994, John Wiley & Sons,
Inc.
[0013] Immune related diseases could be treated by suppressing the
immune response. Using soluble receptors and/or neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0014] Interleukin-17 (IL-17A) has been identified as a cellular
ortholog of a protein encoded by the T lymphotropic Herpes virus
Saimiri (HSV) [see, Rouvier et al., J. Immunol., 150(12): 5445-5456
(19993); Yao et al., J. Immunol., 122(12):5483-5486 (1995) and Yao
et al., Immunity, 3(6):811-821 (1995)]. Subsequent characterization
has shown that this protein is a potent cytokine that acts to
induce proinflammatory responses in a wide variety of peripheral
tissues. IL-17A is a disulfide-linked homodimeric cytokine of about
32 kDa which is synthesized and secreted only by CD4+ activated
memory T cells (reviewed in Fossiez et al., Int. Rev. Immunol., 16:
541-551 [1998]). Specifically, IL-17 is synthesized as a precursor
polypeptide of 155 amino acids with an N-terminal signal sequence
of 19-23 residues and is secreted as a disulfide-linked homodimeric
glycoprotein. Il-17A is disclosed in WO9518826 (1995), WO9715320
(1997) and WO9704097 (1997), as well as U.S. Pat. No.
6,063,372.
[0015] Despite its restricted tissue distribution, IL-17A exhibits
pleitropic biological activities on various types of cells. IL-17A
has been found to stimulate the production of many cytokines. It
induces the secretion of IL-6, IL-8, IL-12, leukemia inhibitory
factor (LIF), prostaglandin E2, MCP-1 and G-CSF by adherent cells
like fibroblasts, keratinocytes, epithelial and endothelial cells.
IL-17A also has the ability to induce ICAM-1 surface expression,
proliferation of T cells, and growth and differentiation of
CD34.sup.+ human progenitors into neutrophils. IL-17A has also been
implicated in bone metabolism, and has been suggested to play an
important role in pathological conditions characterized by the
presence of activated T cells and TNF-.alpha. production such as
rheumatoid arthritis and loosening of bone implants (Van Bezooijen
et al., J. Bone Miner. Res. 14: 1513-1521 [1999]). Activated T
cells of synovial tissue derived from rheumatoid arthritis patients
were found to secrete higher amounts of IL-17A than those derived
from normal individuals or osteoarthritis patients (Chabaud et al.,
Arthritis Rheum. 42: 963-970 [1999]). It was suggested that this
proinflammatory cytokine actively contributes to synovial
inflammation in rheumatoid arthritis. Apart from its
proinflammatory role, IL-17A seems to contribute to the pathology
of rheumatoid arthritis by yet another mechanism. For example,
IL-17A has been shown to induce the expression of osteoclast
differentiation factor (ODF) mRNA in osteoblasts (Kotake et al., J.
Clin. Invest., 103: 1345-1352 [1999]). ODF stimulates
differentiation of progenitor cells into osteoclasts, the cells
involved in bone resorption.
[0016] Since the level of IL-17A is significantly increased in
synovial fluid of rheumatoid arthritis patients, it appears that
IL-17A induced osteoclast formation plays a crucial role in bone
resorption in rheumatoid arthritis. IL-17A is also believed to play
a key role in certain other autoimmune disorders such as multiple
sclerosis (Matusevicius et al., Mult. Scler., 5: 101-104 [1999]).
IL-17A has further been shown, by intracellular signalling, to
stimulate Ca.sup.2+ influx and a reduction in [cAMP], in human
macrophages (Jovanovic et al., J. Immunol., 160:3513 [1998]).
Fibroblasts treated with IL-17A induce the activation of
NF-.kappa.B, [Yao et al., Immunity, 3:811 (1995), Jovanovic et al.,
supra], while macrophages treated with it activate NF-.kappa.B and
mitogen-activated protein kinases (Shalom-Barek et al., J. Biol.
Chem., 273:27467 [1998]).
[0017] Additionally, IL-17A also shares sequence similarity with
mammalian cytokine-like factor 7 that is involved in bone and
cartilage growth. Other proteins with which IL-17A polypeptides
share sequence similarity are human embryo-derived
interleukin-related factor (EDIRF) and interleukin-20.
[0018] Consistent with IL-17A's wide-range of effects, the cell
surface receptor for IL-17A has been found to be widely expressed
in many tissues and cell types (Yao et al., Cytokine, 9:794
[1997]). While the amino acid sequence of the human IL-17A receptor
(IL-17R) (866 amino acids) predicts a protein with a single
transmembrane domain and a long, 525 amino acid intracellular
domain, the receptor sequence is unique and is not similar to that
of any of the receptors from the cytokine/growth factor receptor
family. This coupled with the lack of similarity of IL-17A itself
to other known proteins indicates that IL-17A and its receptor may
be part of a novel family of signalling proteins and receptors. It
has been demonstrated that IL-17A activity is mediated through
binding to its unique cell surface receptor, wherein previous
studies have shown that contacting T cells with a soluble form of
the IL-17A receptor polypeptide inhibited T cell proliferation and
IL-2 production induced by PHA, concanavalin A and anti-TCR
monoclonal antibody (Yao et al., J. Immunol., 155:5483-5486
[1995]). As such, there is significant interest in identifying and
characterizing novel polypeptides having homology to the known
cytokine receptors, specifically IL-17A receptors.
[0019] The expression pattern of IL-17F appears to be similar to
that of IL-17A, such that it includes only activated CD4+ T cells
and monocytes (Starnes et al. J. Immunol. 167: 4137-4140 [2001]).
IL-17F has been demonstrated to induce G-CSF, IL-6, and IL-8 in
fibroblasts (Hymowitz et al, EMBO J. 20:5322-5341 [2001]) and TGF-b
in endothelial cells (Starnes et al. J. Immunol. 167: 4137-4140
[2001]). It has recently been reported that IL-23, a cytokine
produced by dendritic cell, can mediate the production of both
IL-17A and IL-17F, primarily in memory T cells (Aggarwal et al. J.
Biol. Chem. 278:1910-1914 [2003]).
[0020] Moreover, over expression or upregulation of both IL-17A and
IL-17F have been shown in arthritic and asthmatic individuals
(reviewed in Moseley et al. Cytokine Growth Factor Rev 14:155-174
[2003]). With regards to arthritis, these cytokines act in a manner
characteristic to the cartilage and joint destruction that is
associated with rheumatoid- and osteo-arthritis. For example,
IL-17A and IL-17F have been demonstrated to enhance matrix
degradation in articular cartilage explants via release of
cartilage proteoglycan glycosaminoglycans and collagen fragments,
while inhibiting the synthesis of new proteoglycans and collagens
(Cai et al. Cytokine 16:10-21 [2001]; Attur et al Arthritis Rheum
44:2078-2083 [2001]).
[0021] Similar to IL-17A, overexpression of IL-17F in mice has also
been shown to increase lung neutrophil recruitment and result in
increased expression of Th1-associated cytokines in the lung,
including IL-6, IFN-gamma, IP-10 and MIG (Starnes et al. J.
Immunol. 167: 4137-4140 [2001]). IL-17F was also upregulated in T
cells from allergen-challenged asthmatics (Kawaguchi et al J.
Immunol 167:4430-4435 [2001]), and found to induce IL-6 and IL-8
production in NHBE. In contrast to IL-17A, IL-17F appears to
inhibit angiogenesis in vitro (Starnes et al. J. Immunol. 167:
4137-4140 [2001]).
[0022] IL-17F mRNA was not detected by northern blot in various
human tissues but was dramatically induced upon activation of CD4+
T cells and monocytes. Id. In mice, Th2 cells and mastr cells were
found to express IL-17F upon activation. See Dumont, Expert Opin.
Ther. Patents 13(3) (2003). Like IL-17A, the expression of IL-17F
was alos found to be upregulated by IL-23 in mouse.
[0023] The Il-17 cytokine/receptor families appear to represent a
unique signaling system within the cytokine network that will offer
innovative approaches to the manipulation of immune and
inflammatory responses. Accordingly, the present invention is based
on the discovery of a new IL-17 family receptor, ZcytoR14 and its
ability to bind both IL-17A and IL-17F.
[0024] As such, antagonists to IL-17F and IL-17A activity, such as
ZcytoR14 soluble receptors and antibodies thereto including the
anti-human-ZcytoR14 monoclonal and neutralizing antibodies of the
present invention, are useful in therapeutic treatment of
inflammatory diseases, particularly as antagonists to both IL-17F
and IL-17A singly or together in the treatment of psoriasis.
Moreover, antagonists to IL-17F activity, such as ZcytoR14 soluble
receptors and antibodies thereto including the anti-human-ZcytoR14
monoclonal and neutralizing antibodies of the present invention,
are useful in therapeutic treatment of other inflammatory diseases
for example as bind, block, inhibit, reduce, antagonize or
neutralize IL-17F and IL-17A (either individually or together) in
the treatment of atopic and contact dermatitis, IBD, colitis,
Endotoxemia, arthritis, rheumatoid arthritis, psoriatic arthritis,
adult respiratory disease (ARD), septic shock, multiple organ
failure, inflammatory lung injury such as asthma, chronic
obstructive pulmonary disease (COPD), airway hyper-responsiveness,
chronic bronchitis, allergic asthma, bacterial pneumonia,
psoriasis, eczema, and inflammatory bowel disease such as
ulcerative colitis and Crohn's disease, helicobacter pylori
infection. intraabdominal adhesions and/or abscesses as results of
peritoneal inflammation (i.e. from infection, injury, etc.),
systemic lupus erythematosus (SLE), multiple sclerosis, systemic
sclerosis, nephrotic syndrome, organ allograft rejection, graft vs.
host disease (GVHD), kidney, lung, heart, etc. transplant
rejection, streptococcal cell wall (SCW)-induced arthritis,
osteoarthritis, gingivitis/periodontitis, herpetic stromal
keratitis, cancers including prostate, renal, colon, ovarian,
cervical, leukemia, angiogenesis, restenosis and kawasaki
disease.
[0025] Cytokine receptors subunits are characterized by a
multi-domain structure comprising a ligand-binding domain and an
effector domain that is typically involved in signal transduction.
Multimeric cytokine receptors include monomers, homodimers (e.g.,
PDGF receptor .alpha..alpha. and .beta..beta. isoforms,
erythropoietin receptor, MPL [thrombopoietin receptor], and G-CSF
receptor), heterodimers whose subunits each have ligand-binding and
effector domains (e.g., PDGF receptor .alpha..beta. isoform), and
multimers having component subunits with disparate functions (e.g.,
IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and GM-CSF receptors). Some
receptor subunits are common to a plurality of receptors. For
example, the AIC2B subunit, which cannot bind ligand on its own but
includes an intracellular signal transduction domain, is a
component of IL-3 and GM-CSF receptors. Many cytokine receptors can
be placed into one of four related families on the basis of their
structures and functions. Class I hematopoietic receptors, for
example, are characterized by the presence of a domain containing
conserved cysteine residues and the WSXWS motif (SEQ ID NO:10).
Additional domains, including protein kinase domains; fibronectin
type III domains; and immunoglobulin domains, which are
characterized by disulfide-bonded loops, are present in certain
hematopoietic receptors. Cytokine receptor structure has been
reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 and
Cosman, Cytokine 5:95-106, 1993. It is generally believed that
under selective pressure for organisms to acquire new biological
functions, new receptor family members arose from duplication of
existing receptor genes leading to the existence of multi-gene
families. Family members thus contain vestiges of the ancestral
gene, and these characteristic features can be exploited in the
isolation and identification of additional family members.
[0026] Amongst other inventions, the present invention provides
novel uses for a soluble receptor, designated "ZcytoR14" or
"soluble ZcytoR14" or "sZcytoR14", all of which may be used herein
interchangeably, or and neutralizing antibodies to ZcytoR14
cytokine receptors. The present invention also provides soluble
ZcytoR14 polypeptide fragments and fusion proteins, for use in
human inflammatory and autoimmune diseases. The anti-ZcytoR14
antibodies, and soluble ZcytoR14 receptors of the present
invention, including the neutralizing anti-ZcytoR14 antibodies of
the present invention, can be used to block, inhibit, reduce,
antagonize or neutralize the activity of either IL-17F or IL-17A,
or both IL-17A and IL-17F in the treatment of inflammation and
inflammatory diseases such as psoriasis, psoriatic arthritis,
rheumatoid arthritis, endotoxemia, inflammatory bowel disease
(IBD), colitis, asthma, allograft rejection, immune mediated renal
diseases, hepatobiliary diseases, multiple sclerosis,
atherosclerosis, promotion of tumor growth, or degenerative joint
disease and other inflammatory conditions disclosed herein.
[0027] An illustrative nucleotide sequence that encodes human
ZcytoR14 is provided by SEQ ID NO:1; the encoded polypeptide is
shown in SEQ ID NO:2. ZcytoR14 functions as a receptor for both
IL-17A (SEQ ID NOS:13 & 14) and IL-17F (SEQ ID NOS:15 &
16). ZcytoR14 can act as a monomer, a homodimer or a heterodimer.
Preferably, ZcytoR14 acts as a homodimeric receptor for both IL-17A
and/or IL-17F. ZcytoR14 can also act as a heterodimeric receptor
subunit for a IL-17-related cytokine. ZcytoR14 is disclosed in
commonly owned U.S. patent application Ser. No. 10/458,647, and
commonly owned WIPO publication WO 01/04304, both of which are
incorporated herein in their entirety by reference. Analysis of a
human cDNA clone encoding ZcytoR14 (SEQ ID NO:1) revealed an open
reading frame encoding 692 amino acids (SEQ ID NO:2) comprising a
putative signal sequence of approximately 20 amino acid residues
(amino acid residues 1 to 20 of SEQ ID NO:2), an extracellular
ligand-binding domain of approximately 431 amino acid residues
(amino acid residues 21-452 of SEQ ID NO:2; SEQ ID NO:3), a
transmembrane domain of approximately 20 amino acid residues (amino
acid residues 453-473 of SEQ ID NO:2), and an intracellular domain
of approximately 203 amino acid residues (amino acid residues 474
to 677 of SEQ ID NO:2). Furthermore, a ligand binding domain is
represented by SEQ ID NO:22.
[0028] Yet another illustrative nucleotide sequence that encodes a
variant human ZcytoR14, designated as "ZcytoR14-1" is provided by
SEQ ID NO:4, the encoded polypeptide is shown in SEQ ID NO:5.
ZcytoR14-1 is disclosed in commonly owned U.S. patent application
Ser. No. 10/458,647, and commonly owned WIPO publication WO
01/04304, both of which are incorporated herein in their entirety
by reference. Sequence analysis revealed that Zcytor14-1 is a
truncated form of receptor polypeptide. That is, Zcytor14-1 lacks
amino acid residues 1-113 of SEQ ID NO:2. SEQ ID NO: 10 presents an
amino acid sequence of a Zcytor14-1 polypeptide that includes the
N-terminal portion of Zcytor14.
[0029] A comparison of the Zcytor14 and Zcytor14-1 amino acid
sequences also indicated that the two polypeptides represent
alternatively spliced variants. The amino acid sequence of Zcytor14
includes a 17 amino acid segment (amino acid residues 339 to 355 of
SEQ ID NO:2), which Zcytor14-1 lacks, while Zcytor14 lacks,
following amino acid 479, a 13 amino acid segment found in
Zcytor14-1 (amino acid residues 350 to 362 of SEQ ID NO:5). A
polypeptide that contains both amino acid segments is provided by
SEQ ID NO:11, whereas SEQ ID NO:12 presents the amino acid sequence
of a polypeptide that lacks both 13 and 17 amino acid segments.
[0030] Yet another illustrative nucleotide sequence that encodes a
variant human ZcytoR14, designated as "ZcytoR14-6" is provided by
SEQ ID NO:23, the encoded polypeptide is shown in SEQ ID NO:24.
ZcytoR14-6 contains a 25 amino acid residue deletion as compared to
ZcytoR14 as embodied in SEQ ID NO:2. Specifically, ZcytoR14-6 does
not contain amino acid residue 94 to amino acid residue 118 of SEQ
ID NO:2. Analysis of a human cDNA clone encoding ZcytoR14-6 (SEQ ID
NO:23) revealed an extracellular ligand-binding domain of
approximately 427 amino acid residues (amino acid residues 1-427 of
SEQ ID NO:24), a transmembrane domain of approximately 20 amino
acid residues (amino acid residues 428-448 of SEQ ID NO:24), and an
intracellular domain of approximately 218 amino acid residues
(amino acid residues 449 to 667 of SEQ ID NO:24).
[0031] An illustrative nucleotide sequence that encodes a variant
murine ZcytoR14 is provided by SEQ ID NO:25; the encoded
polypeptide is shown in SEQ ID NO:26. Murine ZcytoR14 functions as
a receptor for both murine IL-17A (SEQ ID NOS:17 & 18) and
murine IL-17F (SEQ ID NOS:19 & 20). Analysis of a murine cDNA
clone encoding ZcytoR14 (SEQ ID NO:1) revealed an extracellular
ligand-binding domain of approximately 449 amino acid residues SEQ
ID NO:27). Furthermore, a ligand binding domain is represented by
SEQ ID NO:28.
[0032] Yet another illustrative nucleotide sequence that encodes a
variant murine ZcytoR14 is provided by SEQ ID NO:29; the encoded
polypeptide is shown in SEQ ID NO:30.
[0033] The Zcytor14 gene resides in chromosome 3p25-3p24. As
discussed below, this region is associated with various disorders
and diseases.
[0034] Northern analyses indicate that there is strong expression
of the Zcytor14 gene in thyroid, adrenal gland, prostate, and liver
tissues, and less expression in heart, small intestine, stomach,
and trachea tissues. In contrast, there is little or no expression
in brain, placenta, lung, skeletal muscle, kidney, pancreas,
spleen, thymus, testis, ovary, colon, peripheral blood leukocytes,
spinal cord, lymph node, and bone marrow. These observations show
that Zcytor14 sequences can be used differentiate between various
tissues.
[0035] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 80%, or at least 90%, or greater than 95%, such as
96%, 97%, 98%, or greater than 99% or more identical to a reference
amino acid sequence of 21-692 of SEQ ID NO:2, wherein the isolated
polypeptide specifically binds with an antibody that specifically
binds with a polypeptide comprising the amino acid sequence of SEQ
ID NO:2. The present invention also provides isolated polypeptides
comprising an amino acid sequence that is at least 70%, at least
80%, or at least 90% identical to a reference amino acid sequence
selected from the group consisting of: (a) amino acid residues 21
to 452 of SEQ ID NO:2, (b) amino acid residues 21 to 435 of SEQ ID
NO:10, (c) amino acid residues 21 to 677 of SEQ ID NO:2, and (d)
amino acid residues 1 to 692 of SEQ ID NO:2, wherein the isolated
polypeptide specifically binds with an antibody that specifically
binds with a polypeptide consisting of either the amino acid
sequence of SEQ ID NO:2, or the amino acid sequence of SEQ ID
NO:10. Illustrative polypeptides include a polypeptide comprising
the amino acid sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11,
or SEQ ID NO:12.
[0036] The present invention also provides isolated polypeptides
comprising an extracellular domain, wherein the extracellular
domain comprises either amino acid residues 21 to 452 of the amino
acid sequence of SEQ ID NO:2 or amino acid residues 21 to 435 of
the amino acid sequence of SEQ ID NO:10. Such polypeptides may
further comprise a transmembrane domain that resides in a
carboxyl-terminal position relative to the extracellular domain,
wherein the transmembrane domain comprises amino acid residues 453
to 473 of SEQ ID NO:2. These polypeptides may also comprise an
intracellular domain that resides in a carboxyl-terminal position
relative to the transmembrane domain, wherein the intracellular
domain comprises either amino acid residues 474 to 677 of SEQ ID
NO:2, or amino acid residues 457 to 673 of SEQ ID NO:10, and
optionally, a signal secretory sequence that resides in an
amino-terminal position relative to the extracellular domain,
wherein the signal secretory sequence comprises amino acid residues
1 to 20 of the amino acid sequence of SEQ ID NO:2.
[0037] The present invention also includes variant Zcytor14
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with the amino acid sequence of SEQ
ID NO:2 selected from the group consisting of at least 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, or greater than 95% identity, and wherein any
difference between the amino acid sequence of the variant
polypeptide and the amino acid sequence of SEQ ID NO:2 is due to
one or more conservative amino acid substitutions.
[0038] Moreover, the present invention also provides isolated
polypeptides as disclosed above that bind IL-17F (e.g., human
IL-17F polypeptide sequence as shown in SEQ ID NO:16). The human
IL-17F polynucleotide sequence is shown in SEQ ID NO:15. The mouse
IL-17F polynucleotide sequence is shown in SEQ ID NO:19, and
corresponding polypeptide is shown in SEQ ID NO:20. The present
invention also provides isolated polypeptides as disclosed above
that bind IL-17A (e.g., human IL-17A polypeptide sequence as shown
in SEQ ID NO:14). The human IL-17A polynucleotide sequence is shown
in SEQ ID NO:13. The mouse IL-17A polynucleotide sequence is shown
in SEQ ID NO:17, and corresponding polypeptide is shown in SEQ ID
NO:18.
[0039] The present invention also provides isolated polypeptides
and epitopes comprising at least 15 contiguous amino acid residues
of an amino acid sequence of SEQ ID NO:2 or 3. Illustrative
polypeptides include polypeptides that either comprise, or consist
of SEQ ID NO:2 or 3, an antigenic epitope thereof, or a functional
IL-17A or IL-17F binding fragment thereof. Moreover, the present
invention also provides isolated polypeptides as disclosed above
that bind to, block, inhibit, reduce, antagonize or neutralize the
activity of IL-17F or IL-17A.
[0040] The present invention also includes variant ZcytoR14
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with the amino acid residues of SEQ
ID NO:2 selected from the group consisting of at least 70%
identity, at least 80% identity, at least 90% identity, at least
95% identity, or greater than 95% identity, such as 96%, 97%, 98%,
or greater than 99% or more identity, and wherein any difference
between the amino acid sequence of the variant polypeptide and the
corresponding amino acid sequence of SEQ ID NO:2 is due to one or
more conservative amino acid substitutions. Such conservative amino
acid substitutions are described herein. Moreover, the present
invention also provides isolated polypeptides as disclosed above
that bind to, block, inhibit, reduce, antagonize or neutralize the
activity of IL-17F or IL-17A.
[0041] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include neutralizing antibodies, polyclonal
antibodies, murine monoclonal antibodies, humanized antibodies
derived from murine monoclonal antibodies, and human monoclonal
antibodies. Illustrative antibody fragments include F(ab').sub.2,
F(ab).sub.2, Fab', Fab, Fv, scFv, and minimal recognition units.
Neutralizing antibodies preferably bind ZcytoR14 such that the
interaction of IL-17A and IL-17F with ZcytoR14 is blocked,
inhibited, reduced, antagonized or neutralized; anti-ZcytoR14
neutralizing antibodies such that the binding of either IL-17A or
IL-17F to ZcytoR14 is blocked, inhibited, reduced, antagonized or
neutralized are also encompassed by the present invention. That is,
the neutralizing anti-ZcytoR14 antibodies of the present invention
can either bind, block, inhibit, reduce, antagonize or neutralize
each of IL-17A or IL-17F singly, or bind, block, inhibit, reduce,
antagonize or neutralize IL-17A and IL-17F together. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, or antibody described herein.
[0042] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors. The present invention further
includes pharmaceutical compositions, comprising a pharmaceutically
acceptable carrier and a polypeptide or antibody described
herein.
[0043] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a
fragment thereof. An exemplary anti-idiotype antibody binds with an
antibody that specifically binds a polypeptide consisting of SEQ ID
NO:2.
[0044] The present invention also provides fusion proteins,
comprising a ZcytoR14 polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.c
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0045] The present invention also provides polyclonal and
monoclonal antibodies that bind to polypeptides comprising an
ZcytoR14 extracellular domain such as monomeric, homodimeric,
heterodimeric and multimeric receptors, including soluble
receptors. Moreover, such antibodies can be used antagonize the
binding of ZcytoR14 ligands, IL-17F (SEQ ID NO:16), and IL-17A (SEQ
ID NO:14), individually or together to the ZcytoR14 receptor.
[0046] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
B) Definitions
[0047] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0048] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0049] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0050] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid molecule that encodes a
polypeptide. Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0051] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0052] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0053] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0054] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0055] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0056] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0057] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0058] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0059] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0060] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0061] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0062] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0063] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0064] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0065] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0066] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces ZcytoR14 from an expression vector. In
contrast, ZcytoR14 can be produced by a cell that is a "natural
source" of ZcytoR14, and that lacks an expression vector.
[0067] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0068] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a ZcytoR14 polypeptide fused with a polypeptide that binds an
affinity matrix. Such a fusion protein provides a means to isolate
large quantities of ZcytoR14 using affinity chromatography.
[0069] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. Receptors can be
membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3
receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor
and IL-6 receptor). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0070] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0071] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains, and other linkage to the cell membrane such as
via glycophosphoinositol (gpi). Soluble receptors can comprise
additional amino acid residues, such as affinity tags that provide
for purification of the polypeptide or provide sites for attachment
of the polypeptide to a substrate, or immunoglobulin constant
region sequences. Many cell-surface receptors have naturally
occurring, soluble counterparts that are produced by proteolysis or
translated from alternatively spliced mRNAs. Soluble receptors can
be monomeric, homodimeric, heterodimeric, or multimeric, with
multimeric receptors generally not comprising more than 9 subunits,
preferably not comprising more than 6 subunits, and most preferably
not comprising more than 3 subunits. Receptor polypeptides are said
to be substantially free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively. Soluble receptors of cytokine receptors generally
comprise the extracellular cytokine binding domain free of a
transmembrane domain and intracellular domain. For example,
representative soluble receptors include soluble receptors for
IL-17R as shown in SEQ ID NO:21. It is well within the level of one
of skill in the art to delineate what sequences of a known cytokine
receptor sequence comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain.
Moreover, one of skill in the art using the genetic code can
readily determine polynucleotides that encode such soluble receptor
polypeptides.
[0072] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0073] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, such as 96%, 97%, or
98% or more pure, or greater than 99% pure. One way to show that a
particular protein preparation contains an isolated polypeptide is
by the appearance of a single band following sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis of the protein preparation
and Coomassie Brilliant Blue staining of the gel. However, the term
"isolated" does not exclude the presence of the same polypeptide in
alternative physical forms, such as dimers or alternatively
glycosylated or derivatized forms.
[0074] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0075] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0076] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0077] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and the like, and synthetic
analogs of these molecules.
[0078] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of less than 10.sup.9 M.sup.-1.
[0079] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-ZcytoR14 antibody, and thus, an anti-idiotype antibody
mimics an epitope of ZcytoR14.
[0080] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-ZcytoR14
monoclonal antibody fragment binds with an epitope of ZcytoR14.
[0081] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0082] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0083] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain. Construction of
humanized antibodies for therapeutic use in humans that are derived
from murine antibodies, such as those that bind to or neutralize a
human protein, is within the skill of one in the art.
[0084] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0085] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0086] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith
and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). DNA molecules encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0087] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0088] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0089] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0090] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
ZcytoR14 polypeptide component. Examples of an antibody fusion
protein include a protein that comprises a ZcytoR14 extracellular
domain, and either an Fc domain or an antigen-binding region.
[0091] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0092] An "antigenic peptide" is a peptide which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0093] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0094] An "anti-sense oligonucleotide specific for ZcytoR14" or a
"ZcytoR14 anti-sense oligonucleotide" is an oligonucleotide having
a sequence (a) capable of forming a stable triplex with a portion
of the ZcytoR14 gene, or (b) capable of forming a stable duplex
with a portion of an mRNA transcript of the ZcytoR14 gene.
[0095] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0096] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0097] The term "variant ZcytoR14 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2. Such variants include
naturally-occurring polymorphisms of ZcytoR14 genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:2. Additional variant forms
of ZcytoR14 genes are nucleic acid molecules that contain
insertions or deletions of the nucleotide sequences described
herein. A variant ZcytoR14 gene can be identified, for example, by
determining whether the gene hybridizes with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 OR SEQ ID
NO:4, or its complement, under stringent conditions.
[0098] Alternatively, variant ZcytoR14 genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0099] Regardless of the particular method used to identify a
variant ZcytoR14 gene or variant ZcytoR14 polypeptide, a variant
gene or polypeptide encoded by a variant gene may be functionally
characterized the ability to bind specifically to an anti-ZcytoR14
antibody. A variant ZcytoR14 gene or variant ZcytoR14 polypeptide
may also be functionally characterized the ability to bind to its
ligand, for example, IL-17A and/or IL-17F, using a biological or
biochemical assay described herein.
[0100] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0101] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of specification.
[0102] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0103] The present invention includes functional fragments of
ZcytoR14 genes. Within the context of this invention, a "functional
fragment" of a ZcytoR14 gene refers to a nucleic acid molecule that
encodes a portion of a ZcytoR14 polypeptide which is a domain
described herein or at least specifically binds with an
anti-ZcytoR14 antibody.
[0104] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
C) Production of ZcytoR14 Polynucleotides or Genes
[0105] Nucleic acid molecules encoding a human ZcytoR14 gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:1 OR SEQ ID NO:4. These
techniques are standard and well-established, and may be
accomplished using cloning kits available by commercial suppliers.
See, for example, Ausubel et al. (eds.), Short Protocols in
Molecular Biology, 3.sup.rd Edition, John Wiley & Sons 1995; Wu
et al., Methods in Gene Biotechnology, CRC Press, Inc. 1997; Aviv
and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Huynh et al.,
"Constructing and Screening cDNA Libraries in .lamda.gt10 and
.lamda.gt11," in DNA Cloning: A Practical Approach Vol. 1, Glover
(ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
[0106] Nucleic acid molecules that encode a human ZcytoR14 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the ZcytoR14 gene or cDNA. General
methods for screening libraries with PCR are provided by, for
example, Yu et al., "Use of the Polymerase Chain Reaction to Screen
Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR
Protocols: Current Methods and Applications, White (ed.), Humana
Press, Inc., 1993. Moreover, techniques for using PCR to isolate
related genes are described by, for example, Preston, "Use of
Degenerate Oligonucleotide Primers and the Polymerase Chain
Reaction to Clone Gene Family Members," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), Humana Press, Inc. 1993. As an alternative, a ZcytoR14
gene can be obtained by synthesizing nucleic acid molecules using
mutually priming long oligonucleotides and the nucleotide sequences
described herein (see, for example, Ausubel (1995)). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)). For reviews on polynucleotide synthesis, see, for
example, Glick and Pasternak, Molecular Biotechnology, Principles
and Applications of Recombinant DNA (ASM Press 1994), Itakura et
al., Ann. Rev. Biochem. 53:323 (1984), and Climie et al., Proc.
Nat'l Acad. Sci. USA 87:633 (1990).
D) Production of ZcytoR14 Gene Variants
[0107] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the
ZcytoR14 polypeptides disclosed herein. Those skilled in the art
will readily recognize that, in view of the degeneracy of the
genetic code, considerable sequence variation is possible among
these polynucleotide molecules. Moreover, the present invention
also provides isolated soluble monomeric, homodimeric,
heterodimeric and multimeric receptor polypeptides that comprise at
least one ZcytoR14 receptor subunit that is substantially
homologous to the receptor polypeptide of SEQ ID NO:2. Thus, the
present invention contemplates ZcytoR14 polypeptide-encoding
nucleic acid molecules comprising degenerate nucleotides of SEQ ID
NO:1 or SEQ ID NO:4, and their RNA equivalents.
[0108] Those skilled in the art will readily recognize that, in
view of the degeneracy of the genetic code, considerable sequence
variation is possible among these polynucleotide molecules. SEQ ID
NO:7 is a degenerate nucleotide sequence that encompasses all
nucleic acid molecules that encode the Zcytor14 polypeptide of SEQ
ID NO:2. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO:7 also provides all RNA sequences
encoding SEQ ID NO:2, by substituting U for T. Thus, the present
invention contemplates Zcytor14 polypeptide-encoding nucleic acid
molecules comprising nucleotide 154 to nucleotide 2229 of SEQ ID
NO:1, and their RNA equivalents. Similarly, the Zcytor14-1
degenerate sequence of SEQ ID NO:6 also provides all RNA sequences
encoding SEQ ID NO:5, by substituting U for T.
[0109] Table 1 sets forth the one-letter codes to denote degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by
a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C. TABLE-US-00001 TABLE 1
Nucleotide Resolution Complement Resolution A A T T C C G G G G C C
T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G
W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T
H A|C|T N A|C|G|T N A|C|G|T
[0110] The degenerate codons, encompassing all possible codons for
a given amino acid, are set forth in Table 2. TABLE-US-00002 TABLE
2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGT
TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro
P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG
GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q
CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys
K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG
CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y
TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY
Glu|Gln Z SAR Any X NNN
[0111] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences of SEQ ID NO:3.
Variant sequences can be readily tested for functionality as
described herein.
[0112] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0113] A ZcytoR14-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial human cDNA
or with one or more sets of degenerate probes based on the
disclosed sequences. A cDNA can also be cloned using the polymerase
chain reaction with primers designed from the representative human
ZcytoR14 sequences disclosed herein. In addition, a cDNA library
can be used to transform or transfect host cells, and expression of
the cDNA of interest can be detected with an antibody to ZcytoR14
polypeptide.
[0114] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
ZcytoR14, and that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the
nucleotide sequences disclosed herein, including those containing
silent mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of the amino acid sequences
disclosed herein. cDNA molecules generated from alternatively
spliced mRNAs, which retain the properties of the ZcytoR14
polypeptide are included within the scope of the present invention,
as are polypeptides encoded by such cDNAs and mRNAs. Allelic
variants and splice variants of these sequences can be cloned by
probing cDNA or genomic libraries from different individuals or
tissues according to standard procedures known in the art.
[0115] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that comprise a
soluble ZcytoR14 receptor subunit that is substantially homologous
to either SEQ ID NO:1 or SEQ ID NO:4, or that encodes amino acids
of either SEQ ID NO:2 or SEQ ID NO:5, or allelic variants thereof
and retain the ligand-binding properties of the wild-type ZcytoR14
receptor. Such polypeptides may also include additional polypeptide
segments as generally disclosed herein.
[0116] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1 OR SEQ ID NO:4, or to nucleic
acid molecules comprising a nucleotide sequence complementary to
SEQ ID NO:1 OR SEQ ID NO:4, or fragments thereof.
[0117] In general, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (T.sub.m) for the
specific sequence at a defined ionic strength and pH. The T.sub.m
is the temperature (under defined ionic strength and pH) at which
50% of the target sequence hybridizes to a perfectly matched probe.
Following hybridization, the nucleic acid molecules can be washed
to remove non-hybridized nucleic acid molecules under stringent
conditions, or under highly stringent conditions. See, for example,
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition (Cold Spring Harbor Press 1989); Ausubel et al., (eds.),
Current Protocols in Molecular Biology (John Wiley and Sons, Inc.
1987); Berger and Kimmel (eds.), Guide to Molecular Cloning
Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.
Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such
as OLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0
(Premier Biosoft International; Palo Alto, Calif.), as well as
sites on the Internet, are available tools for analyzing a given
sequence and calculating T.sub.m based on user-defined criteria. It
is well within the abilities of one skilled in the art to adapt
hybridization and wash conditions for use with a particular
polynucleotide hybrid.
[0118] The present invention also provides isolated ZcytoR14
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95%, such as 96%, 97%, 98%, or greater than 95% sequence
identity to the sequences shown in SEQ ID NO:2, or their orthologs.
For example, variant and orthologous ZcytoR14 receptors can be used
to generate an immune response and raise cross-reactive antibodies
to human ZcytoR14. Such antibodies can be humanized, and modified
as described herein, and used therapeutically to treat psoriasis,
psoriatic arthritis, IBD, colitis, endotoxemia as well as in other
therapeutic applications described herein.
[0119] The present invention also contemplates ZcytoR14 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay. Such ZcytoR14 variants include nucleic acid molecules (1)
that remain hybridized with a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1 OR SEQ ID NO:4 (or its
complement) under stringent washing conditions, in which the wash
stringency is equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at
55-65.degree. C., and (2) that encode a polypeptide having at least
70%, at least 80%, at least 90%, at least 95%, or greater than 95%
such as 96%, 97%, 98%, or 99%, sequence identity to the amino acid
sequence of SEQ ID NO:2. Alternatively, ZcytoR14 variants can be
characterized as nucleic acid molecules (1) that remain hybridized
with a nucleic acid molecule having the nucleotide sequence of SEQ
ID NO:1 OR SEQ ID NO:4 (or its complement) under highly stringent
washing conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95%, such as 96%, 97%, 98%,
or 99% or greater, sequence identity to the amino acid sequence of
SEQ ID NO:2.
[0120] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0121] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative ZcytoR14 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID NO:2
or SEQ ID NO:3) and a test sequence that have either the highest
density of identities (if the ktup variable is 1) or pairs of
identities (if ktup=2), without considering conservative amino acid
substitutions, insertions, or deletions. The ten regions with the
highest density of identities are then rescored by comparing the
similarity of all paired amino acids using an amino acid
substitution matrix, and the ends of the regions are "trimmed" to
include only those residues that contribute to the highest score.
If there are several regions with scores greater than the "cutoff"
value (calculated by a predetermined formula based upon the length
of the sequence and the ktup value), then the trimmed initial
regions are examined to determine whether the regions can be joined
to form an approximate alignment with gaps. Finally, the highest
scoring regions of the two amino acid sequences are aligned using a
modification of the Needleman-Wunsch-Sellers algorithm (Needleman
and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl.
Math. 26:787 (1974)), which allows for amino acid insertions and
deletions. Illustrative parameters for FASTA analysis are: ktup=1,
gap opening penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62. These parameters can be introduced into a FASTA
program by modifying the scoring matrix file ("SMATRIX"), as
explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63
(1990).
[0122] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as described above.
[0123] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a ZcytoR14 amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a ZcytoR14 amino acid sequence, a sulfur-containing
amino acid is substituted for a sulfur-containing amino acid in a
ZcytoR14 amino acid sequence, a hydroxy-containing amino acid is
substituted for a hydroxy-containing amino acid in a ZcytoR14 amino
acid sequence, an acidic amino acid is substituted for an acidic
amino acid in a ZcytoR14 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a ZcytoR14 amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a ZcytoR14 amino acid
sequence. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. The BLOSUM62 table is an amino acid
substitution matrix derived from about 2,000 local multiple
alignments of protein sequence segments, representing highly
conserved regions of more than 500 groups of related proteins
(Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915
(1992)). Accordingly, the BLOSUM62 substitution frequencies can be
used to define conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present invention.
Although it is possible to design amino acid substitutions based
solely upon chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3). Particular variants of ZcytoR14 are characterized
by having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% such as 96%, 97%, 98%, or 99% or greater sequence
identity to the corresponding amino acid sequence (e.g., SEQ ID
NO:2), wherein the variation in amino acid sequence is due to one
or more conservative amino acid substitutions.
[0124] Conservative amino acid changes in a ZcytoR14 gene can be
introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1 OR SEQ ID NO:4. Such
"conservative amino acid" variants can be obtained by
oligonucleotide-directed mutagenesis, linker-scanning mutagenesis,
mutagenesis using the polymerase chain reaction, and the like (see
Ausubel (1995); and McPherson (ed.), Directed Mutagenesis: A
Practical Approach (IRL Press 1991)). A variant ZcytoR14
polypeptide can be identified by the ability to specifically bind
anti-ZcytoR14 antibodies.
[0125] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0126] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0127] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for ZcytoR14 amino acid residues.
[0128] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity to
identify amino acid residues that are critical to the activity of
the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699
(1996).
[0129] Although sequence analysis can be used to further define the
ZcytoR14 ligand binding region, amino acids that play a role in
ZcytoR14 binding activity (such as binding of ZcytoR14 to either
Il-17A or IL-17F, or to an anti-ZcytoR14 antibody) can also be
determined by physical analysis of structure, as determined by such
techniques as nuclear magnetic resonance, crystallography, electron
diffraction or photoaffinity labeling, in conjunction with mutation
of putative contact site amino acids. See, for example, de Vos et
al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899
(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).
[0130] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
ZcytoR14 labeled with biotin or FITC can be used for expression
cloning of ZcytoR14 ligands.
[0131] Variants of the disclosed ZcytoR14 nucleotide and
polypeptide sequences can also be generated through DNA shuffling
as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc.
Nat'l Acad. Sci. USA 91:10747 (1994), and international publication
No. WO 97/20078. Briefly, variant DNA molecules are generated by in
vitro homologous recombination by random fragmentation of a parent
DNA followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent DNA molecules, such as allelic variants or DNA
molecules from different species, to introduce additional
variability into the process. Selection or screening for the
desired activity, followed by additional iterations of mutagenesis
and assay provides for rapid "evolution" of sequences by selecting
for desirable mutations while simultaneously selecting against
detrimental changes.
[0132] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-ZcytoR14 antibodies, can be
recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0133] The present invention also includes "functional fragments"
of ZcytoR14 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a ZcytoR14 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO:1 OR SEQ ID NO:4 can be digested with Bal31 nuclease to
obtain a series of nested deletions. The fragments are then
inserted into expression vectors in proper reading frame, and the
expressed polypeptides are isolated and tested for the ability to
bind anti-ZcytoR14 antibodies. One alternative to exonuclease
digestion is to use oligonucleotide-directed mutagenesis to
introduce deletions or stop codons to specify production of a
desired fragment. Alternatively, particular fragments of a ZcytoR14
gene can be synthesized using the polymerase chain reaction.
[0134] This general approach is exemplified by studies on the
truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0135] The present invention also contemplates functional fragments
of a ZcytoR14 gene that have amino acid changes, compared with an
amino acid sequence disclosed herein. A variant ZcytoR14 gene can
be identified on the basis of structure by determining the level of
identity with disclosed nucleotide and amino acid sequences, as
discussed above. An alternative approach to identifying a variant
gene on the basis of structure is to determine whether a nucleic
acid molecule encoding a potential variant ZcytoR14 gene can
hybridize to a nucleic acid molecule comprising a nucleotide
sequence, such as SEQ ID NO:1 OR SEQ ID NO:4.
[0136] The present invention also includes using functional
fragments of ZcytoR14 polypeptides, antigenic epitopes,
epitope-bearing portions of ZcytoR14 polypeptides, and nucleic acid
molecules that encode such functional fragments, antigenic
epitopes, epitope-bearing portions of ZcytoR14 polypeptides. Such
fragments are used to generate polypeptides for use in generating
antibodies and binding partners that bind, block, inhibit, reduce,
antagonize or neutralize activity of IL-17A or IL-17F or both
IL-17A and IL-17F. A "functional" ZcytoR14 polypeptide or fragment
thereof as defined herein is characterized by its ability to block,
inhibit, reduce, antagonize or neutralize IL-17A or IL-17F
inflammatory, proliferative or differentiating activity, by its
ability to induce or inhibit specialized cell functions, or by its
ability to bind specifically to an anti-ZcytoR14 antibody, cell,
IL-17A or IL-17F. As previously described herein, ZcytoR14 is
characterized by a unique cytokine receptor structure and domains
as described herein. Thus, the present invention further
contemplates using fusion proteins encompassing: (a) polypeptide
molecules comprising one or more of the domains described above;
and (b) functional fragments comprising one or more of these
domains. The other polypeptide portion of the fusion protein may be
contributed by another cytokine receptor, such as IL-10R, IL-13R,
IL-17R, IL-10RB (CRF2-4), or by a non-native and/or an unrelated
secretory signal peptide that facilitates secretion of the fusion
protein.
[0137] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a ZcytoR14
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0138] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides, antigenic
peptides, epitopes, and polypeptides of the present invention are
useful to raise antibodies that bind with the polypeptides
described herein, as well as to identify and screen anti-ZcytoR14
monoclonal antibodies that are neutralizing, and that may bind,
block, inhibit, reduce, antagonize or neutralize the activity of
IL-17F and IL-17A (individually or together). Such neutralizing
monoclonal antibodies of the present invention can bind to an
ZcytoR14 antigenic epitope. Hopp/Woods hydrophilicity profiles can
be used to determine regions that have the most antigenic potential
within SEQ ID NO:2 or 4 (Hopp et al., Proc. Natl. Acad. Sci.
78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and
Triquier et al., Protein Engineering 11:153-169, 1998). The profile
is based on a sliding six-residue window. Buried G, S, and T
residues and exposed H, Y, and W residues were ignored. In ZcytoR14
these regions can be determined by one of skill in the art.
Moreover, ZcytoR14 antigenic epitopes within SEQ ID NO:2 or 4 as
predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean
program (DNASTAR, Inc., Madison, Wis.) serve as preferred antigenic
epitopes, and can be determined by one of skill in the art. The
results of this analysis indicated that the following amino acid
sequences of SEQ ID NO:2 would provide suitable antigenic peptides:
amino acids 26 to 33 ("antigenic peptide 1"), amino acids 41 to 46
("antigenic peptide 2"), 74 to 81 ("antigenic peptide 3"), amino
acids 95 to 105 ("antigenic peptide 4"), amino acids 109 to 119
("antigenic peptide 5"), amino acids 95 to 119 ("antigenic peptide
6"), amino acids 178 to 185 ("antigenic peptide 7"), amino acids
200 to 206 ("antigenic peptide 8"), amino acids 231 to 238
("antigenic peptide 9"), amino acids 231 to 241 ("antigenic peptide
10"), amino acids 264 to 270 ("antigenic peptide 11"), amino acids
274 to 281 ("antigenic peptide 12"), amino acids 317 to 324
("antigenic peptide 13"), amino acids 357 to 363 ("antigenic
peptide 14"), amino acids 384 to 392 ("antigenic peptide 15"),
amino acids 398 to 411 ("antigenic peptide 16"), amino acids 405 to
411 ("antigenic peptide 17"), amino acids 423 to 429 ("antigenic
peptide 18"), and amino acids 434 to 439 ("antigenic peptide 19").
The present invention contemplates the use of any one of antigenic
peptides 1 to 19 to generate antibodies to Zcytor14. The present
invention also contemplates polypeptides comprising at least one of
antigenic peptides 1 to 19.
[0139] In preferred embodiments, antigenic epitopes to which
neutralizing antibodies of the present invention bind would contain
residues of SEQ ID NO:2 (and corresponding residues of SEQ ID NO:3)
or SEQ ID NO:5 that are important to ligand-receptor binding, for
example, with ZcytoR14 and IL-17A or IL-17F (individually or
together).
[0140] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a ZcytoR14 polypeptide,
or by chemical peptide synthesis, as described herein. Moreover,
epitopes can be selected by phage display of random peptide
libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol.
5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616
(1996)). Standard methods for identifying epitopes and producing
antibodies from small peptides that comprise an epitope are
described, for example, by Mole, "Epitope Mapping," in Methods in
Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The Humana
Press, Inc. 1992), Price, "Production and Characterization of
Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
[0141] For any ZcytoR14 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise
ZcytoR14 variants based upon the nucleotide and amino acid
sequences described herein.
E) Production of ZcytoR14 Polypeptides
[0142] The polypeptides of the present invention, including
full-length polypeptides; soluble monomeric, homodimeric,
heterodimeric and multimeric receptors; full-length receptors;
receptor fragments (e.g. ligand-binding fragments and antigenic
epitopes), functional fragments, and fusion proteins, can be
produced in recombinant host cells following conventional
techniques. To express a ZcytoR14 gene, a nucleic acid molecule
encoding the polypeptide must be operably linked to regulatory
sequences that control transcriptional expression in an expression
vector and then, introduced into a host cell. In addition to
transcriptional regulatory sequences, such as promoters and
enhancers, expression vectors can include translational regulatory
sequences and a marker gene which is suitable for selection of
cells that carry the expression vector.
[0143] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, an ZcytoR14
expression vector may comprise a ZcytoR14 gene and a secretory
sequence derived from any secreted gene.
[0144] ZcytoR14 proteins of the present invention may be expressed
in mammalian cells. Examples of suitable mammalian host cells
include African green monkey kidney cells (Vero; ATCC CRL 1587),
human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell.
Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (1H-3T3; ATCC CRL 1658).
[0145] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from mammalian viral sources, for
example, adenovirus, bovine papilloma virus, simian virus, or the
like, in which the regulatory signals are associated with a
particular gene which has a high level of expression. Suitable
transcriptional and translational regulatory sequences also can be
obtained from mammalian genes, for example, actin, collagen,
myosin, and metallothionein genes.
[0146] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Natl. Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0147] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
ZcytoR14 gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.
Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res.
19:4485 (1991)).
[0148] In certain embodiments, a DNA sequence encoding a ZcytoR14
soluble receptor polypeptide, or a fragment of ZcytoR14 polypeptide
is operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers. Multiple
components of a soluble receptor complex can be co-transfected on
individual expression vectors or be contained in a single
expression vector. Such techniques of expressing multiple
components of protein complexes are well known in the art.
[0149] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0150] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
selection is carried out in the presence of a neomycin-type drug,
such as G-418 or the like. Selection systems can also be used to
increase the expression level of the gene of interest, a process
referred to as "amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of the
selective agent and then increasing the amount of selective agent
to select for cells that produce high levels of the products of the
introduced genes. A suitable amplifiable selectable marker is
dihydrofolate reductase (DHFR), which confers resistance to
methotrexate. Other drug resistance genes (e.g., hygromycin
resistance, multi-drug resistance, puromycin acetyltransferase) can
also be used. Alternatively, markers that introduce an altered
phenotype, such as green fluorescent protein, or cell surface
proteins such as CD4, CD8, Class I MHC, placental alkaline
phosphatase may be used to sort transfected cells from
untransfected cells by such means as FACS sorting or magnetic bead
separation technology.
[0151] ZcytoR14 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, retroviruses, herpesvirus,
vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994), and
Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages
of the adenovirus system include the accommodation of relatively
large DNA inserts, the ability to grow to high-titer, the ability
to infect a broad range of mammalian cell types, and flexibility
that allows use with a large number of available vectors containing
different promoters.
[0152] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0153] ZcytoR14 can also be expressed in other higher eukaryotic
cells, such as avian, fungal, insect, yeast, or plant cells. The
baculovirus system provides an efficient means to introduce cloned
ZcytoR14 genes into insect cells. Suitable expression vectors are
based upon the Autographa californica multiple nuclear polyhedrosis
virus (AcMNPV), and contain well-known promoters such as Drosophila
heat shock protein (hsp) 70 promoter, Autographa californica
nuclear polyhedrosis virus immediate-early gene promoter (ie-1) and
the delayed early 39K promoter, baculovirus p10 promoter, and the
Drosophila metallothionein promoter. A second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, et al., J. Virol. 67:4566 (1993)).
This system, which utilizes transfer vectors, is sold in the
BAC-to-BAC kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, PFASTBAC (Life Technologies) containing
a Tn7 transposon to move the DNA encoding the ZcytoR14 polypeptide
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol.
71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), and
Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In
addition, transfer vectors can include an in-frame fusion with DNA
encoding an epitope tag at the C- or N-terminus of the expressed
ZcytoR14 polypeptide, for example, a Glu-Glu epitope tag
(Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using
a technique known in the art, a transfer vector containing a
ZcytoR14 gene is transformed into E. coli, and screened for bacmids
which contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is then isolated using common techniques.
[0154] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native ZcytoR14 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native ZcytoR14 secretory signal
sequence.
[0155] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sj21AE, and Sj21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0156] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0157] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No.
4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S.
Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, and
Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e.g., leucine). A suitable vector system for
use in Saccharomyces cerevisiae is the POT1 vector system disclosed
by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows
transformed cells to be selected by growth in glucose-containing
media. Additional suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et al., U.S. Pat. No.
4,615,974, and Bitter, U.S. Pat. No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154,
5,139,936, and 4,661,454.
[0158] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0159] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica, the promoter and terminator in the
plasmid can be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A suitable selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), and which allows ade2 host cells to grow in the absence
of adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, host cells can be used
in which both methanol utilization genes (AUG1 and AUG2) are
deleted. For production of secreted proteins, host cells can be
deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed by
electroporation using an exponentially decaying, pulsed electric
field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
[0160] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0161] Alternatively, ZcytoR14 genes can be expressed in
prokaryotic host cells. Suitable promoters that can be used to
express ZcytoR14 polypeptides in a prokaryotic host are well-known
to those of skill in the art and include promoters capable of
recognizing the T4, T3, Sp6 and T7 polymerases, the P.sub.R and
P.sub.L promoters of bacteriophage lambda, the trp, recA, heat
shock, lacUV5, tac, lpp-lacSpr, phoA, and lacZ promoters of E.
coli, promoters of B. subtilis, the promoters of the bacteriophages
of Bacillus, Streptomyces promoters, the int promoter of
bacteriophage lambda, the bla promoter of pBR322, and the CAT
promoter of the chloramphenicol acetyl transferase gene.
Prokaryotic promoters have been reviewed by Glick, J. Ind.
Microbiol. 1:277 (1987), Watson et al., Molecular Biology of the
Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al.
(1995).
[0162] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0163] When expressing a ZcytoR14 polypeptide in bacteria such as
E. coli, the polypeptide may be retained in the cytoplasm,
typically as insoluble granules, or may be directed to the
periplasmic space by a bacterial secretion sequence. In the former
case, the cells are lysed, and the granules are recovered and
denatured using, for example, guanidine isothiocyanate or urea. The
denatured polypeptide can then be refolded and dimerized by
diluting the denaturant, such as by dialysis against a solution of
urea and a combination of reduced and oxidized glutathione,
followed by dialysis against a buffered saline solution. In the
latter case, the polypeptide can be recovered from the periplasmic
space in a soluble and functional form by disrupting the cells (by,
for example, sonication or osmotic shock) to release the contents
of the periplasmic space and recovering the protein, thereby
obviating the need for denaturation and refolding.
[0164] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0165] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0166] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0167] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al., Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0168] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:2 or 5. As an illustration, polypeptides can
comprise at least six, at least nine, or at least 15 contiguous
amino acid residues of SEQ ID NO:2 or 5. Within certain embodiments
of the invention, the polypeptides comprise 20, 30, 40, 50, 100, or
more contiguous residues of these amino acid sequences. Nucleic
acid molecules encoding such peptides and polypeptides are useful
as polymerase chain reaction primers and probes.
[0169] Moreover, ZcytoR14 polypeptides and fragments thereof can be
expressed as monomers, homodimers, heterodimers, or multimers
within higher eukaryotic cells. Such cells can be used to produce
ZcytoR14 monomeric, homodimeric, heterodimeric and multimeric
receptor polypeptides that comprise at least one ZcytoR14
polypeptide ("ZcytoR14-comprising receptors" or
"ZcytoR14-comprising receptor polypeptides"), or can be used as
assay cells in screening systems. Within one aspect of the present
invention, a polypeptide of the present invention comprising the
ZcytoR14 extracellular domain is produced by a cultured cell, and
the cell is used to screen for ligands for the receptor, including
the natural ligand, IL-17F, as well as IL-17A, or even agonists and
antagonists of the natural ligand. To summarize this approach, a
cDNA or gene encoding the receptor is combined with other genetic
elements required for its expression (e.g., a transcription
promoter), and the resulting expression vector is inserted into a
host cell. Cells that express the DNA and produce functional
receptor are selected and used within a variety of screening
systems. Each component of the monomeric, homodimeric,
heterodimeric and multimeric receptor complex can be expressed in
the same cell. Moreover, the components of the monomeric,
homodimeric, heterodimeric and multimeric receptor complex can also
be fused to a transmembrane domain or other membrane fusion moiety
to allow complex assembly and screening of transfectants as
described above.
[0170] To assay the IL-17A and IL-17F antagonist polypeptides and
antibodies of the present invention, mammalian cells suitable for
use in expressing ZcytoR14-comprising receptors or other receptors
known to bind IL-17A or IL-17F (e.g., cells expressing IL-17R) and
transducing a receptor-mediated signal include cells that express
other receptor subunits that may form a functional complex with
ZcytoR14. It is also preferred to use a cell from the same species
as the receptor to be expressed. Within a preferred embodiment, the
cell is dependent upon an exogenously supplied hematopoietic growth
factor for its proliferation. Preferred cell lines of this type are
the human TF-1 cell line (ATCC number CRL-2003) and the AML-193
cell line (ATCC number CRL-9589), which are GM-CSF-dependent human
leukemic cell lines and BaF3 (Palacios and Steinmetz, Cell 41:
727-734, (1985)) which is an IL-3 dependent murine pre-B cell line.
Other cell lines include BHK, COS-1 and CHO cells. Suitable host
cells can be engineered to produce the necessary receptor subunits
or other cellular component needed for the desired cellular
response. This approach is advantageous because cell lines can be
engineered to express receptor subunits from any species, thereby
overcoming potential limitations arising from species specificity.
Species orthologs of the human receptor cDNA can be cloned and used
within cell lines from the same species, such as a mouse cDNA in
the BaF3 cell line. Cell lines that are dependent upon one
hematopoietic growth factor, such as GM-CSF or IL-3, can thus be
engineered to become dependent upon another cytokine that acts
through the ZcytoR14 receptor, such as IL-17F or IL-17A.
[0171] Cells expressing functional receptor are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, and the assay detects
activation of transcription of the reporter gene. A preferred
promoter element in this regard is a serum response element, or
SRE. See, e.g., Shaw et al., Cell 56:563-572, (1989). A preferred
such reporter gene is a luciferase gene (de Wet et al., Mol. Cell.
Biol. 7:725, (1987)). Expression of the luciferase gene is detected
by luminescence using methods known in the art (e.g., Baumgartner
et al., J. Biol. Chem. 269:29094-29101, (1994); Schenborn and
Goiffin, Promega.sub.--Notes 41:11, 1993). Luciferase activity
assay kits are commercially available from, for example, Promega
Corp., Madison, Wis. Target cell lines of this type can be used to
screen libraries of chemicals, cell-conditioned culture media,
fungal broths, soil samples, water samples, and the like. For
example, a bank of cell-conditioned media samples can be assayed on
a target cell to identify cells that produce ligand. Positive cells
are then used to produce a cDNA library in a mammalian expression
vector, which is divided into pools, transfected into host cells,
and expressed. Media samples from the transfected cells are then
assayed, with subsequent division of pools, re-transfection,
subculturing, and re-assay of positive cells to isolate a cloned
cDNA encoding the ligand.
[0172] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. Within the first
class, the intracellular domain of ZcytoR14, is joined to the
ligand-binding domain of a second receptor. A second class of
hybrid receptor polypeptides comprise the extracellular
(ligand-binding) domain of ZcytoR14 (SEQ ID NO:3) with an
intracellular domain of a second receptor, preferably a
hematopoietic cytokine receptor, and a transmembrane domain. Hybrid
ZcytoR14 monomers, homodimers, heterodimers and multimers of the
present invention receptors of this second class are expressed in
cells known to be capable of responding to signals transduced by
the second receptor. Together, these two classes of hybrid
receptors enable the identification of a responsive cell type for
the development of an assay for detecting IL-17F or IL-17A.
Moreover, such cells can be used in the presence of IL-17F or
IL-17A to assay the soluble receptor antagonists of the present
invention in a competition-type assay. In such assay, a decrease in
the proliferation or signal transduction activity of IL-17F or
IL-17A in the presence of a soluble receptor of the present
invention demonstrates antagonistic activity. Moreover
ZcytoR14-soluble receptor binding assays, an cell-based assays, can
also be used to assess whether a soluble receptor binds, blocks,
inhibits, reduces, antagonizes or neutralizes IL-17F or IL-17A
activity.
F) Production of ZcytoR14 Fusion Proteins and Conjugates
[0173] One general class of ZcytoR14 analogs are variants having an
amino acid sequence that is a mutation of the amino acid sequence
disclosed herein. Another general class of ZcytoR14 analogs is
provided by anti-idiotype antibodies, and fragments thereof, as
described below. Moreover, recombinant antibodies comprising
anti-idiotype variable domains can be used as analogs (see, for
example, Monfardini et al., Proc. Assoc. Am. Physicians 108:420
(1996)). Since the variable domains of anti-idiotype ZcytoR14
antibodies mimic ZcytoR14, these domains can provide ZcytoR14
binding activity. Methods of producing anti-idiotypic catalytic
antibodies are known to those of skill in the art (see, for
example, Joron et al., Ann. NY Acad. Sci. 672:216 (1992), Friboulet
et al., Appl. Biochem. Biotechnol. 47:229 (1994), and Avalle et
al., Ann. NY Acad. Sci. 864:118 (1998)).
[0174] Another approach to identifying ZcytoR14 analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0175] ZcytoR14 polypeptides have both in vivo and in vitro uses.
As an illustration, a soluble form of ZcytoR14 can be added to cell
culture medium to inhibit the effects of the ZcytoR14 ligand (i.e.
IL-17F, IL-17A or both) produced by the cultured cells.
[0176] Fusion proteins of ZcytoR14 can be used to express ZcytoR14
in a recombinant host, and to isolate the produced ZcytoR14. As
described below, particular ZcytoR14 fusion proteins also have uses
in diagnosis and therapy. One type of fusion protein comprises a
peptide that guides a ZcytoR14 polypeptide from a recombinant host
cell. To direct a ZcytoR14 polypeptide into the secretory pathway
of a eukaryotic host cell, a secretory signal sequence (also known
as a signal peptide, a leader sequence, prepro sequence or pre
sequence) is provided in the ZcytoR14 expression vector. While the
secretory signal sequence may be derived from ZcytoR14, a suitable
signal sequence may also be derived from another secreted protein
or synthesized de novo. The secretory signal sequence is operably
linked to a ZcytoR14-encoding sequence such that the two sequences
are joined in the correct reading frame and positioned to direct
the newly synthesized polypeptide into the secretory pathway of the
host cell. Secretory signal sequences are commonly positioned 5' to
the nucleotide sequence encoding the polypeptide of interest,
although certain secretory signal sequences may be positioned
elsewhere in the nucleotide sequence of interest (see, e.g., Welch
et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0177] Although the secretory signal sequence of ZcytoR14 or
another protein produced by mammalian cells (e.g., tissue-type
plasminogen activator signal sequence, as described, for example,
in U.S. Pat. No. 5,641,655) is useful for expression of ZcytoR14 in
recombinant mammalian hosts, a yeast signal sequence is preferred
for expression in yeast cells. Examples of suitable yeast signal
sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0178] ZcytoR14 soluble receptor polypeptides can be prepared by
expressing a truncated DNA encoding the extracellular domain, for
example, a polypeptide which contains SEQ ID NO:3, or the
corresponding region of a non-human receptor. It is preferred that
the extracellular domain polypeptides be prepared in a form
substantially free of transmembrane and intracellular polypeptide
segments. To direct the export of the receptor domain from the host
cell, the receptor DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide. To facilitate
purification of the secreted receptor domain, a C-terminal
extension, such as a poly-histidine tag, substance P, Flag.TM.
peptide (Hopp et al., Biotechnology 6:1204-1210, (1988); available
from Eastman Kodak Co., New Haven, Conn.) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the receptor polypeptide. Moreover,
ZcytoR14 antigenic epitopes from the extracellular cytokine binding
domains are also prepared as described above.
[0179] In an alternative approach, a receptor extracellular domain
of ZcytoR14 or other cytokine receptor component can be expressed
as a fusion with immunoglobulin heavy chain constant regions,
typically an F.sub.C fragment, which contains two constant region
domains and a hinge region but lacks the variable region (See,
Sledziewski, A Z et al., U.S. Pat. Nos. 6,018,026 and 5,750,375).
The soluble ZcytoR14 polypeptides of the present invention include
such fusions. One such fusion is shown in SEQ ID NO:64. Such
fusions are typically secreted as multimeric molecules wherein the
Fc portions are disulfide bonded to each other and two receptor
polypeptides are arrayed in closed proximity to each other. Fusions
of this type can be used to affinity purify the cognate ligand from
solution, as an in vitro assay tool, to block, inhibit or reduce
signals in vitro by specifically titrating out ligand, and as
antagonists in vivo by administering them parenterally to bind
circulating ligand and clear it from the circulation. To purify
ligand, a ZcytoR14-Ig chimera is added to a sample containing the
ligand (e.g., cell-conditioned culture media or tissue extracts)
under conditions that facilitate receptor-ligand binding (typically
near-physiological temperature, pH, and ionic strength). The
chimera-ligand complex is then separated by the mixture using
protein A, which is immobilized on a solid support (e.g., insoluble
resin beads). The ligand is then eluted using conventional chemical
techniques, such as with a salt or pH gradient. In the alternative,
the chimera itself can be bound to a solid support, with binding
and elution carried out as above. The chimeras may be used in vivo
to regulate inflammatory responses including acute phase responses
such as serum amyloid A (SAA), C-reactive protein (CRP), and the
like. Chimeras with high binding affinity are administered
parenterally (e.g., by intramuscular, subcutaneous or intravenous
injection). Circulating molecules bind ligand and are cleared from
circulation by normal physiological processes. For use in assays,
the chimeras are bound to a support via the F.sub.c region and used
in an ELISA format.
[0180] To assist in isolating anti-ZcytoR14 and binding partners of
the present invention, an assay system that uses a ligand-binding
receptor (or an antibody, one member of a
complement/anti-complement pair) or a binding fragment thereof, and
a commercially available biosensor instrument (BIAcore, Pharmacia
Biosensor, Piscataway, N.J.) may be advantageously employed. Such
receptor, antibody, member of a complement/anti-complement pair or
fragment is immobilized onto the surface of a receptor chip. Use of
this instrument is disclosed by Karlsson, J. Immunol. Methods
145:229-40, 1991 and Cunningham and Wells, J. Mol. Biol.
234:554-63, 1993. A receptor, antibody, member or fragment is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within the flow cell.
A test sample is passed through the cell. If a ligand, epitope, or
opposite member of the complement/anti-complement pair is present
in the sample, it will bind to the immobilized receptor, antibody
or member, respectively, causing a change in the refractive index
of the medium, which is detected as a change in surface plasmon
resonance of the gold film. This system allows the determination of
on- and off-rates, from which binding affinity can be calculated,
and assessment of stoichiometry of binding. Alternatively,
ligand/receptor binding can be analyzed using SELDI.TM. technology
(Ciphergen, Inc., Palo Alto, Calif.). Moreover, BIACORE technology,
described above, can be used to be used in competition experiments
to determine if different monoclonal antibodies bind the same or
different epitopes on the ZcytoR14 polypeptide, and as such, be
used to aid in epitope mapping of neutralizing antibodies of the
present invention that bind, block, inhibit, reduce, antagonize or
neutralize IL-17F or both IL-17A and IL-17F.
[0181] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et
al., Science 245:821-25, 1991).
[0182] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a soluble ZcytoR14
receptor can be prepared as a fusion to a dimerizing protein as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant
region domains, e.g., IgG.gamma.1, and the human .kappa. light
chain. Immunoglobulin-soluble ZcytoR14 fusions can be expressed in
genetically engineered cells to produce a variety of multimeric
ZcytoR14 receptor analogs. Auxiliary domains can be fused to
soluble ZcytoR14 receptor to target them to specific cells,
tissues, or macromolecules (e.g., collagen, or cells expressing the
ZcytoR14 ligands, IL-17F or IL-17A). A ZcytoR14 polypeptide can be
fused to two or more moieties, such as an affinity tag for
purification and a targeting domain. Polypeptide fusions can also
comprise one or more cleavage sites, particularly between domains.
See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
[0183] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, ZcytoR14 can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferase fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a ZcytoR14 fusion protein comprising a
maltose binding protein polypeptide can be isolated with an amylose
resin column, while a fusion protein comprising the C-terminal end
of a truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wis.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0184] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0185] Another form of fusion protein comprises a ZcytoR14
polypeptide and an immunoglobulin heavy chain constant region,
typically an Fc fragment, which contains two or three constant
region domains and a hinge region but lacks the variable region. As
an illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:9). In this fusion protein, an
illustrative Fc moiety is a human .gamma.4 chain, which is stable
in solution and has little or no complement activating activity.
Accordingly, the present invention contemplates a ZcytoR14 fusion
protein that comprises a ZcytoR14 moiety and a human Fc fragment,
wherein the C-terminus of the ZcytoR14 moiety is attached to the
N-terminus of the Fc fragment via a peptide linker, such as a
peptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID
NO:5. The ZcytoR14 moiety can be a ZcytoR14 molecule or a fragment
thereof. For example, a fusion protein can comprise the amino acid
of SEQ ID NO:3 and an Fc fragment (e.g., a human Fc fragment) (SEQ
ID NO:64).
[0186] In another variation, a ZcytoR14 fusion protein comprises an
IgG sequence, a ZcytoR14 moiety covalently joined to the
aminoterminal end of the IgG sequence, and a signal peptide that is
covalently joined to the aminoterminal of the ZcytoR14 moiety,
wherein the IgG sequence consists of the following elements in the
following order: a hinge region, a CH.sub.2 domain, and a CH.sub.3
domain. Accordingly, the IgG sequence lacks a CH.sub.1 domain. The
ZcytoR14 moiety displays a ZcytoR14 activity, as described herein,
such as the ability to bind with a ZcytoR14 ligand. This general
approach to producing fusion proteins that comprise both antibody
and nonantibody portions has been described by LaRochelle et al.,
EP 742830 (WO 95/21258).
[0187] Fusion proteins comprising a ZcytoR14 moiety and an Fc
moiety can be used, for example, as an in vitro assay tool. For
example, the presence of a ZcytoR14 ligand in a biological sample
can be detected using a ZcytoR14-immunoglobulin fusion protein, in
which the ZcytoR14 moiety is used to bind the ligand, and a
macromolecule, such as Protein A or anti-Fc antibody, is used to
bind the fusion protein to a solid support. Such systems can be
used to identify agonists and antagonists that interfere with the
binding of a ZcytoR14 ligands, e.g., IL-17F or both IL-17A and
IL-17F, to their receptor.
[0188] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a ZcytoR14
fragment that contains a ZcytoR14 extracellular domain. Such
molecules can be used to target particular tissues for the benefit
of ZcytoR14 binding activity.
[0189] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between ZcytoR14 of
the present invention with the functionally equivalent domain(s)
from another member of the cytokine receptor family. Polypeptide
fusions can be expressed in recombinant host cells to produce a
variety of ZcytoR14 fusion analogs. A ZcytoR14 polypeptide can be
fused to two or more moieties or domains, such as an affinity tag
for purification and a targeting domain. Polypeptide fusions can
also comprise one or more cleavage sites, particularly between
domains. See, for example, Tuan et al., Connective Tissue Research
34:1 (1996).
[0190] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0191] ZcytoR14 binding domains can be further characterized by
physical analysis of structure, as determined by such techniques as
nuclear magnetic resonance, crystallography, electron diffraction
or photoaffinity labeling, in conjunction with mutation of putative
contact site amino acids of ZcytoR14 ligand agonists. See, for
example, de Vos et al., Science 255:306 (1992), Smith et al., J.
Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59
(1992).
[0192] The present invention also contemplates chemically modified
ZcytoR14 compositions, in which a ZcytoR14 polypeptide is linked
with a polymer. Illustrative ZcytoR14 polypeptides are soluble
polypeptides that lack a functional transmembrane domain, such as a
polypeptide consisting of amino acid residues SEQ ID NO:3.
Typically, the polymer is water soluble so that the ZcytoR14
conjugate does not precipitate in an aqueous environment, such as a
physiological environment. An example of a suitable polymer is one
that has been modified to have a single reactive group, such as an
active ester for acylation, or an aldehyde for alkylation. In this
way, the degree of polymerization can be controlled. An example of
a reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C.sub.1-C.sub.10) alkoxy, or aryloxy derivatives thereof
(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The
polymer may be branched or unbranched. Moreover, a mixture of
polymers can be used to produce ZcytoR14 conjugates.
[0193] ZcytoR14 conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
Suitable water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C.sub.1-C.sub.10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A
ZcytoR14 conjugate can also comprise a mixture of such
water-soluble polymers.
[0194] One example of a ZcytoR14 conjugate comprises a ZcytoR14
moiety and a polyalkyl oxide moiety attached to the N-terminus of
the ZcytoR14 moiety. PEG is one suitable polyalkyl oxide. As an
illustration, ZcytoR14 can be modified with PEG, a process known as
"PEGylation." PEGylation of ZcytoR14 can be carried out by any of
the PEGylation reactions known in the art (see, for example, EP 0
154 316, Delgado et al., Critical Reviews in Therapeutic Drug
Carrier Systems 9:249 (1992), Duncan and Spreafico, Clin.
Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol
68:1 (1998)). For example, PEGylation can be performed by an
acylation reaction or by an alkylation reaction with a reactive
polyethylene glycol molecule. In an alternative approach, ZcytoR14
conjugates are formed by condensing activated PEG, in which a
terminal hydroxy or amino group of PEG has been replaced by an
activated linker (see, for example, Karasiewicz et al., U.S. Pat.
No. 5,382,657).
[0195] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a ZcytoR14 polypeptide. An
example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between ZcytoR14 and a water
soluble polymer: amide, carbamate, urethane, and the like. Methods
for preparing PEGylated ZcytoR14 by acylation will typically
comprise the steps of (a) reacting a ZcytoR14 polypeptide with PEG
(such as a reactive ester of an aldehyde derivative of PEG) under
conditions whereby one or more PEG groups attach to ZcytoR14, and
(b) obtaining the reaction product(s). Generally, the optimal
reaction conditions for acylation reactions will be determined
based upon known parameters and desired results. For example, the
larger the ratio of PEG:ZcytoR14, the greater the percentage of
polyPEGylated ZcytoR14 product.
[0196] The product of PEGylation by acylation is typically a
polyPEGylated ZcytoR14 product, wherein the lysine .epsilon.-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting ZcytoR14
will be at least 95% mono-, di-, or tri-pegylated, although some
species with higher degrees of PEGylation may be formed depending
upon the reaction conditions. PEGylated species can be separated
from unconjugated ZcytoR14 polypeptides using standard purification
methods, such as dialysis, ultrafiltration, ion exchange
chromatography, affinity chromatography, and the like.
[0197] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with ZcytoR14 in the presence
of a reducing agent. PEG groups can be attached to the polypeptide
via a --CH.sub.2--NH group.
[0198] Moreover, anti-ZcytoR14 antibodies or antibody fragments of
the present invention can be PEGylated using methods in the art and
described herein.
[0199] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
.epsilon.-amino groups of the lysine residues and the .alpha.-amino
group of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups. The
present invention provides a substantially homogenous preparation
of ZcytoR14 monopolymer conjugates.
[0200] Reductive alkylation to produce a substantially homogenous
population of monopolymer ZcytoR14 conjugate molecule can comprise
the steps of: (a) reacting a ZcytoR14 polypeptide with a reactive
PEG under reductive alkylation conditions at a pH suitable to
permit selective modification of the .alpha.-amino group at the
amino terminus of the ZcytoR14, and (b) obtaining the reaction
product(s). The reducing agent used for reductive alkylation should
be stable in aqueous solution and able to reduce only the Schiff
base formed in the initial process of reductive alkylation.
Illustrative reducing agents include sodium borohydride, sodium
cyanoborohydride, dimethylamine borane, trimethylamine borane, and
pyridine borane.
[0201] For a substantially homogenous population of monopolymer
ZcytoR14 conjugates, the reductive alkylation reaction conditions
are those that permit the selective attachment of the water-soluble
polymer moiety to the N-terminus of ZcytoR14. Such reaction
conditions generally provide for pKa differences between the lysine
amino groups and the .alpha.-amino group at the N-terminus. The pH
also affects the ratio of polymer to protein to be used. In
general, if the pH is lower, a larger excess of polymer to protein
will be desired because the less reactive the N-terminal
.alpha.-group, the more polymer is needed to achieve optimal
conditions. If the pH is higher, the polymer:ZcytoR14 need not be
as large because more reactive groups are available. Typically, the
pH will fall within the range of 3 to 9, or 3 to 6. This method can
be employed for making ZcytoR14-comprising homodimeric,
heterodimeric or multimeric soluble receptor conjugates.
[0202] Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of
water-soluble polymer to ZcytoR14 will generally be in the range of
1:1 to 100:1. Typically, the molar ratio of water-soluble polymer
to ZcytoR14 will be 1:1 to 20:1 for polyPEGylation, and 1:1 to 5:1
for monoPEGylation.
[0203] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)). This method can be employed for making
ZcytoR14-comprising homodimeric, heterodimeric or multimeric
soluble receptor conjugates.
[0204] The present invention contemplates compositions comprising a
peptide or polypeptide, such as a soluble receptor or antibody
described herein. Such compositions can further comprise a carrier.
The carrier can be a conventional organic or inorganic carrier.
Examples of carriers include water, buffer solution, alcohol,
propylene glycol, macrogol, sesame oil, corn oil, and the like.
G) Isolation of ZcytoR14 Polypeptides
[0205] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95%, such as 96%, 97%, 98%,
or greater than 99% purity with respect to contaminating
macromolecules, particularly other proteins and nucleic acids, and
free of infectious and pyrogenic agents. The polypeptides of the
present invention may also be purified to a pharmaceutically pure
state, which is greater than 99.9% pure. In certain preparations,
purified polypeptide is substantially free of other polypeptides,
particularly other polypeptides of animal origin.
[0206] Fractionation and/or conventional purification methods can
be used to obtain preparations of ZcytoR14 purified from natural
sources (e.g., human tissue sources), synthetic ZcytoR14
polypeptides, and recombinant ZcytoR14 polypeptides and fusion
ZcytoR14 polypeptides purified from recombinant host cells. In
general, ammonium sulfate precipitation and acid or chaotrope
extraction may be used for fractionation of samples. Exemplary
purification steps may include hydroxyapatite, size exclusion, FPLC
and reverse-phase high performance liquid chromatography. Suitable
chromatographic media include derivatized dextrans, agarose,
cellulose, polyacrylamide, specialty silicas, and the like. PEI,
DEAE, QAE and Q derivatives are suitable. Exemplary chromatographic
media include those media derivatized with phenyl, butyl, or octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl
650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso
Haas) and the like. Suitable solid supports include glass beads,
silica-based resins, cellulosic resins, agarose beads, cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide
resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties.
[0207] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0208] Additional variations in ZcytoR14 isolation and purification
can be devised by those of skill in the art. For example,
anti-ZcytoR14 antibodies, obtained as described below, can be used
to isolate large quantities of protein by immunoaffinity
purification.
[0209] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification. Moreover, the ligand-binding properties of ZcytoR14
extracellular domain can be exploited for purification, for
example, of ZcytoR14-comprising soluble receptors; for example, by
using affinity chromatography wherein IL-17F ligand is bound to a
column and the ZcytoR14-comprising receptor is bound and
subsequently eluted using standard chromatography methods.
[0210] ZcytoR14 polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. ZcytoR14
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; PEGylated or non-PEGylated; and may or may not
include an initial methionine amino acid residue.
H) Production of Antibodies to ZcytoR14 Proteins
[0211] Antibodies to ZcytoR14 can be obtained, for example, using
the product of a ZcytoR14 expression vector or ZcytoR14 isolated
from a natural source as an antigen. Particularly useful
anti-ZcytoR14 antibodies "bind specifically" with ZcytoR14.
Antibodies are considered to be specifically binding if the
antibodies exhibit at least one of the following two properties:
(1) antibodies bind to ZcytoR14 with a threshold level of binding
activity, and (2) antibodies do not significantly cross-react with
polypeptides related to ZcytoR14.
[0212] With regard to the first characteristic, antibodies
specifically bind if they bind to a ZcytoR14 polypeptide, peptide
or epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1
or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci.
51:660 (1949)). With regard to the second characteristic,
antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect ZcytoR14, but
not presently known polypeptides using a standard Western blot
analysis. Examples of known related polypeptides include known
cytokine receptors.
[0213] Anti-ZcytoR14 antibodies can be produced using antigenic
ZcytoR14 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, or between 15 to about 30
amino acids contained within SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5
or another amino acid sequence disclosed herein. However, peptides
or polypeptides comprising a larger portion of an amino acid
sequence of the invention, containing from 30 to 50 amino acids, or
any length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are useful for inducing
antibodies that bind with ZcytoR14. It is desirable that the amino
acid sequence of the epitope-bearing peptide is selected to provide
substantial solubility in aqueous solvents (i.e., the sequence
includes relatively hydrophilic residues, while hydrophobic
residues are typically avoided). Moreover, amino acid sequences
containing proline residues may be also be desirable for antibody
production.
[0214] As an illustration, potential antigenic sites in ZcytoR14
were identified using the Jameson-Wolf method, Jameson and Wolf,
CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0215] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; .alpha. region threshold=103;
.beta. region threshold=105; Garnier-Robson parameters: .alpha. and
.beta. decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible. Hopp/Woods
hydrophilicity profiles can be used to determine regions that have
the most antigenic potential within SEQ ID NO:3 (Hopp et al., Proc.
Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18,
1986 and Triquier et al., Protein Engineering 11:153-169, 1998).
The profile is based on a sliding six-residue window. Buried G, S,
and T residues and exposed H, Y, and W residues were ignored.
Moreover, ZcytoR14 antigenic epitopes within SEQ ID NO:3 as
predicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean
program (DNASTAR, Inc., Madison, Wis.) serve as preferred antigenic
epitopes, and can be determined by one of skill in the art. Such
antigenic epitopes include (1) amino acid residue 73 to amino acid
residue 82 of SEQ ID NO:3; (2) amino acid residue 95 to amino acid
residue 104 of SEQ ID NO:3; (3) amino acid residue 111 to amino
acid residue 119 of SEQ ID NO:3; (4) amino acid residue 179 to
amino acid residue 186 of SEQ ID NO:3; (5) amino acid residue 200
to amino acid residue 205 of SEQ ID NO:3; (6) amino acid residue
229 to amino acid residue 236 of SEQ ID NO:3; (7) amino acid
residue 264 to amino acid residue 268 of SEQ ID NO:3; and (8) amino
acid residue 275 to amino acid residue 281 of SEQ ID NO:3. The
present invention contemplates the use of any one of antigenic
peptides X to Y to generate antibodies to ZcytoR14 or as a tool to
screen or identify neutralizing monoclonal antibodies of the
present invention. The present invention also contemplates
polypeptides comprising at least one of antigenic peptides X to Y.
The present invention contemplates the use of any antigenic
peptides or epitopes described herein to generate antibodies to
ZcytoR14, as well as to identify and screen anti-ZcytoR14
monoclonal antibodies that are neutralizing, and that may bind,
block, inhibit, reduce, antagonize or neutralize the activity of
IL-17F and IL-17A (individually or together).
[0216] Moreover, suitable antigens also include the ZcytoR14
polypeptides comprising a ZcytoR14 cytokine binding, or
extracellular domain disclosed above in combination with another
cytokine extracellular domain, such as a class I or II cytokine
receptor domain, such as those that may form soluble ZcytoR14
heterodimeric or multimeric polypeptides, and the like.
[0217] Polyclonal antibodies to recombinant ZcytoR14 protein or to
ZcytoR14 isolated from natural sources can be prepared using
methods well-known to those of skill in the art. See, for example,
Green et al., "Production of Polyclonal Antisera," in
Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press
1992), and Williams et al., "Expression of foreign proteins in E.
coli using plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a ZcytoR14 polypeptide can be increased through
the use of an adjuvant, such as alum (aluminum hydroxide) or
Freund's complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of
ZcytoR14 or a portion thereof with an immunoglobulin polypeptide or
with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0218] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-ZcytoR14 antibody of the
present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310
(1990).
[0219] Alternatively, monoclonal anti-ZcytoR14 antibodies can be
generated. Rodent mono-clonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0220] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a ZcytoR14 gene product,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0221] In addition, an anti-ZcytoR14 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0222] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0223] For particular uses, it may be desirable to prepare
fragments of anti-ZcytoR14 antibodies. Such antibody fragments can
be obtained, for example, by proteolytic hydrolysis of the
antibody. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. As an
illustration, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0224] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0225] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0226] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0227] As an illustration, a scFV can be obtained by exposing
lymphocytes to ZcytoR14 polypeptide in vitro, and selecting
antibody display libraries in phage or similar vectors (for
instance, through use of immobilized or labeled ZcytoR14 protein or
peptide). Genes encoding polypeptides having potential ZcytoR14
polypeptide binding domains can be obtained by screening random
peptide libraries displayed on phage (phage display) or on
bacteria, such as E. coli. Nucleotide sequences encoding the
polypeptides can be obtained in a number of ways, such as through
random mutagenesis and random polynucleotide synthesis. These
random peptide display libraries can be used to screen for peptides
which interact with a known target which can be a protein or
polypeptide, such as a ligand or receptor, a biological or
synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display
libraries are known in the art (Ladner et al., U.S. Pat. No.
5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,
U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,
and Kay et al., Phage Display of Peptides and Proteins (Academic
Press, Inc. 1996)) and random peptide display libraries and kits
for screening such libraries are available commercially, for
instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.),
Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.
(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,
N.J.). Random peptide display libraries can be screened using the
ZcytoR14 sequences disclosed herein to identify proteins which bind
to ZcytoR14.
[0228] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0229] Alternatively, an anti-ZcytoR14 antibody may be derived from
a "humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0230] Moreover, anti-ZcytoR14 antibodies or antibody fragments of
the present invention can be PEGylated using methods in the art and
described herein.
[0231] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-ZcytoR14 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-ZcytoR14 antibodies or antibody fragments as immunogens with
the techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.
77:1875 (1996).
[0232] An anti-ZcytoR14 antibody can be conjugated with a
detectable label to form an anti-ZcytoR14 immunoconjugate. Suitable
detectable labels include, for example, a radioisotope, a
fluorescent label, a chemiluminescent label, an enzyme label, a
bioluminescent label or colloidal gold. Methods of making and
detecting such detectably-labeled immunoconjugates are well-known
to those of ordinary skill in the art, and are described in more
detail below.
[0233] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0234] Anti-ZcytoR14 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0235] Alternatively, anti-ZcytoR14 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0236] Similarly, a bioluminescent compound can be used to label
anti-ZcytoR14 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0237] Alternatively, anti-ZcytoR14 immunoconjugates can be
detectably labeled by linking an anti-ZcytoR14 antibody component
to an enzyme. When the anti-ZcytoR14-enzyme conjugate is incubated
in the presence of the appropriate substrate, the enzyme moiety
reacts with the substrate to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0238] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-ZcytoR14 antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0239] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-ZcytoR14 antibodies that
have been conjugated with avidin, streptavidin, and biotin (see,
for example, Wilchek et al. (eds.), "Avidin-Biotin Technology,"
Methods In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et
al., "Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0240] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0241] The present invention also contemplates kits for performing
an immunological diagnostic assay for ZcytoR14 gene expression.
Such kits comprise at least one container comprising an
anti-ZcytoR14 antibody, or antibody fragment. A kit may also
comprise a second container comprising one or more reagents capable
of indicating the presence of ZcytoR14 antibody or antibody
fragments. Examples of such indicator reagents include detectable
labels such as a radioactive label, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label,
colloidal gold, and the like. A kit may also comprise a means for
conveying to the user that ZcytoR14 antibodies or antibody
fragments are used to detect ZcytoR14 protein. For example, written
instructions may state that the enclosed antibody or antibody
fragment can be used to detect ZcytoR14. The written material can
be applied directly to a container, or the written material can be
provided in the form of a packaging insert.
I) Use of Anti-ZcytoR14 Antibodies to Antagonize ZcytoR14 Binding
to IL-17F or both IL-17A and IL-17F
[0242] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble ZcytoR14 receptor polypeptides or fragments thereof,
such as antigenic epitopes, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled soluble ZcytoR14 receptor polypeptides or
fragments thereof, such as antigenic epitopes). Genes encoding
polypeptides having potential binding domains such as soluble
ZcytoR14 receptor polypeptides or fragments thereof, such as
antigenic epitopes can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides that interact with a
known target that can be a protein or polypeptide, such as a ligand
or receptor, a biological or synthetic macromolecule, or organic or
inorganic substances. Techniques for creating and screening such
random peptide display libraries are known in the art (Ladner et
al., U.S. Pat. No. 5,223,409; Ladner et al., U.S. Pat. No.
4,946,778; Ladner et al., U.S. Pat. No. 5,403,484 and Ladner et
al., U.S. Pat. No. 5,571,698) and random peptide display libraries
and kits for screening such libraries are available commercially,
for instance from Clontech (Palo Alto, Calif.), Invitrogen Inc.
(San Diego, Calif.), New England Biolabs, Inc. (Beverly, Mass.) and
Pharmacia LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide
display libraries can be screened using the soluble ZcytoR14
receptor polypeptides or fragments thereof, such as antigenic
epitope polypeptide sequences disclosed herein to identify proteins
which bind to ZcytoR14-comprising receptor polypeptides. These
"binding polypeptides," which interact with soluble
ZcytoR14-comprising receptor polypeptides, can be used for tagging
cells; for isolating homolog polypeptides by affinity purification;
they can be directly or indirectly conjugated to drugs, toxins,
radionuclides and the like. These binding polypeptides can also be
used in analytical methods such as for screening expression
libraries and neutralizing activity, e.g., for binding, blocking,
inhibiting, reducing, antagonizing or neutralizing interaction
between IL-17A and IL-17F (individually or together) and ZcytoR14,
or viral binding to a receptor. The binding polypeptides can also
be used for diagnostic assays for determining circulating levels of
soluble ZcytoR14-comprising receptor polypeptides; for detecting or
quantitating soluble or non-soluble ZcytoR14-comprising receptors
as marker of underlying pathology or disease. These binding
polypeptides can also act as "antagonists" to block or inhibit
soluble or membrane-bound ZcytoR14 monomeric receptor or ZcytoR14
homodimeric, heterodimeric or multimeric polypeptide binding (e.g.
to ligand) and signal transduction in vitro and in vivo. Again,
these binding polypeptides serve as anti-ZcytoR14 monomeric
receptor or anti-ZcytoR14 homodimeric, heterodimeric or multimeric
polypeptides and are useful for inhibiting IL-17F or both IL-17A
and IL-17F activity, as well as receptor activity or
protein-binding. Antibodies raised to the natural receptor
complexes of the present invention, and ZcytoR14-epitope-binding
antibodies, and anti-ZcytoR14 neutralizing monoclonal antibodies
may be preferred embodiments, as they may act more specifically
against the ZcytoR14 and can inhibit IL-17F or both IL-17A and
IL-17F. Moreover, the antagonistic and binding activity of the
antibodies of the present invention can be assayed in an IL-17A or
IL-17F proliferation, signal trap, luciferase or binding assays in
the presence of IL-17A or IL-17F respectively, and
ZcytoR14-comprising soluble receptors, and other biological or
biochemical assays described herein.
[0243] Antibodies to soluble ZcytoR14 receptor polypeptides (e.g.,
antibodies to SEQ ID NO:3) or fragments thereof, such as antigenic
epitopes may be used for inhibiting the inflammatory effects of
IL-17A, IL-17F, or both IL-17A and IL-17F in vivo, for therapeutic
use against inflammation and inflammatory diseases such as
psoriasis, psoriatic arthritis, rheumatoid arthritis, endotoxemia,
inflammatory bowel disease (IBD), colitis, asthma, allograft
rejection, immune mediated renal diseases, hepatobiliary diseases,
multiple sclerosis, atherosclerosis, promotion of tumor growth, or
degenerative joint disease and other inflammatory conditions
disclosed herein; tagging cells that express ZcytoR14 receptors;
for isolating soluble ZcytoR14-comprising receptor polypeptides by
affinity purification; for diagnostic assays for determining
circulating levels of soluble ZcytoR14-comprising receptor
polypeptides; for detecting or quantitating soluble
ZcytoR14-comprising receptors as marker of underlying pathology or
disease; in analytical methods employing FACS; for screening
expression libraries; for generating anti-idiotypic antibodies that
can act as IL-17F or IL-17A agonists; and as neutralizing
antibodies or as antagonists to bind, block, inhibit, reduce, or
antagonize ZcytoR14 receptor function, or to bind, block, inhibit,
reduce, antagonize or neutralize IL-17F and/or IL-17A activity
(either individually or together) in vitro and in vivo. Suitable
direct tags or labels include radionuclides, enzymes, substrates,
cofactors, biotin, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like; indirect
tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to soluble ZcytoR14-comprising receptor polypeptides, or
fragments thereof may be used in vitro to detect denatured or
non-denatured ZcytoR14-comprising receptor polypeptides or
fragments thereof in assays, for example, Western Blots or other
assays known in the art.
[0244] Antibodies to soluble ZcytoR14 receptor or soluble ZcytoR14
homodimeric, heterodimeric or multimeric receptor polypeptides are
useful for tagging cells that express the corresponding receptors
and assaying their expression levels, for affinity purification,
within diagnostic assays for determining circulating levels of
receptor polypeptides, analytical methods employing
fluorescence-activated cell sorting. Moreover, divalent antibodies,
and anti-idiotypic antibodies may be used as agonists to mimic the
effect of the ZcytoR14 ligand, IL-17F or IL-17A.
[0245] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
soluble ZcytoR14 receptor or soluble ZcytoR14 homodimeric,
heterodimeric or multimeric receptor polypeptides can be used to
identify or treat tissues or organs that express a corresponding
anti-complementary molecule (i.e., a ZcytoR14-comprising soluble or
membrane-bound receptor). More specifically, antibodies to soluble
ZcytoR14-comprising receptor polypeptides, or bioactive fragments
or portions thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues or organs
that express the ZcytoR14-comprising receptor such as
ZcytoR14-expressing cancers.
[0246] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind ZcytoR14-comprising receptor
polypeptides, such as "binding polypeptides," (including binding
peptides disclosed above), antibodies, or bioactive fragments or
portions thereof. Suitable detectable molecules include
radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like. Suitable cytotoxic molecules may be directly or
indirectly attached to the polypeptide or antibody, and include
bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas exotoxin, ricin, abrin and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188 or
yttrium-90 (either directly attached to the polypeptide or
antibody, or indirectly attached through means of a chelating
moiety, for instance). Binding polypeptides or antibodies may also
be conjugated to cytotoxic drugs, such as adriamycin. For indirect
attachment of a detectable or cytotoxic molecule, the detectable or
cytotoxic molecule can be conjugated with a member of a
complementary/anticomplementary pair, where the other member is
bound to the binding polypeptide or antibody portion. For these
purposes, biotin/streptavidin is an exemplary
complementary/anticomplementary pair.
[0247] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues). Alternatively, if the binding polypeptide
has multiple functional domains (i.e., an activation domain or a
ligand binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for directing a
detectable molecule, a cytotoxic molecule or a complementary
molecule to a cell or tissue type of interest. In instances where
the fusion protein including only a single domain includes a
complementary molecule, the anti-complementary molecule can be
conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0248] In another embodiment, ZcytoR14 binding polypeptide-cytokine
or antibody-cytokine fusion proteins can be used for enhancing in
vivo killing of target tissues (for example, spleen, pancreatic,
blood, lymphoid, colon, and bone marrow cancers), if the binding
polypeptide-cytokine or anti-ZcytoR14 receptor antibody targets the
hyperproliferative cell (See, generally, Hornick et al., Blood
89:4437-47, 1997). The described fusion proteins enable targeting
of a cytokine to a desired site of action, thereby providing an
elevated local concentration of cytokine. Suitable anti-ZcytoR14
monomer, homodimer, heterodimer or multimer antibodies target an
undesirable cell or tissue (i.e., a tumor or a leukemia), and the
fused cytokine mediates improved target cell lysis by effector
cells. Suitable cytokines for this purpose include interleukin 2
and granulocyte-macrophage colony-stimulating factor (GM-CSF), for
instance.
[0249] Alternatively, ZcytoR14 receptor binding polypeptides or
antibody fusion proteins described herein can be used for enhancing
in vivo killing of target tissues by directly stimulating a
ZcytoR14 receptor-modulated apoptotic pathway, resulting in cell
death of hyperproliferative cells expressing ZcytoR14-comprising
receptors.
J) Therapeutic Uses of Polypeptides Having ZcytoR14 Activity or
Antibodies to ZcytoR14
[0250] Amino acid sequences having soluble ZcytoR14 activity can be
used to modulate the immune system by binding ZcytoR14 ligands
IL-17A and IL-17F (either singly or together), and thus, preventing
the binding of ZcytoR14 ligand with endogenous ZcytoR14 receptor.
ZcytoR14 antagonists, such as soluble ZcytoR14 or anti-ZcytoR14
antibodies, can also be used to modulate the immune system by
inhibiting the binding of ZcytoR14 ligand with the endogenous
ZcytoR14 receptor. Accordingly, the present invention includes the
use of proteins, polypeptides, and peptides having ZcytoR14
activity (such as soluble ZcytoR14 polypeptides, ZcytoR14
polypeptide fragments, ZcytoR14 analogs (e.g., anti-ZcytoR14
anti-idiotype antibodies), and ZcytoR14 fusion proteins) to a
subject which lacks an adequate amount of this polypeptide, or
which produces an excess of ZcytoR14 ligand. ZcytoR14 antagonists
(e.g., anti-ZcytoR14 antibodies) can be also used to treat a
subject which produces an excess of either ZcytoR14 ligand or
ZcytoR14. Suitable subjects include mammals, such as humans. For
example, such ZcytoR14 polypeptides and anti-ZcytoR14 antibodies
are useful in binding, blocking, inhibiting, reducing, antagonizing
or neutralizing IL-17A and IL-17F (either singly or together), in
the treatment of inflammation and inflammatory diseases such as
psoriasis, psoriatic arthritis, rheumatoid arthritis, endotoxemia,
inflammatory bowel disease (IBD), colitis, asthma, allograft
rejection, immune mediated renal diseases, hepatobiliary diseases,
multiple sclerosis, atherosclerosis, promotion of tumor growth, or
degenerative joint disease and other inflammatory conditions
disclosed herein.
[0251] Within preferred embodiments, the soluble receptor form of
ZcytoR14, SEQ ID NO:3) is a monomer, homodimer, heterodimer, or
multimer that binds to, blocks, inhibits, reduces, antagonizes or
neutralizes IL-17F and IL-17A (individually or together) in vivo.
Antibodies and binding polypeptides to such ZcytoR14 monomer,
homodimer, heterodimer, or multimers also serve as antagonists of
ZcytoR14 activity, and as IL-17A and IL-17F antagonists (singly or
together), as described herein.
[0252] In addition, we have described herein that both polyclonal
and monoclonal neutralizing anti-IL-17F antibodies bind to, block,
inhibit, reduce, antagonize or neutralize IL-17F and IL-17A
activity in cell based neutralization assays. Analysis of the
tissue distribution of the mRNA corresponding ZcytoR14 cDNA showed
that mRNA the ZcytoR14 gene is strongly expressed in thyroid,
adrenal gland, prostate, and liver tissues, and expressed to a
lesser extent in heart, small intestine, stomach, and trachea
tissues. In particular, ZcytoR14 is consistently expressed in non-T
cell peripheral blood cell lines, including monocytes, B-cells, and
cells of the myeloid lineage. Also, ZcytoR14 mRNA is reliably
expressed in cell lines derived from skin. Other cell lines that
express ZcytoR14 are all 5 of the large intestine cell lines that
were present on the array. In contrast, there is little or no
expression in brain, placenta, lung, skeletal muscle, kidney,
pancreas, spleen, thymus, testis, ovary, colon, peripheral blood
leukocytes, spinal cord, lymph node, and bone marrow. The ligand to
which ZcytoR14 binds (IL-17F and/or IL-17A) is implicated in
inducing inflammatory response and contributing to inflammatory
diseases, primarily via its ability to enhance production of
inflammatory mediators, including IL-1b, IL-6 and TNF-a, as well as
those mediators that are involved in the proliferation, maturation
and chemotaxis of neutrophils (reviewed in Witowski et al. Cell.
Mol. Life Sci. 61:567-579 [2004]).
[0253] Thus, particular embodiments of the present invention are
directed toward use of soluble ZcytoR14 and anti-ZcytoR14
antibodies as antagonists in inflammatory and immune diseases or
conditions such as psoriasis, psoriatic arthritis, atopic
dermatitis, inflammatory skin conditions, rheumatoid arthritis,
inflammatory bowel disease (IBD), Crohn's Disease, diverticulosis,
asthma, pancreatitis, type I diabetes (IDDM), pancreatic cancer,
pancreatitis, Graves Disease, colon and intestinal cancer,
autoimmune disease, sepsis, organ or bone marrow transplant;
inflammation due to endotoxemia, trauma, surgery or infection;
amyloidosis; splenomegaly; graft versus host disease; and where
inhibition of inflammation, immune suppression, reduction of
proliferation of hematopoietic, immune, inflammatory or lymphoid
cells, macrophages, T-cells (including Th1 and Th2 cells),
suppression of immune response to a pathogen or antigen, or other
instances where inhibition of IL-17F or IL-17A cytokines is
desired.
[0254] Moreover, antibodies or binding polypeptides that bind
ZcytoR14 polypeptides described herein, and ZcytoR14 polypeptides
themselves are useful to:
[0255] 1) Block, inhibit, reduce, antagonize or neutralize
signaling via IL-17A or IL-17F receptors in the treatment of acute
inflammation, inflammation as a result of trauma, tissue injury,
surgery, sepsis or infection, and chronic inflammatory diseases
such as asthma, inflammatory bowel disease (IBD), chronic colitis,
splenomegaly, rheumatoid arthritis, recurrent acute inflammatory
episodes (e.g., tuberculosis), and treatment of amyloidosis, and
atherosclerosis, Castleman's Disease, asthma, and other diseases
associated with the induction of acute-phase response.
[0256] 2) Block, inhibit, reduce, antagonize or neutralize
signaling via IL-17A or IL-17F receptors in the treatment of
autoimmune diseases such as IDDM, multiple sclerosis (MS), systemic
Lupus erythematosus (SLE), myasthenia gravis, rheumatoid arthritis,
and IBD to prevent or inhibit signaling in immune cells (e.g.
lymphocytes, monocytes, leukocytes) via ZcytoR14. Alternatively
antibodies, such as monoclonal antibodies (MAb) to
ZcytoR14-comprising receptors, can also be used as an antagonist to
deplete unwanted immune cells to treat autoimmune disease. Asthma,
allergy and other atopic disease may be treated with an MAb
against, for example, soluble ZcytoR14 soluble receptors to inhibit
the immune response or to deplete offending cells. Blocking,
inhibiting, reducing, or antagonizing signaling via ZcytoR14, using
the polypeptides and antibodies of the present invention, may also
benefit diseases of the pancreas, kidney, pituitary and neuronal
cells. IDDM, NIDDM, pancreatitis, and pancreatic carcinoma may
benefit. ZcytoR14 may serve as a target for MAb therapy of cancer
where an antagonizing MAb inhibits cancer growth and targets
immune-mediated killing. (Holliger P, and Hoogenboom, H: Nature
Biotech. 16: 1015-1016, 1998). Mabs to soluble ZcytoR14 may also be
useful to treat nephropathies such as glomerulosclerosis,
membranous neuropathy, amyloidosis (which also affects the kidney
among other tissues), renal arteriosclerosis, glomerulonephritis of
various origins, fibroproliferative diseases of the kidney, as well
as kidney dysfunction associated with SLE, IDDM, type II diabetes
(NIDDM), renal tumors and other diseases.
[0257] 3) Agonize, enhance, increase or initiate signaling via
IL-17A or IL-17F receptors in the treatment of autoimmune diseases
such as IDDM, MS, SLE, myasthenia gravis, rheumatoid arthritis, and
IBD. Anti-ZcytoR14 neutralizing and monoclonal antibodies may
signal lymphocytes or other immune cells to differentiate, alter
proliferation, or change production of cytokines or cell surface
proteins that ameliorate autoimmunity. Specifically, modulation of
a T-helper cell response to an alternate pattern of cytokine
secretion may deviate an autoimmune response to ameliorate disease
(Smith J A et al., J. Immunol. 160:4841-4849, 1998). Similarly,
agonistic anti-soluble ZcytoR14 monomers, homodimers, heterodimers
and multimer monoclonal antibodies may be used to signal, deplete
and deviate immune cells involved in asthma, allergy and atopic
disease. Signaling via ZcytoR14 may also benefit diseases of the
pancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,
pancreatitis, and pancreatic carcinoma may benefit. ZcytoR14 may
serve as a target for MAb therapy of pancreatic cancer where a
signaling MAb inhibits cancer growth and targets immune-mediated
killing (Tutt, A L et al., J. Immunol. 161: 3175-3185, 1998).
Similarly renal cell carcinoma may be treated with monoclonal
antibodies to ZcytoR14-comprising soluble receptors of the present
invention.
[0258] Soluble ZcytoR14 polypeptides described herein can be used
to bind, block, inhibit, reduce, antagonize or neutralize IL-17F or
IL-17A activity, either singly or together, in the treatment of
autoimmune disease, atopic disease, NIDDM, pancreatitis and kidney
dysfunction as described above. A soluble form of ZcytoR14 may be
used to promote an antibody response mediated by Th cells and/or to
promote the production of IL-4 or other cytokines by lymphocytes or
other immune cells.
[0259] The soluble ZcytoR14-comprising receptors of the present
invention are useful as antagonists of IL-17A or IL-17F cytokine.
Such antagonistic effects can be achieved by direct neutralization
or binding of IL-17A or IL-17F. In addition to antagonistic uses,
the soluble receptors of the present invention can bind IL-17F and
act as carrier proteins for IL-17A or IL-17F cytokine, in order to
transport the ligand to different tissues, organs, and cells within
the body. As such, the soluble receptors of the present invention
can be fused or coupled to molecules, polypeptides or chemical
moieties that direct the soluble-receptor-Ligand complex to a
specific site, such as a tissue, specific immune cell, or tumor.
For example, in acute infection or some cancers, benefit may result
from induction of inflammation and local acute phase response
proteins by the action of IL-17F. Thus, the soluble receptors of
the present invention can be used to specifically direct the action
of IL-17A or IL-17F. See, Cosman, D. Cytokine 5: 95-106, 1993; and
Fernandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.
[0260] Moreover, the soluble receptors of the present invention can
be used to stabilize the IL-17F or IL-17A, to increase the
bioavailability, therapeutic longevity, and/or efficacy of the
Ligand by stabilizing the Ligand from degradation or clearance, or
by targeting the ligand to a site of action within the body. For
example the naturally occurring IL-6/soluble IL-6R complex
stabilizes IL-6 and can signal through the gp130 receptor. See,
Cosman, D. supra., and Fernandez-Botran, R. supra. Moreover,
ZcytoR14 may be combined with a cognate ligand such as IL-17F to
comprise a ligand/soluble receptor complex. Such complexes may be
used to stimulate responses from cells presenting a companion
receptor subunit such as, for example, pDIRS1 (IL-17ARB) or CRF2-4
(IL-10RB). The cell specificity of ZcytoR14/ligand complexes may
differ from that seen for the ligand administered alone.
Furthermore the complexes may have distinct pharmacokinetic
properties such as affecting half-life, dose/response and organ or
tissue specificity. ZcytoR14/IL-17F or ZcytoR14/IL-17A complexes
thus may have agonist activity to enhance an immune response or
stimulate mesangial cells or to stimulate hepatic cells.
Alternatively only tissues expressing a signaling subunit the
heterodimerizes with the complex may be affected analogous to the
response to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l.
Acad. Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.)
"The Cytokine Handbook", 3.sup.rd Ed., p. 208-209). Soluble
receptor/cytokine complexes for IL-12 and CNTF display similar
activities.
[0261] Moreover Inflammation is a protective response by an
organism to fend off an invading agent. Inflammation is a cascading
event that involves many cellular and humoral mediators. On one
hand, suppression of inflammatory responses can leave a host
immunocompromised; however, if left unchecked, inflammation can
lead to serious complications including chronic inflammatory
diseases (e.g., psoriasis, arthritis, rheumatoid arthritis,
multiple sclerosis, inflammatory bowel disease and the like),
septic shock and multiple organ failure. Importantly, these diverse
disease states share common inflammatory mediators. The collective
diseases that are characterized by inflammation have a large impact
on human morbidity and mortality. Therefore it is clear that
anti-inflammatory proteins, such as ZcytoR14, and anti-ZcytoR14
antibodies, could have crucial therapeutic potential for a vast
number of human and animal diseases, from asthma and allergy to
autoimmunity and septic shock.
[0262] 1. Arthritis
[0263] Arthritis, including osteoarthritis, rheumatoid arthritis,
arthritic joints as a result of injury, and the like, are common
inflammatory conditions which would benefit from the therapeutic
use of anti-inflammatory proteins, such as ZcytoR14 polypeptides of
the present invention. For example, rheumatoid arthritis (RA) is a
systemic disease that affects the entire body and is one of the
most common forms of arthritis. It is characterized by the
inflammation of the membrane lining the joint, which causes pain,
stiffness, warmth, redness and swelling. Inflammatory cells release
enzymes that may digest bone and cartilage. As a result of
rheumatoid arthritis, the inflamed joint lining, the synovium, can
invade and damage bone and cartilage leading to joint deterioration
and severe pain amongst other physiologic effects. The involved
joint can lose its shape and alignment, resulting in pain and loss
of movement.
[0264] Rheumatoid arthritis (RA) is an immune-mediated disease
particularly characterized by inflammation and subsequent tissue
damage leading to severe disability and increased mortality. A
variety of cytokines are produced locally in the rheumatoid joints.
Numerous studies have demonstrated that IL-1 and TNF-alpha, two
prototypic pro-inflammatory cytokines, play an important role in
the mechanisms involved in synovial inflammation and in progressive
joint destruction. Indeed, the administration of TNF-alpha and IL-1
inhibitors in patients with RA has led to a dramatic improvement of
clinical and biological signs of inflammation and a reduction of
radiological signs of bone erosion and cartilage destruction.
However, despite these encouraging results, a significant
percentage of patients do not respond to these agents, suggesting
that other mediators are also involved in the pathophysiology of
arthritis (Gabay, Expert. Opin. Biol. Ther. 2(2):135-149, 2002).
One of those mediators could be IL-17A or IL-17F, and as such a
molecule that binds or inhibits IL-17F or IL-17A activity, such as
soluble ZcytoR14, ZcytoR14 polypeptides, or anti ZcytoR14
antibodies or binding partners, could serve as a valuable
therapeutic to reduce inflammation in rheumatoid arthritis, and
other arthritic diseases.
[0265] There are several animal models for rheumatoid arthritis
known in the art. For example, in the collagen-induced arthritis
(CIA) model, mice develop chronic inflammatory arthritis that
closely resembles human rheumatoid arthritis. Since CIA shares
similar immunological and pathological features with RA, this makes
it an ideal model for screening potential human anti-inflammatory
compounds. The CIA model is a well-known model in mice that depends
on both an immune response, and an inflammatory response, in order
to occur. The immune response comprises the interaction of B-cells
and CD4+ T-cells in response to collagen, which is given as
antigen, and leads to the production of anti-collagen antibodies.
The inflammatory phase is the result of tissue responses from
mediators of inflammation, as a consequence of some of these
antibodies cross-reacting to the mouse's native collagen and
activating the complement cascade. An advantage in using the CIA
model is that the basic mechanisms of pathogenesis are known. The
relevant T-cell and B-cell epitopes on type II collagen have been
identified, and various immunological (e.g., delayed-type
hypersensitivity and anti-collagen antibody) and inflammatory
(e.g., cytokines, chemokines, and matrix-degrading enzymes)
parameters relating to immune-mediated arthritis have been
determined, and can thus be used to assess test compound efficacy
in the CIA model (Wooley, Curr. Opin. Rheum. 3:407-20, 1999;
Williams et al., Immunol. 89:9784-788, 1992; Myers et al., Life
Sci. 61:1861-78, 1997; and Wang et al., Immunol. 92:8955-959,
1995).
[0266] One group has shown that an anti-mouse IL-17 antibody
reduces symptoms in a mouse CIA-model relative to control mice,
thus showing conceptually that soluble Zcytor14-Fc may be
beneficial in treating human disease. The administration of a
single mouse-IL-17-specific rat antisera reduced the symptoms of
arthritis in the animals when introduced prophylactically or after
symptoms of arthritis were already present in the model (Lubberts
et al, Arthritis Rheum. 50:650-9, 2004). Therefore, Zcytor14-Fc can
be used to neutralize IL-17A and/or IL-17F in the treatment of
specific human diseases such as arthritis, psoriasis, psoriatic
arthritis, endotoxemia, inflammatory bowel disease (IBD), colitis,
and other inflammatory conditions disclosed herein.
[0267] The administration of soluble ZcytoR14 comprising
polypeptides (ZcytoR14), such as ZcytoR14-Fc4 or other ZcytoR14
soluble and fusion proteins to these CIA model mice is used to
evaluate the use of soluble ZcytoR14 as an antagonist to IL-17F
used to ameliorate symptoms and alter the course of disease.
Moreover, results showing inhibition of IL-17F by ZcytoR14 would
provide proof of concept that other IL-17F antagonists, such as
soluble ZcytoR14 or neutralizing antibodies thereto, can also be
used to ameliorate symptoms and alter the course of disease.
Furthermore, since IL-17A and/or IL-17F induces production of IL-1b
and TNF-a, both of which are implicated in the pathogenesis and
progression of rheumatoid arthritis, the systemic or local
administration of soluble ZcytoR14 comprising polypeptides, such as
ZcytoR14-Fc4 or other IL-17F soluble receptors (e.g., ZcytoR14; SEQ
ID NO:3) and anti-ZcytoR14 antibodies, and fusion proteins can
potentially suppress the inflammatory response in RA. By way of
example and without limitation, the injection of 10-200 ug
Zcytor14-Fc per mouse (one to seven times a week for up to but not
limited to 4 weeks via s.c., i.p., or i.m route of administration)
can significantly reduce the disease score (paw score, incident of
inflammation, or disease). Depending on the initiation of
Zcytor14-Fc administration (e.g. prior to or at the time of
collagen immunization, or at any time point following the second
collagen immunization, including those time points at which the
disease has already progressed), Zcytor14 can be efficacious in
preventing rheumatoid arthritis, as well as preventing its
progression. Other potential therapeutics include ZcytoR14
polypeptides, anti-ZcytoR14 antibodies, or anti IL-17F antibodies
or binding partners, and the like.
[0268] 2. Endotoxemia
[0269] Endotoxemia is a severe condition commonly resulting from
infectious agents such as bacteria and other infectious disease
agents, sepsis, toxic shock syndrome, or in immunocompromised
patients subjected to opportunistic infections, and the like.
Therapeutically useful of anti-inflammatory proteins, such as
ZcytoR14 polypeptides and antibodies of the present invention,
could aid in preventing and treating endotoxemia in humans and
animals. ZcytoR14 polypeptides, or anti-ZcytoR14 antibodies or
binding partners, could serve as a valuable therapeutic to reduce
inflammation and pathological effects in endotoxemia.
[0270] Lipopolysaccharide (LPS) induced endotoxemia engages many of
the proinflammatory mediators that produce pathological effects in
the infectious diseases and LPS induced endotoxemia in rodents is a
widely used and acceptable model for studying the pharmacological
effects of potential pro-inflammatory or immunomodulating agents.
LPS, produced in gram-negative bacteria, is a major causative agent
in the pathogenesis of septic shock (Glausner et al., Lancet
338:732, 1991). A shock-like state can indeed be induced
experimentally by a single injection of LPS into animals. Molecules
produced by cells responding to LPS can target pathogens directly
or indirectly. Although these biological responses protect the host
against invading pathogens, they may also cause harm. Thus, massive
stimulation of innate immunity, occurring as a result of severe
Gram-negative bacterial infection, leads to excess production of
cytokines and other molecules, and the development of a fatal
syndrome, septic shock syndrome, which is characterized by fever,
hypotension, disseminated intravascular coagulation, and multiple
organ failure (Dumitru et al. Cell 103:1071-1083, 2000).
[0271] These toxic effects of LPS are mostly related to macrophage
activation leading to the release of multiple inflammatory
mediators. Among these mediators, TNF appears to play a crucial
role, as indicated by the prevention of LPS toxicity by the
administration of neutralizing anti-TNF antibodies (Beutler et al.,
Science 229:869, 1985). It is well established that 1 ug injection
of E. coli LPS into a C57B1/6 mouse will result in significant
increases in circulating IL-6, TNF-alpha, IL-1, and acute phase
proteins (for example, SAA) approximately 2 hours post injection.
The toxicity of LPS appears to be mediated by these cytokines as
passive immunization against these mediators can result in
decreased mortality (Beutler et al., Science 229:869, 1985). The
potential immunointervention strategies for the prevention and/or
treatment of septic shock include anti-TNF mAb, IL-1 receptor
antagonist, LIF, IL-10, and G-CSF.
[0272] The administration of soluble ZcytoR14 comprising
polypeptides, such as ZcytoR14-Fc4 or other ZcytoR14 soluble and
fusion proteins to these LPS-induced model may be used to evaluate
the use of ZcytoR14 to ameliorate symptoms and alter the course of
LPS-induced disease. Moreover, results showing inhibition of IL-17F
by ZcytoR14 provide proof of concept that other IL-17F antagonists,
such as soluble ZcytoR14 or antibodies thereto, can also be used to
ameliorate symptoms in the LPS-induced model and alter the course
of disease. The model will show induction of IL-17F by LPS
injection and the potential treatment of disease by ZcytoR14
polypeptides. Since LPS induces the production of pro-inflammatory
factors possibly contributing to the pathology of endotoxemia, the
neutralization of IL-17F activity or other pro-inflammatory factors
by an antagonist ZcytoR14 polypeptide can be used to reduce the
symptoms of endotoxemia, such as seen in endotoxic shock. Other
potential therapeutics include ZcytoR14 polypeptides, anti-ZcytoR14
antibodies, or binding partners, and the like.
[0273] 3. Inflammatory Bowel Disease IBD
[0274] In the United States approximately 500,000 people suffer
from Inflammatory Bowel Disease (IBD) which can affect either colon
and rectum (Ulcerative colitis) or both, small and large intestine
(Crohn's Disease). The pathogenesis of these diseases is unclear,
but they involve chronic inflammation of the affected tissues.
ZcytoR14 polypeptides, anti-ZcytoR14 antibodies, or binding
partners, could serve as a valuable therapeutic to reduce
inflammation and pathological effects in IBD and related
diseases.
[0275] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon, characterized by
inflammation and ulceration of the mucosa or innermost lining of
the colon. This inflammation causes the colon to empty frequently,
resulting in diarrhea. Symptoms include loosening of the stool and
associated abdominal cramping, fever and weight loss. Although the
exact cause of UC is unknown, recent research suggests that the
body's natural defenses are operating against proteins in the body
which the body thinks are foreign (an "autoimmune reaction").
Perhaps because they resemble bacterial proteins in the gut, these
proteins may either instigate or stimulate the inflammatory process
that begins to destroy the lining of the colon. As the lining of
the colon is destroyed, ulcers form releasing mucus, pus and blood.
The disease usually begins in the rectal area and may eventually
extend through the entire large bowel. Repeated episodes of
inflammation lead to thickening of the wall of the intestine and
rectum with scar tissue. Death of colon tissue or sepsis may occur
with severe disease. The symptoms of ulcerative colitis vary in
severity and their onset may be gradual or sudden. Attacks may be
provoked by many factors, including respiratory infections or
stress.
[0276] Although there is currently no cure for UC available,
treatments are focused on suppressing the abnormal inflammatory
process in the colon lining. Treatments including corticosteroids
immunosuppressives (eg. azathioprine, mercaptopurine, and
methotrexate) and aminosalicytates are available to treat the
disease. However, the long-term use of immunosuppressives such as
corticosteroids and azathioprine can result in serious side effects
including thinning of bones, cataracts, infection, and liver and
bone marrow effects. In the patients in whom current therapies are
not successful, surgery is an option. The surgery involves the
removal of the entire colon and the rectum.
[0277] There are several animal models that can partially mimic
chronic ulcerative colitis. The most widely used model is the
2,4,6-trinitrobenesulfonic acid/ethanol (TNBS) induced colitis
model, which induces chronic inflammation and ulceration in the
colon. When TNBS is introduced into the colon of susceptible mice
via intra-rectal instillation, it induces T-cell mediated immune
response in the colonic mucosa, in this case leading to a massive
mucosal inflammation characterized by the dense infiltration of
T-cells and macrophages throughout the entire wall of the large
bowel. Moreover, this histopathologic picture is accompanies by the
clinical picture of progressive weight loss (wasting), bloody
diarrhea, rectal prolapse, and large bowel wall thickening (Neurath
et al. Intern. Rev. Immunol. 19:51-62, 2000).
[0278] Another colitis model uses dextran sulfate sodium (DSS),
which induces an acute colitis manifested by bloody diarrhea,
weight loss, shortening of the colon and mucosal ulceration with
neutrophil infiltration. DSS-induced colitis is characterized
histologically by infiltration of inflammatory cells into the
lamina propria, with lymphoid hyperplasia, focal crypt damage, and
epithelial ulceration. These changes are thought to develop due to
a toxic effect of DSS on the epithelium and by phagocytosis of
lamina propria cells and production of TNF-alpha and IFN-gamma.
Despite its common use, several issues regarding the mechanisms of
DSS about the relevance to the human disease remain unresolved. DSS
is regarded as a T cell-independent model because it is observed in
T cell-deficient animals such as SCID mice.
[0279] The administration of soluble ZcytoR14 comprising
polypeptides, such as ZcytoR14-Fc4 or other ZcytoR14 soluble and
fusion proteins to these TNBS or DSS models can be used to evaluate
the use of soluble ZcytoR14 to ameliorate symptoms and alter the
course of gastrointestinal disease. Moreover, the results showing
inhibition of IL-17F by ZcytoR14 provide proof of concept that
other IL-17F antagonists, such as soluble ZcytoR14 or antibodies
thereto, can also be used to ameliorate symptoms in the colitis/IBD
models and alter the course of disease.
[0280] 4. Psoriasis
[0281] Psoriasis is a chronic skin condition that affects more than
seven million Americans. Psoriasis occurs when new skin cells grow
abnormally, resulting in inflamed, swollen, and scaly patches of
skin where the old skin has not shed quickly enough. Plaque
psoriasis, the most common form, is characterized by inflamed
patches of skin ("lesions") topped with silvery white scales.
Psoriasis may be limited to a few plaques or involve moderate to
extensive areas of skin, appearing most commonly on the scalp,
knees, elbows and trunk. Although it is highly visible, psoriasis
is not a contagious disease. The pathogenesis of the diseases
involves chronic inflammation of the affected tissues. ZcytoR14
polypeptides, anti-ZcytoR14 antibodies, or binding partners, could
serve as a valuable therapeutic to reduce inflammation and
pathological effects in psoriasis, other inflammatory skin
diseases, skin and mucosal allergies, and related diseases.
[0282] Psoriasis is a T-cell mediated inflammatory disorder of the
skin that can cause considerable discomfort. It is a disease for
which there is no cure and affects people of all ages. Psoriasis
affects approximately two percent of the populations of European
and North America. Although individuals with mild psoriasis can
often control their disease with topical agents, more than one
million patients worldwide require ultraviolet or systemic
immunosuppressive therapy. Unfortunately, the inconvenience and
risks of ultraviolet radiation and the toxicities of many therapies
limit their long-term use. Moreover, patients usually have
recurrence of psoriasis, and in some cases rebound, shortly after
stopping immunosuppressive therapy.
[0283] ZcytoR14 soluble receptor polypeptides and antibodies
thereto may also be used within diagnostic systems for the
detection of circulating levels of IL-17F or IL-17A ligand, and in
the detection of IL-17F associated with acute phase inflammatory
response. Within a related embodiment, antibodies or other agents
that specifically bind to ZcytoR14 soluble receptors of the present
invention can be used to detect circulating receptor polypeptides;
conversely, ZcytoR14 soluble receptors themselves can be used to
detect circulating or locally-acting IL-17F or IL-17A polypeptides.
Elevated or depressed levels of ligand or receptor polypeptides may
be indicative of pathological conditions, including inflammation or
cancer. IL-17F is known to induce associated acute phase
inflammatory response. Moreover, detection of acute phase proteins
or molecules such as IL-17A or IL-17F can be indicative of a
chronic inflammatory condition in certain disease states (e.g.,
asthma, psoriasis, rheumatoid arthritis, colitis, IBD). Detection
of such conditions serves to aid in disease diagnosis as well as
help a physician in choosing proper therapy.
[0284] In utero administration of soluble ZcytoR14 can be used to
show efficacy in vivo in disease models by reducing or eliminating
the phenotype associated with IL-17F transgenic pups which over
express IL-17F, or IL-17A transgenic pups which over express
IL-17A. There are precedents in the art for in utero treatment with
antagonists such as neutralizing monoclonal antibodies (mAbs). In
one case, the development of the B-1 subset of B cells was
dramatically affected by treating pregnant female mice with a mAb
specific for the B cell-specific molecule, CD19 (e.g., Krop I. Et
al., Eur. J. Immunol. 26(1):238-42, 1996). Krop et al. injected
timed pregnant mice intraperitoneally with 500 ug of rat anti-mouse
CD19 mAb (or a rat isotype-matched control Ab) in PBS beginning on
day 9 of gestation, with subsequent injections every other day
until birth. Pups were also injected once with 500 ug of these
antibodies at 10 days of age. In another case, Tanaka et al., found
that in utero treatment with monoclonal antibody to IL-2 receptor
beta-chain completely abrogates development of Thy-1+ dendritic
epidermal cells. The two distinct subunits of the IL-2 receptor,
i.e. the alpha-chain (IL-2R alpha) and the beta-chain (IL-2R beta),
are expressed in an almost mutually exclusive fashion throughout
fetal thymus ontogeny. Blocking IL-2R beta, a signal transducing
component of IL-2R, by administering a neutralizing mAb to IL-2R
beta, resulted in the complete and selective disappearance of
Thy-1+ skin dendritic epidermal cells. Development of any other T
cell subsets was uncompromised. This indicated that IL-2 plays a
crucial role in the development of fetal V gamma 5+ cells and their
descendants (see, Tanaka, T. et al., Int Immunol. 4(4):487-9,
1992). In addition, Schattemann G C et al., showed that PDGF-A is
required for normal murine cardiovascular development using an in
utero system. Several lines of evidence suggest that
platelet-derived growth factor A chain (PDGF-A) is required for
normal embryonic cardiovascular development. Introduction of
anti-PDGF-A neutralizing antibodies into mouse deciduas in utero
resulted in the selective disruption of PDGF-A ligand-receptor
interactions in vivo for a period of 18-24 hr and allowed
assessment of whether PDGF-A is required for cardiovascular
development and when it is required (see, Schattemann G C et al.,
Dev. Biol. 176(1):133-42, 1996). These results, as well as others
described in the art, provide evidence that antagonists such as
neutralizing mAbs or soluble receptors can elicit strong effects in
utero. Similarly, data showing the efficacy of soluble receptors
and/or neutralizing IL-17A or IL-17F with monoclonal antibodies in
vivo in disease models to reduce or eliminate the skin phenotype
found in IL-17A and IL-17F transgenic pups which over express
IL-17A and IL-17F respectively can be shown.
[0285] In addition to other disease models described herein, the
activity of soluble ZcytoR14 and/or anti-ZcytoR14 antibodies on
inflammatory tissue derived from human psoriatic lesions can be
measured in vivo using a severe combined immune deficient (SCID)
mouse model. Several mouse models have been developed in which
human cells are implanted into immunodeficient mice (collectively
referred to as xenograft models); see, for example, Cattan A R,
Douglas E, Leuk. Res. 18:513-22, 1994 and Flavell, D J,
Hematological Oncology 14:67-82, 1996. As an in vivo xenograft
model for psoriasis, human psoriatic skin tissue is implanted into
the SCID mouse model, and challenged with an appropriate
antagonist. Moreover, other psoriasis animal models in the art may
be used to evaluate IL-17A and IL-17F antagonists, such as human
psoriatic skin grafts implanted into AGR129 mouse model, and
challenged with an appropriate antagonist (e.g., see, Boyman, O. et
al., J. Exp. Med. Online publication #20031482, 2004, incorporated
herein by reference). Soluble ZcytoR14 or Anti-ZcytoR14 antibodies
that bind, block, inhibit, reduce, antagonize or neutralize the
activity of IL-17F or both IL-17A and IL-17F are preferred
antagonists, however, anti-IL-17A and anti-IL-22 antibodies (alone
or in combination), soluble ZcytoR14, as well as other IL-17A and
IL-17F antagonists can be used in this model. Similarly, tissues or
cells derived from human colitis, IBD, arthritis, or other
inflammatory lesions can be used in the SCID model to assess the
anti-inflammatory properties of the IL-17A and IL-17F antagonists
described herein.
[0286] Therapies designed to abolish, retard, or reduce
inflammation using soluble ZcytoR14, anti-ZcytoR14 antibodies or
its derivatives, agonists, conjugates or variants can be tested by
administration of anti-ZcytoR14 antibodies or soluble ZcytoR14
compounds to SCID mice bearing human inflammatory tissue (e.g.,
psoriatic lesions and the like), or other models described herein.
Efficacy of treatment is measured and statistically evaluated as
increased anti-inflammatory effect within the treated population
over time using methods well known in the art. Some exemplary
methods include, but are not limited to measuring for example, in a
psoriasis model, epidermal thickness, the number of inflammatory
cells in the upper dermis, and the grades of parakeratosis. Such
methods are known in the art and described herein. For example, see
Zeigler, M. et al. Lab Invest 81:1253, 2001; Zollner, T. M. et al.
J. Clin. Invest. 109:671, 2002; Yamanaka, N. et al. Microbiol.
Immunol. 45:507, 2001; Raychaudhuri, S. P. et al. Br. J. Dermatol.
144:931, 2001; Boehncke, W. H et al. Arch. Dermatol. Res. 291:104,
1999; Boehncke, W. H et al. J. Invest. Dermatol. 116:596, 2001;
Nickoloff, B. J. et al. Am. J. Pathol. 146:580, 1995; Boehncke, W.
H et al. J. Cutan. Pathol. 24:1, 1997; Sugai, J., M. et al. J.
Dermatol. Sci. 17:85, 1998; and Villadsen L. S. et al. J. Clin.
Invest. 112:1571, 2003. Inflammation may also be monitored over
time using well-known methods such as flow cytometry (or PCR) to
quantitate the number of inflammatory or lesional cells present in
a sample, score (weight loss, diarrhea, rectal bleeding, colon
length) for IBD, paw disease score and inflammation score for CIA
RA model. For example, therapeutic strategies appropriate for
testing in such a model include direct treatment using soluble
ZcytoR14, anti-ZcytoR14 antibodies, other IL-17A and IL-17F
antagonists (singly or together), or related conjugates or
antagonists based on the disrupting interaction of soluble ZcytoR14
with its ligands IL-17A and IL-17F, or for cell-based therapies
utilizing soluble ZcytoR14 or anti-ZcytoR14 antibodies or its
derivatives, agonists, conjugates or variants.
[0287] Moreover, Psoriasis is a chronic inflammatory skin disease
that is associated with hyperplastic epidermal keratinocytes and
infiltrating mononuclear cells, including CD4+ memory T cells,
neutrophils and macrophages (Christophers, Int. Arch. Allergy
Immunol., 110:199, 1996). It is currently believed that
environmental antigens play a significant role in initiating and
contributing to the pathology of the disease. However, it is the
loss of tolerance to self-antigens that is thought to mediate the
pathology of psoriasis. Dendritic cells and CD4.sup.+ T cells are
thought to play an important role in antigen presentation and
recognition that mediate the immune response leading to the
pathology. We have recently developed a model of psoriasis based on
the CD4+CD45RB transfer model (Davenport et al., Internat.
Immunopharmacol., 2:653-672). Soluble ZcytoR14 or anti-ZcytoR14
antibodies of the present invention are administered to the mice.
Inhibition of disease scores (skin lesions, inflammatory cytokines)
indicates the effectiveness of IL-17A and IL-17F antagonists in
psoriasis, e.g., anti-ZcytoR14 antibodies or ZcytoR14 soluble
receptors.
[0288] 5. Atopic Dermatitis.
[0289] AD is a common chronic inflammatory disease that is
characterized by hyperactivated cytokines of the helper T cell
subset 2 (Th2). Although the exact etiology of AD is unknown,
multiple factors have been implicated, including hyperactive Th2
immune responses, autoimmunity, infection, allergens, and genetic
predisposition. Key features of the disease include xerosis
(dryness of the skin), pruritus (itchiness of the skin),
conjunctivitis, inflammatory skin lesions, Staphylococcus aureus
infection, elevated blood eosinophilia, elevation of serum IgE and
IgG1, and chronic dermatitis with T cell, mast cell, macrophage and
eosinophil infiltration. Colonization or infection with S. aureus
has been recognized to exacerbate AD and perpetuate chronicity of
this skin disease.
[0290] AD is often found in patients with asthma and allergic
rhinitis, and is frequently the initial manifestation of allergic
disease. About 20% of the population in Western countries suffer
from these allergic diseases, and the incidence of AD in developed
countries is rising for unknown reasons. AD typically begins in
childhood and can often persist through adolescence into adulthood.
Current treatments for AD include topical corticosteroids, oral
cyclosporin A, non-corticosteroid immunosuppressants such as
tacrolimus (FK506 in ointment form), and interferon-gamma. Despite
the variety of treatments for AD, many patients' symptoms do not
improve, or they have adverse reactions to medications, requiring
the search for other, more effective therapeutic agents. The
soluble ZcytoR14 polypeptides and anti-ZcytoR14 antibodies of the
present invention, including the neutralizing anti-human ZcytoR14
antibodies of the present invention, can be used to neutralize
IL-17F and IL-17A in the treatment of specific human diseases such
as atoptic dermatitis, inflammatory skin conditions, and other
inflammatory conditions disclosed herein.
[0291] 6. Asthma
[0292] IL-17 plays an important role in allergen-induced T cell
activation and neutrophilic influx in the airways. The receptor for
IL-17 is expressed in the airways (Yao, et al. Immunity 3:811
(1995)) and IL-17 mediated neutrophil recruitment in allergic
asthma is largely induced by the chemoattractant IL-8, GRO-.alpha.
and macrophage inflammatory protein-2 (MIP-2) produced by IL-17
stimulated human bronchial epithelial cells (HBECs) and human
bronichial fibroblasts (Yao, et al. J Immunol 155:5483 (1995));
Molet, et al. J Allergy Clin Immunol 108:430 (2001)). IL-17 also
stimulates HBECs to release IL-6, a neutrophil-activating factor
(Fossiez, et al, J Exp Med 183:2593 (1996), and Linden, et al. Int
Arch Allergy Immunol 126:179 (2001)) and has been shown to
synergize with TNF-.alpha. to prolong the survival of human
neutrophils in vitro (Laan, et al. Eur Respir J 21:387 (2003)).
Moreover, IL-17 is capable of amplifying the inflammatory responses
in asthma by its ability to enhance the secretion of cytokines
implicated in airway remodeling such as the profibrotic cytokines,
IL-6 and IL-11 and inflammatory mediators granulocyte
colony-stimulating factor (G-CSF) and granulocyte macrophage
colony-stimulating factor (GM-CSF) (Molet, et al. J Allergy Clin
Immunol 108:430 (2001)).
[0293] Clinical evidence shows that acute, severe exacerbations of
asthma are associated with recruitment and activation of
neutrophils in the airways, thus IL-17 is likely to play a
significant role in asthma. Patients with mild asthma display a
detectable increase in the local concentration of free, soluble
IL-17A protein (Molet, et al. J Allergy Clin Immunol 108:430
(2001)) while healthy human volunteers with induced, severe airway
inflammation due to the exposure to a swine confinement, display a
pronounced increase in the concentration of free, soluble IL-17A
protein in the bronchoalveolar space (Fossiez, et al, J Exp Med
183:2593 (1996), and Linden, et al. Int Arch Allergy Immunol
126:179 (2001)). Furthermore, IL-17 levels in sputum have
correlated with individuals who have increased airway
hyper-reactivity Barczyk, et al. Respir Med 97:726 (2003).
[0294] In animal models of airway hyper-responsiveness, chronic
inhalation of ovalbumin by sensitized mice resulted in bronchial
eosinophilic inflammation and early induction of IL-17 mRNA
expression in inflamed lung tissue, together with a bronchial
neutrophilia Hellings, et al. Am J Respir Cell Mol Biol 28:42
(2003). Anti-IL-17 monoclonal antibodies strongly reduced bronchial
neutrophilic influx but significantly enhanced IL-5 levels in both
bronchoalveolar lavage fluid and serum, and aggravated
allergen-induced bronchial eosinophilic influx, suggesting that
IL-17A may be involved in determining the balance between
neutrophil and eosinophil accumulation following antigen insult
Id.
[0295] Among the IL-17 family members, IL-17F is most closely
related to IL-17A. The biological activities mediated by IL-17F are
similar to those of IL-17A, where IL-17F stimulates production of
IL-6, IL-8 and G-CSF Hurst, et al. J Immunol 169:443 (2002). IL-17F
also induces production of IL-2, transforming growth factor
(TGF)-.quadrature., and monocyte chemoattractant protein (MCP) in
endothelial cells Starnes, et al. J Immunol 167:4137 (2001).
Similarly, allergen challenge can increase local IL-17F in patients
with allergic asthma Kawaguchi, et al. J Immunol 167:4430 (2001).
Gene delivery of IL-17F in murine lung increases neutrophils in the
bronchoalveolar space, while mucosal transfer of the IL-17F gene
enhances the levels of Ag-induced pulmonary neutrophilia and airway
responsiveness to methacholine Oda, et al. Am J Respir Crit Care
Med 171:12 (2005).
[0296] Apart from asthma, several chronic inflammatory airway
diseases are characterized by neutrophil recruitment in the airways
and IL-17 has been reported to play an important role in the
pathogenesis of respiratory conditions such as chronic obstructive
pulmonary disease (COPD), bacterial pneumonia and cystic fibrosis
(Linden, et al. Eur Respir J 15:973 (2000), Ye, et al. Am J Respir
Cell Mol Biol 25:335 (2001), Rahman, et al. Clin Immunol 115:268
(2005)). An anti-IL-17A and/or anti-IL-17F therapeutic molecule
could be demonstrated to be efficacious for chronic inflammatory
airway disease in an in vitro model of inflammation. The ability of
antagonists to IL-17F and/or IL-17A activity, such as ZcytoR14
soluble receptors and antibodies thereto including the
anti-human-ZcytoR14 monoclonal and neutralizing antibodies of the
present invention to inhibit IL-17A or and/or IL-17F-induced
cytokine and chemokine production from cultured HBECs or bronchial
fibroblasts could be used as a measure of efficacy for such
antagonists in the prevention of the production of inflammatory
mediators directly resulting from IL-17A and/or F stimulation. If
the addition of antagonists to IL-17F and/or IL-17A activity, such
as ZcytoR14 soluble receptors and antibodies thereto including the
anti-human-ZcytoR14 monoclonal and neutralizing antibodies of the
present invention markedly reduces the production and expression of
inflammatory mediators, it would be expected to be efficacious in
inflammatory aspects associated with chronic airway
inflammation.
[0297] For pharmaceutical use, the soluble ZcytoR14 or
anti-ZcytoR14 antibodies of the present invention are formulated
for parenteral, particularly intravenous or subcutaneous, delivery
according to conventional methods. Intravenous administration will
be by bolus injection, controlled release, e.g, using mini-pumps or
other appropriate technology, or by infusion over a typical period
of one to several hours. In general, pharmaceutical formulations
will include a hematopoietic protein in combination with a
pharmaceutically acceptable vehicle, such as saline, buffered
saline, 5% dextrose in water or the like. Formulations may further
include one or more excipients, preservatives, solubilizers,
buffering agents, albumin to prevent protein loss on vial surfaces,
etc. When utilizing such a combination therapy, the cytokines may
be combined in a single formulation or may be administered in
separate formulations. Methods of formulation are well known in the
art and are disclosed, for example, in Remington's Pharmaceutical
Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990,
which is incorporated herein by reference. Therapeutic doses will
generally be in the range of 0.1 to 100 mg/kg of patient weight per
day, preferably 0.5-20 mg/kg per day, with the exact dose
determined by the clinician according to accepted standards, taking
into account the nature and severity of the condition to be
treated, patient traits, etc. Determination of dose is within the
level of ordinary skill in the art. The proteins will commonly be
administered over a period of up to 28 days following chemotherapy
or bone-marrow transplant or until a platelet count of
>20,000/mm.sup.3, preferably >50,000/mm.sup.3, is achieved.
More commonly, the proteins will be administered over one week or
less, often over a period of one to three days. In general, a
therapeutically effective amount of soluble ZcytoR14 or
anti-ZcytoR14 antibodies of the present invention is an amount
sufficient to produce a clinically significant increase in the
proliferation and/or differentiation of lymphoid or myeloid
progenitor cells, which will be manifested as an increase in
circulating levels of mature cells (e.g. platelets or neutrophils).
Treatment of platelet disorders will thus be continued until a
platelet count of at least 20,000/mm.sup.3, preferably
50,000/mm.sup.3, is reached. The soluble ZcytoR14 or anti-ZcytoR14
antibodies of the present invention can also be administered in
combination with other cytokines such as IL-3, -6 and -11; stem
cell factor; erythropoietin; G-CSF and GM-CSF. Within regimens of
combination therapy, daily doses of other cytokines will in general
be: EPO, 150 U/kg; GM-CSF, 5-15 lg/kg; IL-3, 1-5 lg/kg; and G-CSF,
1-25 lg/kg. Combination therapy with EPO, for example, is indicated
in anemic patients with low EPO levels.
[0298] Generally, the dosage of administered soluble ZcytoR14 (or
ZcytoR14 analog or fusion protein) or anti-ZcytoR14 antibodies will
vary depending upon such factors as the patient's age, weight,
height, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of soluble ZcytoR14 or anti-ZcytoR14 antibodies which is in
the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body
weight of patient), although a lower or higher dosage also may be
administered as circumstances dictate.
[0299] Administration of soluble ZcytoR14 or anti-ZcytoR14
antibodies to a subject can be intravenous, intraarterial,
intraperitoneal, intramuscular, subcutaneous, intrapleural,
intrathecal, by perfusion through a regional catheter, or by direct
intralesional injection. When administering therapeutic proteins by
injection, the administration may be by continuous infusion or by
single or multiple boluses.
[0300] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising soluble ZcytoR14 or anti-ZcytoR14 antibodies can be
prepared and inhaled with the aid of dry-powder dispersers, liquid
aerosol generators, or nebulizers (e.g., Pettit and Gombotz,
TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev. 35:235
(1999)). This approach is illustrated by the AERX diabetes
management system, which is a hand-held electronic inhaler that
delivers aerosolized insulin into the lungs. Studies have shown
that proteins as large as 48,000 kDa have been delivered across
skin at therapeutic concentrations with the aid of low-frequency
ultrasound, which illustrates the feasibility of trascutaneous
administration (Mitragotri et al., Science 269:850 (1995)).
Transdermal delivery using electroporation provides another means
to administer a molecule having ZcytoR14 binding activity (Potts et
al., Pharm. Biotechnol. 10:213 (1997)).
[0301] A pharmaceutical composition comprising a soluble ZcytoR14
or anti-ZcytoR14 antibody can be formulated according to known
methods to prepare pharmaceutically useful compositions, whereby
the therapeutic proteins are combined in a mixture with a
pharmaceutically acceptable carrier. A composition is said to be a
"pharmaceutically acceptable carrier" if its administration can be
tolerated by a recipient patient. Sterile phosphate-buffered saline
is one example of a pharmaceutically acceptable carrier. Other
suitable carriers are well-known to those in the art. See, for
example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th
Edition (Mack Publishing Company 1995).
[0302] For purposes of therapy, soluble ZcytoR14 or anti-ZcytoR14
antibody molecules and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a therapeutic molecule of the present invention and
a pharmaceutically acceptable carrier is said to be administered in
a "therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient. For example, an agent used to
treat inflammation is physiologically significant if its presence
alleviates the inflammatory response.
[0303] A pharmaceutical composition comprising ZcytoR14 (or
ZcytoR14 analog or fusion protein) or neutralizing anti-ZcytoR14
antibody can be furnished in liquid form, in an aerosol, or in
solid form. Liquid forms, are illustrated by injectable solutions
and oral suspensions. Exemplary solid forms include capsules,
tablets, and controlled-release forms. The latter form is
illustrated by miniosmotic pumps and implants (Bremer et al.,
Pharm. Biotechnol. 10:239 (1997); Ranade, "Implants in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 95-123 (CRC Press 1995); Bremer et al., "Protein Delivery
with Infusion Pumps," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 239-254 (Plenum Press 1997);
Yewey et al., "Delivery of Proteins from a Controlled Release
Injectable Implant," in Protein Delivery: Physical Systems, Sanders
and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0304] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0305] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0306] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0307] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull.
20:881 (1997)).
[0308] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0309] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0310] Polypeptides and antibodies can be encapsulated within
liposomes using standard techniques of protein microencapsulation
(see, for example, Anderson et al., Infect. Immun. 31:1099 (1981),
Anderson et al., Cancer Res. 50:1853 (1990), and Cohen et al.,
Biochim. Biophys. Acta 1063:95 (1991), Alving et al. "Preparation
and Use of Liposomes in Immunological Studies," in Liposome
Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page 317 (CRC
Press 1993), Wassef et al., Meth. Enzymol. 149:124 (1987)). As
noted above, therapeutically useful liposomes may contain a variety
of components. For example, liposomes may comprise lipid
derivatives of poly(ethylene glycol) (Allen et al., Biochim.
Biophys. Acta 1150:9 (1993)).
[0311] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0312] The present invention also contemplates chemically modified
polypeptides having binding ZcytoR14 activity such as ZcytoR14
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, and ZcytoR14 antagonists, for example anti-ZcytoR14
antibodies or binding polypeptides, or neutralizing anti-ZcytoR14
antibodies, which a polypeptide is linked with a polymer, as
discussed above.
[0313] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0314] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a ZcytoR14 extracellular domain, e.g., ZcytoR14
monomeric, homodimeric, heterodimeric or multimeric soluble
receptors, or a ZcytoR14 antagonist (e.g., an antibody or antibody
fragment that binds a ZcytoR14 polypeptide, or neutralizing
anti-ZcytoR14 antibody). Therapeutic polypeptides can be provided
in the form of an injectable solution for single or multiple doses,
or as a sterile powder that will be reconstituted before injection.
Alternatively, such a kit can include a dry-powder disperser,
liquid aerosol generator, or nebulizer for administration of a
therapeutic polypeptide. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition. Moreover, such information may include a statement
that the ZcytoR14 composition is contraindicated in patients with
known hypersensitivity to ZcytoR14.
[0315] A pharmaceutical composition comprising Anti-ZcytoR14
antibodies or binding partners (or Anti-ZcytoR14 antibody
fragments, antibody fusions, humanized antibodies and the like), or
ZcytoR14 soluble receptor, can be furnished in liquid form, in an
aerosol, or in solid form. Liquid forms, are illustrated by
injectable solutions, aerosols, droplets, topological solutions and
oral suspensions. Exemplary solid forms include capsules, tablets,
and controlled-release forms. The latter form is illustrated by
miniosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.
10:239 (1997); Ranade, "Implants in Drug Delivery," in Drug
Delivery Systems, Ranade and Hollinger (eds.), pages 95-123 (CRC
Press 1995); Bremer et al., "Protein Delivery with Infusion Pumps,"
in Protein Delivery: Physical Systems, Sanders and Hendren (eds.),
pages 239-254 (Plenum Press 1997); Yewey et al., "Delivery of
Proteins from a Controlled Release Injectable Implant," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages
93-117 (Plenum Press 1997)). Other solid forms include creams,
pastes, other topological applications, and the like.
[0316] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0317] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0318] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0319] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull.
20:881 (1997)).
[0320] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0321] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0322] Anti-ZcytoR14 neutralizing antibodies and binding partners
with IL-17F OR IL-17A binding activity, or ZcytoR14 soluble
receptor, can be encapsulated within liposomes using standard
techniques of protein microencapsulation (see, for example,
Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al.,
Cancer Res. 50:1853 (1990), and Cohen et al., Biochim. Biophys.
Acta 1063:95 (1991), Alving et al. "Preparation and Use of
Liposomes in Immunological Studies," in Liposome Technology, 2nd
Edition, Vol. III, Gregoriadis (ed.), page 317 (CRC Press 1993),
Wassef et al., Meth. Enzymol. 149:124 (1987)). As noted above,
therapeutically useful liposomes may contain a variety of
components. For example, liposomes may comprise lipid derivatives
of poly(ethylene glycol) (Allen et al., Biochim. Biophys. Acta
1150:9 (1993)).
[0323] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0324] The present invention also contemplates chemically modified
Anti-ZcytoR14 antibody or binding partner, for example
anti-Anti-ZcytoR14 antibodies or ZcytoR14 soluble receptor, linked
with a polymer, as discussed above.
[0325] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0326] The present invention contemplates compositions of
anti-IL-17F antibodies, and methods and therapeutic uses comprising
an antibody, peptide or polypeptide described herein. Such
compositions can further comprise a carrier. The carrier can be a
conventional organic or inorganic carrier. Examples of carriers
include water, buffer solution, alcohol, propylene glycol,
macrogol, sesame oil, corn oil, and the like.
K) Production of Transgenic Mice
[0327] Transgenic mice can be engineered to over-express the either
IL-17F, IL-17A or the Zcytor14 gene in all tissues or under the
control of a tissue-specific or tissue preferred regulatory
element. These over-producers can be used to characterize the
phenotype that results from over-expression, and the transgenic
animals can serve as models for human disease caused by excess
IL-17F, IL-17A or Zcytor14. Transgenic mice that over-express any
of these also provide model bioreactors for production of Zcytor14,
such as soluble Zcytor14, in the milk or blood of larger animals.
Methods for producing transgenic mice are well-known to those of
skill in the art (see, for example, Jacob, "Expression and Knockout
of Interferons in Transgenic Mice," in Overexpression and Knockout
of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124
(Academic Press, Ltd. 1994), Monastersky and Robl (eds.),
Strategies in Transgenic Animal Science (ASM Press 1995), and Abbud
and Nilson, "Recombinant Protein Expression in Transgenic Mice," in
Gene Expression Systems: Using Nature for the Art of Expression,
Fernandez and Hoeffler (eds.), pages 367-397 (Academic Press, Inc.
1999)).
[0328] For example, a method for producing a transgenic mouse that
expresses a Zcytor14 gene can begin with adult, fertile males
(studs) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown,
N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic
Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks,
(Taconic Farms)) and adult fertile females (recipients) (B6D2f1,
2-4 months, (Taconic Farms)). The donors are acclimated for one
week and then injected with approximately 8 IU/mouse of Pregnant
Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, Mo.)
I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic
Gonadotropin (hCG (Sigma)) I.P. to induce superovulation. Donors
are mated with studs subsequent to hormone injections. Ovulation
generally occurs within 13 hours of hCG injection. Copulation is
confirmed by the presence of a vaginal plug the morning following
mating.
[0329] Fertilized eggs are collected under a surgical scope. The
oviducts are collected and eggs are released into urinanalysis
slides containing hyaluronidase (Sigma). Eggs are washed once in
hyaluronidase, and twice in Whitten's W640 medium (described, for
example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and
Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are then stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
[0330] Ten to twenty micrograms of plasmid DNA containing a
Zcytor14 encoding sequence is linearized, gel-purified, and
resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a
final concentration of 5-10 nanograms per microliter for
microinjection. For example, the Zcytor14 encoding sequences can
encode a polypeptide comprising amino acid residues 21 to 452 of
SEQ ID NO:2.
[0331] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0332] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pre-gassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0333] The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0334] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy two-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, a nick in the oviduct is made
with a 28 g needle close to the bursa, making sure not to tear the
ampulla or the bursa.
[0335] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs allowed to slide in. The peritoneal wall is
closed with one suture and the skin closed with a wound clip. The
mice recuperate on a 37.degree. C. slide warmer for a minimum of
four hours.
[0336] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0337] Genomic DNA is prepared from the tail snips using, for
example, a QIAGEN DNEASY kit following the manufacturer's
instructions. Genomic DNA is analyzed by PCR using primers designed
to amplify a Zcytor14 gene or a selectable marker gene that was
introduced in the same plasmid. After animals are confirmed to be
transgenic, they are back-crossed into an inbred strain by placing
a transgenic female with a wild-type male, or a transgenic male
with one or two wild-type female(s). As pups are born and weaned,
the sexes are separated, and their tails snipped for
genotyping.
[0338] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the zyphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum and
the left lateral lobe of the liver exteriorized. Using 4-0 silk, a
tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14 ml polypropylene round bottom tube and snap frozen in liquid
nitrogen and then stored on dry ice. The surgical site is closed
with suture and wound clips, and the animal's cage placed on a
37.degree. C. heating pad for 24 hours post operatively. The animal
is checked daily post operatively and the wound clips removed 7-10
days after surgery. The expression level of Zcytor14 mRNA is
examined for each transgenic mouse using an RNA solution
hybridization assay or polymerase chain reaction.
[0339] In addition to producing transgenic mice that over-express
IL-17F, IL-17A or Zcytor14, it is useful to engineer transgenic
mice with either abnormally low or no expression of any of these
genes. Such transgenic mice provide useful models for diseases
associated with a lack of IL-17F, IL-17A or Zcytor14. As discussed
above, Zcytor14 gene expression can be inhibited using anti-sense
genes, ribozyme genes, or external guide sequence genes. To produce
transgenic mice that under-express the Zcytor14 gene, such
inhibitory sequences are targeted to Zcytor14 mRNA. Methods for
producing transgenic mice that have abnormally low expression of a
particular gene are known to those in the art (see, for example, Wu
et al., "Gene Underexpression in Cultured Cells and Animals by
Antisense DNA and RNA Strategies," in Methods in Gene
Biotechnology, pages 205-224 (CRC Press 1997)).
[0340] An alternative approach to producing transgenic mice that
have little or no Zcytor14 gene expression is to generate mice
having at least one normal Zcytor14 allele replaced by a
nonfunctional Zcytor14 gene. One method of designing a
nonfunctional Zcytor14 gene is to insert another gene, such as a
selectable marker gene, within a nucleic acid molecule that encodes
Zcytor14. Standard methods for producing these so-called "knockout
mice" are known to those skilled in the art (see, for example,
Jacob, "Expression and Knockout of Interferons in Transgenic Mice,"
in Overexpression and Knockout of Cytokines in Transgenic Mice,
Jacob (ed.), pages 111-124 (Academic Press, Ltd. 1994), and Wu et
al., "New Strategies for Gene Knockout," in Methods in Gene
Biotechnology, pages 339-365 (CRC Press 1997)).
[0341] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Expression of the ZcytoR14 Gene
[0342] Northern analyses were performed using Human Multiple Tissue
Blots (CLONTECH Laboratories, Inc., Palo Alto, Calif.). Two probes
were generated from gel purified PCR products. The first probe was
made using ZC21798 (5' CGG CGT GGT GGT CTT GCT CTT 3'; SEQ ID NO:8)
and ZC21808 (5' TCC CGT CCC CCG CCC CAG GTC 3'; SEQ ID NO:31) as
primers. The probe was a radioactively labeled using the Multiprime
labeling kit from Amersham (Arlington Heights, Ill.) according to
the manufacturer's protocol. The probe was purified using a NUCTRAP
push column (STRATAGENE, La Jolla, Calif.). EXPRESSHYB (CLONTECH)
solution was used for the prehybridization and hybridization
solutions for the northern blots. Hybridization took place
overnight at 65.quadrature. C. Following hybridization, the blots
were washed for 30 minutes each in solutions that contained 0.1%
SDS and SSC as follows: twice in 2.times.SSC at room temperature,
three times in 0.1.times.SSC at 50 C..degree., once in
0.1.times.SSC at 55.degree. C., and once in 0.1.times.SSC at
65.degree. C. The results demonstrated the ZcytoR14 gene is
strongly expressed in thyroid, adrenal gland, prostate, and liver
tissues, and expressed to a lesser extent in heart, small
intestine, stomach, and trachea tissues. In contrast, there is
little or no expression in brain, placenta, lung, skeletal muscle,
kidney, pancreas, spleen, thymus, testis, ovary, colon, peripheral
blood leukocytes, spinal cord, lymph node, and bone marrow.
Example 2
Distribution of mRNA in Cell Line Panels Using PCR
[0343] Total RNA was purified from resting and stimulated cell
lines grown in-house and purified using a Qiagen (Valencia, Calif.)
RNeasy kit according to the manufacturer's instructions, or an
acid-phenol purification protocol (Chomczynski and Sacchi,
Analytical Biochemistry, 162:156-9, 1987). The quality of the RNA
was assessed by running an aliquot on an Agilent Bioanalyzer. If
the RNA was significantly degraded, it was not used for subsequent
creation of first strand cDNA. Presence of contaminating genomic
DNA was assessed by a PCR assay on an aliquot of the RNA with
zc41011 (5'CTCTCCATCCTTATCTTTCATCAAC 3'; SEQ ID NO:32) and zc41012
(5'CTCTCTGCTGGCTAAACAAAACAC 3'; SEQ ID NO:33), primers that amplify
a single site of intergenic genomic DNA. The PCR conditions for the
contaminating genomic DNA assay were as follows: 2.5 .mu.l
10.times. buffer and 0.5 .mu.l Advantage 2 cDNA polymerase mix (BD
Biosciences Clontech, Palo Alto, Calif.), 2 ul 2.5 mM dNTP mix
(Applied Biosystems, Foster City, Calif.), 2.5 .mu.l 10.times.
Rediload (Invitrogen, Carlsbad, Calif.), and 0.5 .mu.l 20 uM
zc41011 and zc41012, in a final volume of 25 ul. Cycling parameters
were 94.degree. C. 20'', 40 cycles of 94.degree. C. 20'' 60.degree.
C. 1'20'' and one cycle of 72.degree. C. 7'. 10 ul of each reaction
was subjected to agarose gel electrophoresis and gels were examined
for presence of a PCR product from contaminating genomic DNA. If
contaminating genomic DNA was observed, the total RNA was DNAsed
using DNA-free reagents (Ambion, Inc, Austin, Tex.) according to
the manufacturer's instructions, then retested as described above.
Only RNAs which appeared to be free of contaminating genomic DNA
were used for subsequent creation of first strand cDNA.
[0344] 20 .mu.g total RNA from 82 human cell lines were each
brought to 98 .mu.l with H.sub.2O, then split into two 49 ul
aliquots, each containing 10 .mu.g total RNA, and placed in two
96-well PCR plates. To each aliquot was added reagents for first
strand cDNA synthesis (Invitrogen First Strand cDNA Synthesis
System, Carlsbad, Calif.): 20 .mu.l 25 mM MgCl.sub.2, 10 ul
10.times. RT buffer, 10 ul 0.1M DTT, 2 .mu.l oligo dT, 2 ul
RNAseOut. Then, to one aliquot from each cell line 2 .mu.l
Superscript II Reverse Transcriptase was added, and to the
corresponding cell line aliquot 2 .mu.l H.sub.2O was added to make
a minus Reverse Transcriptase negative control. All samples were
incubated as follows: 25.degree. C. 10', 42.degree. C. 50',
70.degree. C. 15'. Samples were arranged in deep well plates and
diluted to 1.7 ml with H.sub.2O. A Multipette (Saigan) robot was
used to aliquot 16.5 .mu.l into each well of a 96-well PCR plate
multiple times, generating numerous one-use PCR panels of the cell
lines, which were then sealed and stored at -20.degree. C. Each
well in these panels represents first strand cDNA from
approximately 100 ng total RNA. The 82 cell lines are spread across
two panels, array #118A and #118B. Quality of first strand cDNA on
the panels was assessed by a multiplex PCR assay on one set of the
panels using primers to two widely expressed, but only moderately
abundant genes, CLTC (clathrin) and TFRC (transferrin receptor C).
0.5 ul each of Clathrin primers zc42901
(5'CTCATATTGCTCAACTGTGTGAAAAG 3'; SEQ ID NO:34), zc42902
(5'TAGAAGCCACCTGAACACAAATCTG3'; SEQ ID NO:35), and TFRC primers
zc42599 (5'ATCTTGCGTTGTATGTTGAAAATCAATT3'; SEQ ID NO:36), zc42600
(5'TTCTCCACCAGGTAAACAAGTCTAC3'; SEQ ID NO:37), were mixed with 2.5
.mu.l 10.times. buffer and 0.5 .mu.l Advantage 2 cDNA polymerase
mix (BD Biosciences Clontech, Palo Alto, Calif.), 2 .mu.l 2.5 mM
dNTP mix (Applied Biosystems, Foster City, Calif.), 2.5 .mu.l
10.times. Rediload (Invitrogen, Carlsbad, Calif.), and added to
each well of a panel of array #118A and array #118B. Cycling
parameters were as follows: 94.degree. C. 20'', 35 cycles of
94.degree. C. 20'', 67.degree. C. 80'', and one cycle of 72.degree.
C. 7'. 10 .mu.l of each reaction was subjected to agarose gel
electrophoresis and gels were scored for the presence of a robust
PCR product for each gene specific to the +RT wells for each cell
line.
[0345] Expression of mRNA in the human first strand cDNA panels for
ZcytoR14 was assayed by PCR with sense oligo ZC42756
(5'ctctccaggcccaagtcgtgctct3'; SEQ ID NO:38) and antisense oligo
ZC42757 (5'ttgtcctgggggcctcgtgtctcc3'; SEQ ID NO:39) under these
PCR conditions per sample: 2.5 .mu.l 10.times. buffer and 0.5 .mu.l
advantage 2 cDNA polymerase mix (BD Biosciences Clontech, Palo
Alto, Calif.), 2 .mu.l 2.5 mM dNTP mix (Applied Biosystems,), 2.5
ul 10.times. Rediload (Invitrogen, Carlsbad, Calif.), and 0.5 .mu.l
20 uM each sense and antisense primer. Cycling conditions were
94.degree. C. 2', 35 cycles of 94.degree. C. 1', 66.degree. C.
30'', 72.degree. C. 1.5.degree., and one cycle of 72.degree. C. 7'.
10 .mu.l of each reaction was subjected to agarose gel
electrophoresis and gels were scored for positive or negative
expression of ZcytoR14.
[0346] ZcytoR14 mRNA is widely expressed in many cell lines
representing a broad spectrum of tissue and cell types. In
particular, ZcytoR14 is consistently expressed in non-T cell
peripheral blood cell lines, including monocytes, B-cells, and
cells of the myeloid lineage. Also, ZcytoR14 mRNA is reliably
expressed in cell lines derived from skin. Other cell lines that
express ZcytoR14 are all 5 of the large intestine cell lines that
were present on the array.
Example 3
Distribution of mRNA in Mouse Cell Line Panels Using RT PCR
[0347] Total RNA was purified from 60 resting and stimulated cell
lines grown in-house and purified using a Qiagen (Valencia, Calif.)
RNeasy kit according to the manufacturer's instructions, an
acid-phenol purification protocol (Chomczynski and Sacchi,
Analytical Biochemistry, 162:156-9, 1987), or a Trizol reagent
protocol (Invitrogen, Carlsbad, Calif.).
[0348] 5 .mu.g of total RNA from each cell line was arranged in a
deep well 96-well plate, 125 .mu.l 3M NaOAc and 100 .mu.l Pellet
Paint (Novagen, Madison, Wis.)) were added to each well, then the
final volume was adjusted to 1.25 ml with H.sub.20. A Multipette
(Saigan) robot was used to aliquot 25 .mu.l of the RNA mixture
followed by 75 ul EtOH into each well of a 96-well PCR plate
multiple times, generating numerous one-use RT PCR panels of the
cell lines, which were then sealed and stored at -20.degree. C. RT
PCR screening was performed by first centrifuging a panel in a
Qiagen (Valencia, Calif.) 96-well centrifuge for 10' at 6000 RPM.
Supernatant was removed by inverting the plate onto absorbent
paper. RNA pellets were washed with 100 .mu.l 70% EtOH, followed by
a 5' centrifugation at 6000 RPM. Supernatant was again removed and
plates allowed to air-dry until the remaining EtOH was evaporated.
RNA pellets were resuspended in 15 .mu.l H.sub.20.
[0349] Expression of ZcytoR14 mRNA in the mouse cell line RNA
panels was assayed by RT PCR with zc38910
(5'acgaagcccaggtaccagaaagag3'; SEQ ID NO:40) and zc38679
(5'aaaagcgccgcagccaagagtagg3'; SEQ ID NO:41) under these RT PCR
conditions per sample: SuperScript One-Step PCR with Platinum Taq
kit, Invitrogen, Carlsbad, Calif. Cycling conditions were: 1 cycle
of 48.degree. C. for 30 minutes, 94.degree. C. for 2 minutes,
followed by 35 cycles of 94.degree. C. for 15 seconds, 55.degree.
C. for 30 seconds, 72.degree. C. for 1.5 minutes, followed by 1
cycle of 72.degree. C. for 7 minutes. 10 .mu.l of each reaction was
subjected to agarose gel electrophoresis and gels were scored for
positive or negative expression of ZcytoR14.
[0350] Murine ZcytoR14mRNA is expressed in several mouse cell
lines, notably in cell lines derived from bone marrow, including
osteoblast, adipocyte, and preadipocyte cell lines. Also, mouse
ZcytoR14 is mRNA is represented in several samples from the
endocrine system, such as pancreas stromal cell lines, pancreas
islet cell lines, and hypothalamus, salivary gland, and testis cell
lines.
Example 4
Refolding and Purification pIL-17F Produced in E. coli
A) Inclusion Body Isolation and Extraction of pIL-17F
[0351] Following induction of protein expression in either batch
ferment or shaker flask culture, the E. coli broth is centrifuged
in 1 liter bottles @ 3000 RPM in a Sorvall swinging bucket rotor.
Washing of the cell paste to remove any broth contaminants is
performed with 50 mM Tris pH 8.0 containing 200 mM NaCl and 5 mM
EDTA until the supernate is clear.
[0352] The cell pellets are then suspended in ice-cold lysis buffer
(50 mM Tris pH 8.0; 5 mM EDTA; 200 mM NaCl, 10% sucrose (w/v); 5 mM
DTT; 5 mM Benzamidine;) to 10-20 Optical Density units at 600 nm.
This slurry is then subjected to 3 passes at 8500-9000 psi in a
chilled APV 2000 Lab Homogenizer producing a disrupted cell lysate.
The insoluble fraction (inclusion bodies) is recovered by
centrifugation of the cell lysate at 20,000.times.G for 1 hour at
4.degree. C.
[0353] The inclusion body pellet resulting from the 20,000.times.G
spin is weighed and then re-suspended in wash buffer (50 mM Tris pH
8 containing 200 mM NaCl, 5 mM EDTA, 5 mM DTT, 5 mM Benzamidine) at
10 ml wash buffer per gram inclusion bodies. Complete dispersion is
achieved by homogenizing with an OMNI international rotor stator
generator. This suspension is centrifuged at 20,000.times.G for 30
minutes at 4.degree. C. The wash cycle is repeated 3-5 times until
the supernatant is clear.
[0354] The final washed pellet is solubilized in 7M Guanidine HCl
in 40 mM Tris buffer at pH 8 containing 0.1M Sodium Sulfite and
0.02 M Sodium Tetrathionate. The extraction and sulfitolysis
reaction is allowed to proceed with gentle stirring at 4.degree. C.
overnight. The resulting pinkish colored solution is centrifuged at
35,000.times.g for 1 hour at 4.degree. C. and the clarified
supernate, containing the soluble pIL-17F, is 0.45 um filtered.
B) pIL-17F Refolding Procedure
[0355] The solubilized, sulfitolyzed pIL-17F is refolded by drop
wise dilution into ice cold refolding buffer containing 55 mM MES,
10.56 mM NaCl, 0.44 mM KCl, 0.055% PEG (3400 K), 1.1 mM EDTA, 20%
Glycerol, 0.5M Guanidine HCl, 0.75 M Arginine and the Glutathione
redox pair at a 1:1 ratio (1 mM GSH:1 mM GSSG). The pH of the
refolding buffer is adjusted to 6.5 with HCl and the pIL-17F is
added to a final concentration of 100 ug/ml. Once diluted, the
mixture is allowed to stir slowly in the cold room for 72
hours.
C) Product Recovery & Purification
[0356] The refolded pIL-17F is concentrated 10.times. vs. a 10 kDa
cutoff membrane on a lab scale TFF system. Next it is filtered
using a 0.45 micron membrane and the pH is adjusted to 5.1 with the
addition of Acetic acid. The pH-adjusted material is captured by
cation exchange chromatography on a Pharmacia SP Fast Flow column
equilibrated in 50 mM Acetate buffer, pH 5.1. The pIL-17F is loaded
by inline proportioning at 1:5 with equilibration buffer at a flow
rate of 190 cm/hr. This dilution lowers the ionic strength enabling
efficient binding of the target to the matrix. After sample loading
is complete, the column is washed to baseline absorbance with
equilibration buffer. The column is washed with 0.4 M NaCl in 50 mM
Acetate buffer at pH 5.1 and then the bound protein is eluted with
a 5 CV gradient from 0.4 M to 1.5 M NaCl in 50 mM Acetate buffer at
pH 5.1. The protein elutes at .about.1M NaCl and is approximately
85% dimeric by SDS PAGE analysis of eluate fractions. The fractions
containing pIL-17F are pooled and concentrated against a 10 kDa
cutoff ultrafiltration membrane using an Amicon stirred cell in
preparation for the final purification and buffer exchange by size
exclusion chromatography.
D) Size Exclusion Buffer Exchange and Formulation
[0357] The concentrated cation pool (at a volume of 3-4% of CV) is
injected at a flow rate of 30 cm/hr onto a Pharmacia Superdex 75
size exclusion column equilibrated in 50 mM Sodium Phosphate buffer
containing 109 mM NaCl, pH 7.2. The symmetric eluate peak
containing the product is diluted to a concentration of 1 mg/ml in
50 mM Sodium Phosphate buffer containing 109 mM NaCl, pH 7.2.
Finally the pIL-17F is 0.2 micron sterile filtered, aliquoted and
stored at -80.degree. C. The final process yield is 20%.
Example 5
Construction of Mammalian Soluble ZcytoR14 Expression Construct
[0358] An expression construct containing human ZcytoR14
[L21-K451]-mFc1 (mouse BALB/c .mu.2a Fc) is constructed via overlap
PCR and homologous recombination using a DNA fragment (SEQ ID
NO:42) encoding a ZcytoR14 polypeptide (SEQ ID NO:43), a DNA
fragment encoding mFc1 (SEQ ID NO:44), and the expression vector
pZMP20. The fragments are generated by PCR amplification.
[0359] The PCR fragment encoding ZcytoR14 [L21-K451] contains a 5'
overlap with the pZMP20 vector sequence in the optimized tissue
plasminogen activator pre-pro secretion leader sequence coding
region, the ZcytoR14 extracellular domain coding [L21-K451], and a
3' overlap with the mFc1 coding region. The PCR amplification
reaction uses the 5' oligonucleotide
[GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGAGGCT
TGTGGGGCCT; SEQ ID NO:46], the 3' oligonucleotide
[TGTGGGCCCTCTGGGCTCCTTGTGGATGTATTTGTC; SEQ ID NO:47], and a
previously generated DNA clone of ZcytoR14 as the template.
[0360] The PCR fragment encoding mFc1 contains a 5' overlap with
the ZcytoR14 sequence, the mFc1 coding region, and a 3' overlap
with the pZMP20 vector in the poliovirus internal ribosome entry
site region. The PCR amplification reaction uses the 5
oligonucleotide [GACAAATACATCCACAAGGAGCCCAGAGGGCCCACA; SEQ ID
NO:48], the 3' oligonucleotide
[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTTACCCGGAGTCCGGGA; SEQ ID
NO:49], and a previously generated DNA clone of mFc1 as the
template.
[0361] The PCR amplification reaction conditions are as follows: 1
cycle, 94.degree. C., 5 minutes; 35 cycles, 94.degree. C., 1
minute, followed by 55.degree. C., 2 minutes, followed by
72.degree. C., 3 minutes; 1 cycle, 72.degree. C., 10 minutes. The
PCR reaction mixtures are run on a 1% agarose gel and the DNA
fragments corresponding to the expected sizes are extracted from
the gel using a QIAquick.TM. Gel Extraction Kit (Qiagen, Cat. No.
28704).
[0362] The two PCR fragments are joined by overlap PCR.
Approximately 1 .mu.l each of the two gel extracted fragments are
combined in a PCR amplification reaction using the 5'
oligonucleotide
[GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGAGGCT
TGTGGGGCCT; SEQ ID NO: 46] and the 3' oligonucleotide
[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTTACCCGGAGTCCGGGA; SEQ ID
NO:49]. PCR conditions used are as follows: 1 cycle, 94.degree. C.,
5 minutes; 35 cycles, 94.degree. C., 1 minute, followed by
55.degree. C., 2 minutes, followed by 72.degree. C., 3 minutes; 1
cycle, 72.degree. C., 10 minutes. The PCR reaction mixture is run
on a 1% agarose gel and the DNA fragment corresponding to the size
of the insert is extracted from the gel using a QIAquick.TM. Gel
Extraction Kit (Qiagen, Cat. No. 28704).
[0363] Plasmid pZMP20 is a mammalian expression vector containing
an expression cassette having the MPSV promoter, a BglII site for
linearization prior to yeast recombination, an otPA signal peptide
sequence, an internal ribosome entry element from poliovirus, the
extracellular domain of CD8 truncated at the C-terminal end of the
transmembrane domain; an E. coli origin of replication; a mammalian
selectable marker expression unit comprising an SV40 promoter,
enhancer and origin of replication, a DHFR gene, and the SV40
terminator; and URA3 and CEN-ARS sequences required for selection
and replication in S. cerevisiae.
[0364] The plasmid pZMP20 is digested with BglII prior to
recombination in yeast with the gel extracted ZcytoR14
[L21-K451]-mFc1 PCR fragment. 100 .mu.l of competent yeast (S.
cerevisiae) cells are combined with 10 .mu.l of the ZcytoR14
[L21-K451]-mFc1 insert DNA and 100 ng of BglII digested pZMP20
vector, and the mix is transferred to a 0.2 cm electroporation
cuvette. The yeast/DNA mixture is electropulsed using power supply
(BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV (5
kV/cm), .infin. ohms, and 25 .mu.F. Six hundred .mu.l of 1.2 M
sorbitol is added to the cuvette, and the yeast is plated in 100
.mu.l and 300 .mu.l aliquots onto two URA-D plates and incubated at
30.degree. C. After about 72 hours, the Ura.sup.+ yeast
transformants from a single plate are resuspended in 1 ml H.sub.2O
and spun briefly to pellet the yeast cells. The cell pellet is
resuspended in 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100
mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five hundred .mu.l of
the lysis mixture is added to an Eppendorf tube containing 250
.mu.l acid-washed glass beads and 300 .mu.l phenol-chloroform, is
vortexed for 3 minutes, and spun for 5 minutes in an Eppendorf
centrifuge at maximum speed. Three hundred .mu.l of the aqueous
phase is transferred to a fresh tube, and the DNA is precipitated
with 600 .mu.l ethanol, followed by centrifugation for 30 minutes
at maximum speed. The tube is decanted and the pellet is washed
with 1 mL of 70% ethanol. The tube is decanted and the DNA pellet
is resuspended in 30 .mu.l 10 mM Tris, pH 8.0, 1 mM EDTA.
[0365] Transformation of electrocompetent E. coli host cells
(DH12S) is done using 5 .mu.l of the yeast DNA preparation and 50
.mu.l of E. coli cells. The cells are electropulsed at 2.0 kV,
25.degree. F., and 400 ohms. Following electroporation, 1 ml SOC
(2% Bacto.TM. Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract
(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4, 20 mM glucose) is added and then the cells are plated
in 50 .mu.l and 200 .mu.l aliquots on two LB AMP plates (LB broth
(Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0366] The inserts of three DNA clones for the construct is
subjected to sequence analysis and one clone containing the correct
sequence is selected. Large scale plasmid DNA is isolated using a
commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen,
Valencia, Calif.) according to manufacturer's instructions.
Example 6
Construction of Mammalian Soluble ZcytoR14 Expression Constructs
that Express ZcytoR14-CEE, ZcytoR14-CHIS, and ZcytoR14-CFLAG
[0367] An expression construct containing human ZcytoR14 [L21-K451]
with a C-terminal tag, either Glu-Glu (CEE), six His (CHIS), or
FLAG (CFLAG), is constructed via PCR and homologous recombination
using a DNA fragment encoding ZcytoR14 [L21-K451] (SEQ ID NO:42)
and the expression vector pZMP20.
[0368] The PCR fragment encoding ZcytoR14CEE contains a 5' overlap
with the pZMP20 vector sequence in the optimized tissue plasminogen
activator pre-pro secretion leader sequence coding region, the
ZcytoR14 extracellular domain coding [L21-K451], the sequence of
the Glu-Glu tag (Glu Glu Tyr Met Pro Met Glu; SEQ ID NO:53), and a
3' overlap with the pZMP20 vector in the poliovirus internal
ribosome entry site region. The PCR amplification reaction uses the
5' oligonucleotide
[GTTTCGCTCAGCCAGGAAATCCATGCCGAGTTGAGACGCTTCCGTAGACTGGAGAGGCT
TGTGGGGCCT; SEQ ID NO:46], the 3' oligonucleotide
[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTATTCCATGGGCATGTATTCTTCCT
TGTGGATGTATTTGTC; SEQ ID NO:50], and a previously generated DNA
clone of ZcytoR14 as the template.
[0369] The PCR amplification reaction condition is as follows: 1
cycle, 94.degree. C., 5 minutes; 35 cycles, 94.degree. C., 1
minute, followed by 55.degree. C., 2 minutes, followed by
72.degree. C., 3 minutes; 1 cycle, 72.degree. C., 10 minutes. The
PCR reaction mixture is run on a 1% agarose gel and the DNA
fragment corresponding to the expected size is extracted from the
gel using a QIAquick.TM. Gel Extraction Kit (Qiagen, Cat. No.
28704).
[0370] The plasmid pZMP20 is digested with BglII prior to
recombination in yeast with the gel extracted ZcytoR14CEE PCR
fragment. One hundred .mu.l of competent yeast (S. cerevisiae)
cells are combined with 10 .mu.l of the ZcytoR14CEE insert DNA and
100 ng of BglII digested pZMP20 vector, and the mix is transferred
to a 0.2 cm electroporation cuvette. The yeast/DNA mixture is
electropulsed using power supply (BioRad Laboratories, Hercules,
Calif.) settings of 0.75 kV (5 kV/cm), .infin. ohms, and 25 .mu.F.
Six hundred .mu.l of 1.2 M sorbitol is added to the cuvette, and
the yeast is plated in 100 .mu.l and 300 .mu.l aliquots onto two
URA-D plates and incubated at 30.degree. C. After about 72 hours,
the Ura.sup.+ yeast transformants from a single plate are
resuspended in 1 ml H.sub.2O and spun briefly to pellet the yeast
cells. The cell pellet is resuspended in 0.5 ml of lysis buffer (2%
Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA).
The five hundred .mu.l of the lysis mixture is added to an
Eppendorf tube containing 250 .mu.l acid-washed glass beads and 300
.mu.l phenol-chloroform, is vortexed for 3 minutes, and spun for 5
minutes in an Eppendorf centrifuge at maximum speed. Three hundred
.mu.l of the aqueous phase is transferred to a fresh tube, and the
DNA is precipitated with 600 .mu.l ethanol, followed by
centrifugation for 30 minutes at maximum speed. The tube is
decanted and the pellet is washed with 1 mL of 70% ethanol. The
tube is decanted and the DNA pellet is resuspended in 30 .mu.l 10
mM Tris, pH 8.0, 1 mM EDTA.
[0371] Transformation of electrocompetent E. coli host cells
(DH12S) is done using 5 .mu.l of the yeast DNA preparation and 50
.mu.l of E. coli cells. The cells are electropulsed at 2.0 kV,
25.degree. F., and 400 ohms. Following electroporation, 1 ml SOC
(2% Bacto.TM. Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract
(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4, 20 mM glucose) is added and then the cells are plated
in 50 .mu.l and 200 .mu.l aliquots on two LB AMP plates (LB broth
(Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0372] The inserts of three DNA clones for the construct is
subjected to sequence analysis and one clone containing the correct
sequence is selected. Large scale plasmid DNA is isolated using a
commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen,
Valencia, Calif.) according to manufacturer's instructions.
[0373] The same process is used to prepare the ZcytoR14 with a
C-terminal his tag, composed of Gly Ser Gly Gly His His His His His
His (ZcytoR14CHIS; SEQ ID NO:51) or the C-terminal FLAG tag,
composed of Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys (ZcytoR14CFLAG;
SEQ ID NO:52). To prepare these constructs, instead of the 3'
oligonucleotide of SEQ ID NO:50; the 3' oligonucleotide
[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTAGTGATGGTGATGGTGATGTCCAC
CAGATCCCTTGTGGATGTATTTGTC; SEQ ID NO:54] is used to generate
ZcytoR14CHIS or the 3' oligonucleotide
[CAACCCCAGAGCTGTTTTAAGGCGCGCCTCTAGATTACTTATCATCATCATCCTTATAAT
CGGATCCCTTGTGGATGTATTTGTC; SEQ ID NO:55] is used to generate
ZcytoR14CFLAG.
Example 7
Transfection and Expression of Soluble ZcytoR14 Receptor Expression
Constructs that Express the ZcytoR14-mFc1 Fusion Protein, and the
ZcytoR14-CEE, ZcytoR14-CHIS, and ZcytoR14-CFLAG C-Terminal Tagged
Proteins
[0374] Three sets of 200 .mu.g of each of the soluble ZcytoR14
fusion or tagged expression constructs are separately digested with
200 units of PvuI at 37.degree. C. for three hours, precipitated
with isopropyl alcohol, and centrifuged in a 1.5 mL microfuge tube.
The supernatant is decanted off the pellet, and the pellet is
washed with 1 mL of 70% ethanol and allowed to incubate for 5
minutes at room temperature. The tube is spun in a microfuge for 10
minutes at 14,000 RPM and the supernatant is decanted off the
pellet. The pellet is then resuspended in 750 .mu.l of CHO cell
tissue culture medium in a sterile environment, allowed to incubate
at 60.degree. C. for 30 minutes, and is allowed to cool to room
temperature. Approximately 5.times.10.sup.6 CHO cells are pelleted
in each of three tubes and are resuspended using the DNA-medium
solution. The DNA/cell mixtures are placed in a 0.4 cm gap cuvette
and electroporated using the following parameters; 950 .mu.F, high
capacitance, at 300 V. The contents of the cuvettes are then
removed, pooled, and diluted to 25 mLs with CHO cell tissue culture
medium and placed in a 125 mL shake flask. The flask is placed in
an incubator on a shaker at 37.degree. C., 6% CO.sub.2 with shaking
at 120 RPM.
[0375] The CHO cells are subjected to nutrient selection followed
by step amplification to 200 nM methotrexate (MTX), and then to 1
.mu.M MTX. Fusion or tagged protein expression is confirmed by
Western blot, and the CHO cell pool is scaled-up for harvests for
protein purification.
Example 8
Expression of Soluble ZcytoR14
[0376] An expression plasmid containing ZcytoR14-Tbx-C(Fc9) (SEQ ID
NO:64) was constructed via homologous recombination using a DNA
fragment of ZcytoR14_Tbx and the expression vector pZMP40. The
fragment was generated by PCR amplification using primers zc44531
and zc44545.
[0377] The PCR fragment ZcytoR14_Tbx contains a partial ZcytoR14
extracellular domain coding region, which was made using a
previously generated clone of ZcytoR14 as the template. The
fragment includes a 5' overlap with the pZMP40 vector sequence in
the otPA coding region, the ZcytoR14 segment (amino acid residue 21
to 451 of SEQ ID NO:2), a linker sequence, a thrombin cleavage
site, and a 3' overlap with the pZMP40 vector in the Fc9 coding
region. PCR conditions used were as follows: 1 cycle, 94.degree.
C., 5 minutes; 35 cycles, 94.degree. C., 1 minute, followed by
55.degree. C., 2 minutes, followed by 72.degree. C., 3 minutes; 1
cycle, 72.degree. C., 10 minutes.
[0378] The PCR reaction mixtures were run on a 1% agarose gel and a
band corresponding to the sizes of the inserts were gel-extracted
using a QIAquick.TM. Gel Extraction Kit (Qiagen, Cat. No.
28704).
[0379] Plasmid pZMP40 is a mammalian expression vector containing
an expression cassette having the MPSV promoter, multiple
restriction sites for insertion of coding sequences, an otPA signal
peptide sequence, and the sequence for Fc9; an internal ribosome
entry site (IRES) element from poliovirus, and the extracellular
domain of CD8 truncated at the C-terminal end of the transmembrane
domain; an E. coli origin of replication; a mammalian selectable
marker expression unit comprising an SV40 promoter, enhancer and
origin of replication, a DHFR gene, and the SV40 terminator; and
URA3 and CEN-ARS sequences required for selection and replication
in S. cerevisiae. It was constructed from pZMP21 (Patent Pub. No.
US 2003/0232414 A1; deposited at the American Type Culture
Collection and designated as ATCC# PTA-5266).
[0380] The plasmid pZMP40 was cut with BglII prior to recombination
in yeast with the PCR fragment. One hundred microliters of
competent yeast (S. cerevisiae) cells were independently combined
with 10 .mu.l of the insert DNA (SEQ ID NO:66) and 100 ng of cut
pZMP40 vector, and the mix was transferred to a 0.2-cm
electroporation cuvette. The yeast/DNA mixture was electropulsed
using power supply (BioRad Laboratories, Hercules, Calif.) settings
of 0.75 kV (5 kV/cm), .infin. ohms, and 25.degree. F. Six hundred
.mu.l of 1.2 M sorbitol was added to the cuvette, and the yeast was
plated in a 100-.mu.l and 300 .mu.l aliquot onto two URA-D plates
and incubated at 30.degree. C. After about 72 hours, the Ura.sup.+
yeast transformants from a single plate were resuspended in 1 ml
H.sub.2O and spun briefly to pellet the yeast cells. The cell
pellet was resuspended in 0.5 ml of lysis buffer (2% Triton X-100,
1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five
hundred microliters of the lysis mixture was added to an Eppendorf
tube containing 250 .mu.l acid-washed glass beads and 300 .mu.l
phenol-chloroform, was vortexed for 3 minutes, and spun for 5
minutes in an Eppendorf centrifuge at maximum speed. Three hundred
microliters of the aqueous phase was transferred to a fresh tube,
and the DNA was precipitated with 600 .mu.l ethanol (EtOH),
followed by centrifugation for 30 minutes at maximum speed. The
tube was decanted and the pellet was washed with 1 mL of 70%
ethanol. The tube was decanted and the DNA pellet was resuspended
in 30 .mu.l TE.
[0381] Transformation of electrocompetent E. coli host cells
(DH12S) was done using 5 .mu.l of the yeast DNA prep and 50 .mu.l
of cells. The cells were electropulsed at 2.0 kV, 25 .mu.F, and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto.TM. Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was
added and then the cells were plated in a 50 .mu.l and a 200 .mu.l
aliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto.TM.
Agar (Difco), 100 mg/L Ampicillin).
[0382] The inserts of three clones for the construct was subjected
to sequence analysis and one clone for each construct, containing
the correct sequence, was selected. Larger scale plasmid DNA was
isolated using a commercially available kit (QIAGEN Plasmid Mega
Kit, Qiagen, Valencia, Calif.) according to manufacturer's
instructions.
[0383] Three sets of 200 .mu.g of the ZcytoR14
[L21-K451]_Tbx_C(Fc9) construct were then each digested with 200
units of Pvu I at 37.degree. C. for three hours and then were
precipitated with IPA and spun down in a 1.5 mL microfuge tube. The
supernatant was decanted off the pellet, and the pellet was washed
with 1 mL of 70% ethanol and allowed to incubate for 5 minutes at
room temperature. The tube was spun in a microfuge for 10 minutes
at 14,000 RPM and the supernatant was decanted off the pellet. The
pellet was then resuspended in 750 .mu.l of PF-CHO media in a
sterile environment, allowed to incubate at 60.degree. C. for 30
minutes, and was allowed to cool to room temperature. 5E6 APFDXB11
cells were spun down in each of three tubes and were resuspended
using the DNA-media solution. The DNA/cell mixtures were placed in
a 0.4 cm gap cuvette and electroporated using the following
parameters: 950.degree. F., high capacitance, and 300 V. The
contents of the cuvettes were then removed, pooled, and diluted to
25 mLs with PF-CHO media and placed in a 125 mL shake flask. The
flask was placed in an incubator on a shaker at 37.degree. C., 6%
CO.sub.2, and shaking at 120 RPM.
[0384] The cell line was subjected to nutrient selection followed
by step amplification to 200 nM methotrexate (MTX), and then to 1
.mu.M MTX. Expression was confirmed by western blot, and the cell
line was scaled-up and protein purification followed.
Example 9
Purification of Soluble ZcytoR14 from CHO Cells
[0385] Conditioned media from CHO cells expressing ZcytoR14-TbX-Fc9
(SEQ ID NO:64) was concentrated approximately 10-fold with a
Pellicon-II tangential flow system against two Biomax 0.1 m.sup.2
30 kD molecular weight cutoff membrane cassettes (Millipore,
Bedford, Mass.). The concentrated media was pH adjusted to 5.5 with
glacial acetic acid, 0.2 .quadrature.m sterile filtered then loaded
onto a Protein G sepharose fast flow resin (Pharmacia, Piscataway,
N.J.) via batch chromatography overnight at 4 C. Prior to loading
the pH adjusted conditioned media, the Protein G resin was
pre-equilibrated with, 5 column volumes (approximately 150 ml) of
25 mM sodium acetate, 150 mM NaCl, pH 5.5. The ratio of filtered,
pH adjusted conditioned media to resin was 33:1 (v/v).
[0386] The batched chromatography process was performed at ambient
room temperature (approximately 21 C). The batched, pH adjusted,
0.22 .mu.m filtered, conditioned media was poured into an empty
5.5.times.20.5 cm glass column (BioRad, Hercules, Calif.) and
packed via gravity. The column was washed with 10 column volumes
(approximately 300 ml) of 25 mM sodium acetate, 150 mM NaCl, pH
5.5. Bound protein was then pH eluted with 100 mM glycine, pH 2.7.
9.0 ml fractions were collected and immediately neutralized with
1.0 ml 2.0M Tris, pH 8.0. The collected fractions were analyzed via
SDS-PAGE Coomassie staining. Fractions containing ZcytoR14-Tbx-Fc9
were pooled and concentrated approximately 6-fold using a 5 kD
molecular weight cutoff Biomax membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0387] The pooled, concentrated fractions were then dialyzed, at 4
C, extensively against 1.times. phosphate buffered saline, pH 7.3
(Sigma, St. Louis, Mo.) using a 7 kD molecular weight cutoff
membrane Slide-A-Lyzer (Pierce, Rockford, Ill.). ZcytoR14-TbX-Fc9
as formulated in 1.times. phosphate buffered saline, pH 7.3 was
0.22 .mu.m sterile filtered prior to aliquoting and storage at -80
C.
Example 10
Binding of IL-17A and IL-17F to Human ZcytoR14
A) Binding of Biotinylated Cytokines to Transfected Cells
[0388] Baby Hamster Kidney (BHK) cells that had been transfected
with expression vectors encoding human IL-17 receptor (SEQ ID
NO:21), human ZcytoR14 (SEQ ID NO:2), or both of these receptors
are assessed for their ability to bind biotinylated human IL-17A
and human IL-17F. Cells are harvested with versene, counted and
diluted to 10.sup.7 cells per ml in staining media (SM), which is
HBSS plus 1 mg/ml bovine serum albumin (BSA), 10 mM Hepes, and 0.1%
sodium azide (w/v). Biotinylated human IL-17A (SEQ ID NO:14) and
human IL-17F (SEQ ID NO:16) are incubated with the cells on ice for
30 minutes at various concentrations. After 30 minutes, excess
cytokine is washed away with SM and the cells are incubated with a
1:100 dilution of streptavidin conjugated to phycoerythrin (SA-PE)
for 30 minutes on ice. Excess SA-PE is washed away and cells are
analyzed by flow cytometry. The amount of cytokine binding was
quantitated from the mean fluorescence intensity of the cytokine
staining. From this analysis, we find that human IL-17A binds both
the human IL-17R and ZcytoR14 to a similar extent. Also, human
IL-17F binds ZcytoR14 to a similar level, but binds IL-17R
detectably, but to a much lower level than was seen with
IL-17A.
B) Binding of Biotinylated Cytokines to Human Peripheral Blood
Mononuclear Cells
[0389] Human peripheral blood mononuclear cells (PBMC) were
prepared from whole blood by ficoll density gradient
centrifugation. PBMC at 10.sup.7 cells per ml were simultaneously
incubated with biotinylated IL-17A or IL-17F at 1 .mu.g/ml and
fluorochrome conjugated antibodies to specific cell surface
proteins that were designed to distinguish various white blood cell
lineages lineages. These markers include CD4, CD8, CD19, CD11b,
CD56 and CD16. Excess antibody and cytokine are washed away, and
specific cytokine binding is detected by incubating with SA-PE as
described above. Samples were analyzed by flow cytometry and from
this analysis, we find that human IL-17A binds to virtually all
PBMC populations examined, but that human IL-17F does not
detectably bind to any population.
C) Inhibition of Specific Binding of Biotinlyated Human IL-17A and
IL-17F with Unlabeled Cytokine
[0390] Binding studies are performed as discussed above, but excess
unlabeled human IL-17A and IL-17F are included in the binding
reaction. In studies with BHK cells, the amount of unlabeled
cytokine was varied over a range of concentrations and we find that
addition of unlabeled IL-17A competed for binding of both IL-17A
and IL-17F to both ZcytoR14 and IL-17R. However, unlabeled IL-17F
competed for binding of both IL-17A and IL-17F to ZcytoR14, but it
did not compete effectively for binding to IL-17R. This indicates
that both IL-17A and IL-17F specifically bind to ZcytoR14, and that
they bind at a site that is either identical or overlaps
significantly since they cross-compete for binding. Also, IL-17A
competes for the relatively weak binding of IL-17F for IL-17R,
indicating these two cytokines also bind to a similar region in the
IL-17R, but IL-17F binds IL-17R with much reduced affinity relative
to ZcytoR14.
D) Inhibition of Specific Binding of Biotinylated Human IL-17A and
IL-17F with Soluble ZcytoR14 and IL-17R
[0391] Binding studies are performed as discussed above, except
that a soluble form of ZcytoR14 or IL-17R are included in the
binding reactions. These soluble receptors are fusion proteins
derived from the extracellular domain of each receptor fused to the
human IgG1 constant (Fc) region. We find that soluble ZcytoR14
inhibits binding of both human IL-17A and IL-17F to both IL-17R and
ZcytoR14 transfected BHK cells. However, soluble IL-17R inhibits
binding of IL-17A to either receptor, but does not effectively
block binding of IL-17F to ZcytoR14, consistent with the poor
binding of IL-17F for the IL-17R.
Example 11
IL-17A and IL-17F Bind to ZcytoR14
A) Binding Inhibition with Cold Ligand
[0392] BHK cells transfected with hZcytoR14 (SEQ ID NO:2) and
IL-17R (SEQ ID NO:21) were plated at 40,000 cells/well in a 24-well
dish (Costar 3527) two days prior to assay. IL-17A (SEQ ID NO:14)
and IL-17F (SEQ ID NO:16) that had been radiolabeled by the
iodobead method were added independently to wells in triplicate at
10 ng/ml with a total of 250 ul/well in binding buffer (RPMI 1640
media (JRH 51502-500M) with 10 mg/ml bovine serum albumin
(Gibco15260-037)). Cold competitors were added in 100 fold molar
excess. Competitors tested included IL-17A, IL-17B, IL-17C, IL-17D,
IL-17E, IL-17F and IL-21. Wells were incubated on ice for 1-hour
followed by two washes with PBS (Invitrogen 20012-027) and one wash
with a high salt solution (1.5M NaCL, 50 mM HEPES pH 7.4). Wells
were extracted with 500 ul of 0.8M NaOH for 30 min. at room
temperature and counts per minute were measured in a gamma counter
(Packard Cobra II A5005).
[0393] The results indicated that 100.times. molar cold IL-17A and
IL-17F were able to reduce binding of .sup.125I I-17A to BHK
hZcytoR14 by approximately 7 fold while IL-17B, C, D, E and IL-21
had no effect on binding. 100.times. molar cold IL-17A reduced the
binding of .sup.125I I-17A to BHK IL-17R by approximately 4 fold
while IL-17B, C, D, E, F and IL-21 had no effect on binding.
100.times. molar cold IL-17A and IL-17F reduced the binding of
.sup.125IL-17F to BHK hZcytoR14 by approximately 4 fold and 5 fold,
respectively, while IL-17B, C, D, E and IL-21 had no effect on
binding.
B) Binding Inhibition with Soluble Receptor:
[0394] Binding to hzytor14 (SEQ ID NO:2) and IL-17R (SEQ ID NO:21)
transfected BHK cells was performed as in one, but 100 fold molar
excess soluble hZcytoR14x1/Fc9 (Example 8) and soluble IL-17R/Fc
(obtained from R&D; Ref. 177-IR) were used in place of cold
ligand in the competition. Cells were washed, extracted and counted
as in part one.
[0395] Soluble hZcytoR14/Fc inhibited binding of .sup.125IL-17F to
BHK hZcytoR14 with an IC.sub.50 of 10.times. molar excess average
from three experiments. Soluble hZcytoR14/Fc inhibition of
.sup.125IIL-17A on the same cell line gave an average IC.sub.50 of
20.times. molar excess and soluble IL-17R/Fc inhibition of
.sup.125I I-17A gave an average IC.sub.50 of 20.times. molar
excess.
C) Binding Saturation
[0396] Transfected BHK cells were plated into 24-well dishes as in
one. Radiolabeled IL-17A and IL-17F were added starting at a
concentration of 4 nM in eight 1:3 dilutions (to a concentration of
1.83 pM) in triplicate with a total of 250 .mu.l/well in binding
buffer. Separately, 100 fold molar excess of cold ligand was added
at each dilution point. Cells were washed, extracted and counted as
in one. Specific counts per minute were plotted against
concentration of radiolabeled ligand added by subtracting the 100
fold excess counts from the uncompeted counts at each dilution
point. These normalized data were plotted to generate saturation
binding curves for each combination of radiolabeled ligand and
transfected BHK cells. Table 4 shows the affinity values calculated
from all three experiments. TABLE-US-00004 TABLE 4 125I IL-17A +
BHK hZcytoR14 125I IL-17A + BHK IL-17R 1. 180 pM 1. 2.5 +/- 0.2 nM
2. 200 pM 2. 4.5 +/- 0.3 nM 3. 370 pM 3. 5.9 +/- 0.1 nM 125I IL-17F
+ BHK hZcytoR14 125I IL-17F + BHK IL-17R 1. 50 pM 1. Very low
affinity 2. 60 pM 2. Very low affinity 3. 80 pM 3. Very low
affinity
One-site binding curve fits agreed most closely with IL-17A &
IL-17F binding to IL-17R. Two-site binding curve fits agreed most
closely with IL-17A and IL-17F binding to hZcytoR14. The high
affinity binding site is the value shown above. The low affinity
binding site had very low affinity and varied widely between the
three experiments.
Example 12
Murine Nih3t3 Cells Respond to Human IL-17A and IL-17F
A) Cell Plating and kz142 Adenovirus Reporter Infection.
[0397] Nih3t3 cells, derived from mouse fibroblasts (described in
ATCC) Nih3t3 were plated at 5000 cells/well in solid white, cell
culture coated 96 well plates, (Cat. #3917. Costar) using DMEM/10%
FBS, containing glutamine and amended with pyruvate and cultured
overnight at 37o C. and 5% C02. On this second day, the plating
media was removed and Kz142 adenovirus particles at a multiplicity
of infection of 5000 particles/cell were prepared in DMEM/1% FBS,
containing glutamine and amended with pyruvate and cultured
overnight at 37o C. and 5% C02.
B) Luciferase Assay Measuring IL-17A and F Activation of kz142
Adenovirus Reporter Infected Nih3t3 Cells.
[0398] Following the overnight incubation with the adenovirus
particle reporter, human IL-17A and IL-17F Ligand treatments were
prepared in serum free media ( )amended to 0.28% BSA. The
adenovirus particles and media were removed and the appropriate
ligand doses were given in triplicates. Incubation at 37.degree. C.
and 5% C02 was continued for 4 hours, after which the media was
removed, cells lysed for 15 minutes and mean fluorescence intensity
(MFI) measured using the luciferase assay system and reagents.
(Cat.#e1531 Promega. Madison, Wis.) and a Microplate luminometer.
Activity was detected at concentrations ranging from 0.1-1000 ng/ml
human IL-17A and IL-17F, generating EC50 values of about 50 ng/ml
for both ligands. These data suggest that nih3t3 cells carry
receptors to these ligands and that IL-17A and IL-17F activate the
NfKb/Ap-1 transcription factor.
Example 13
Murine Nih3t3 Cells Express Both IL-17 Receptor and ZcytoR14
Receptor
[0399] RTPCR analysis of nih3t3 RNA demonstrated that these cells
are positive for both IL-17 Receptor and ZcytoR14 receptor,
consistent with their nfkb/ap1 response to human IL-17A and IL-17F
mediation being mediated through one or both of these
receptors.
RTPCR Details:
A) Mouse cytor14 PCR
[0400] First strand cDNA was prepared from total RNA isolated from
nih3t3 cells using standard methods. PCR was applied using hot star
polymerase and the manufacturer's recommendations (Qiagen,
Valencia, Calif.) using sense primer, zc38910, 5'
ACGAAGCCCAGGTACCAGAAAGAG 3' (SEQ ID NO:56) and antisense primer, zc
38679, 5' AAAAGCGCCGCAGCCAAGAGTAGG 3' (SEQ ID NO:57) and 35 cycles
of amplification. Agarose gel electrophoresis revealed a single,
robust amplicon of the expected, 850 bp size.
B) Mouse IL-17R PCR
[0401] First strand cDNA was prepared from total RNA isolated from
nih3t3 cells using standard methods. PCR was applied using hot star
polymerase and the manufacturer's recommendations (Qiagen,
Valencia, Calif.) using sense primer, zc38520, 5'
CGTAAGCGGTGGCGGTTTTC 3' (SEQ ID NO:58) and antisense primer, zc
38521, 5' TGGGCAGGGCACAGTCACAG 3' (SEQ ID NO:59) and 35 cycles of
amplification. Agarose gel electrophoresis revealed a single,
robust amplicon of the expected, 498 bp size.
Example 14
Creation of a Stable Nih3t3 Assay Clone Expressing the ap1/nfkb
Transcription Factor
[0402] The murine nih3t3 cell line described above was stably
transfected with the kz142 ap1/nfkb reporter construct, containing
a neomycin-selectible marker. The Neo resistant transfection pool
was plated at clonal density. Clones were isolated using cloning
rings and screened by luciferase assay using the human IL-17A
ligand as an inducer. Clones with the highest mean fluorescence
intensity (MFI) (via ap1/NfkB luciferase) and the lowest background
were selected. A stable transfectant cell line was selected and
called nih3t3/kz142.8.
Example 15
Inhibition of Activation by Human IL-17A and IL-17F in Murine
Nih3t3 Cells Using Soluble ZcytoR14 and IL-17 Receptor/FC
Chimeras
[0403] Soluble forms of ZcytoR14 or IL-17R were used as antagonists
of human IL-17A and IL-17F activation of ap1/nfkb elements in a
luciferase assay. These soluble receptors are fusion proteins
derived from the extracellular domain of each receptor fused to the
human IgG1 constant (Fc) region. The soluble human IL-17R F fusion
protein was purchased. (recombinant human IL-17R/FC chimera,
catalog number 177-IR-100, R&D Systems, Inc., Minneapolis, Mn.)
The soluble human ZcytoR14 FC chimera (ZcytoR14sR/FC9) was
constructed as described above. We find that an excess
ZcytoR14sR/FC9 and human IL17RsR/FC chimera inhibit EC50 levels of
both human IL-17A and IL-17F mediation of ap1/nfkb activation of
the murine nih3t3/kz142.8 assay cell line.
[0404] The ZcytoR14sR/FC9 protein showed the greatest potency in
antagonizing IL-17F activation and IL17RsR/FC chimera showed the
greatest potency in antagonizing IL-17A activation.
Example 16
IL-17F mRNA is Upregulated in a Murine Model of Asthma
[0405] IL-17F mRNA levels were measured in a sensitization and
airway challenge model in mice. Groups of mice, 8 to 10 wks of age,
were sensitized by intraperitoneal injection of 10 ug of
recombinant Dermatophagoides pteronyssinus allergen 1 (DerP1)
(Indoor biotechnologies, Cardiff, UK) in 50% Imject Alum (Pierce)
on days 0 and 7. Seven days later, mice were challenged on 3
consecutive days (days 14, 15 and 16) with 20 ug of DerP1 in 50 ul
PBS. There were 4 mice representing this group. Negative controls
included 5 mice given phosphate buffered saline (PBS)
sensitization, followed by PBS challenge. In addition to 3 mice
given DerP1 sensitization, followed by PBS challenge. Forty-eight
hours following allergen, or control challenge whole lung tissue
was harvested and total RNA was isolated.
[0406] First strand cDNA was prepared using identical amounts of
total RNA from each subject. IL-17F PCR was applied using Qiagen
hotstar polymerase (Qiagen, Valencia, Calif.) and the
manufacturer's recommendations. The IL-17F PCR utilized 35 cycles
of amplification with sense primer, zc46098, 5'
ACTTGCCATTCTGAGGGAGGTAGC 3' (SEQ ID NO:60) and antisense primer,
46099, 5' CACAGGTGCAGCCAACTTTTAGGA 3' (SEQ ID NO:61). In order to
establish that the template quality was uniform amongst all
subjects, Beta Actin PCR was applied to the same amount of each
template used in the IL-17F amplification. B actin PCR included 25
cycles of PCR with sense primer, zc44779, 5' GTGGGCCGCTCTAGGCACCA
3' (SEQ ID NO:62) and antisense primer, zcc44776, 5'
CGGTTGGCCTTAGGGTTCAGGGGGG 3' (SEQ ID NO:63).
[0407] All 4 mice from the DerP1 sensitized, DerP1 challenged
treatment group (the asthma simulation) showed robust IL-17F
amplification. In contrast, weak IL-17F amplification was seen from
the negative controls, including 3 of 3 subjects representing the
DerP1 sensitized/PBS challenged treatment group and 5 of 5 subjects
from the PBS sensitized/PBS challenged treatment group. B actin
amplification was at least as robust for the negative controls as
for the asthma-simulated subjects, demonstrating that the weak
negative control IL-17F amplification was not due to template
problems.
Example 17
COS Cell Transfection and Secretion Trap
A) Cos Cell Transfection and Secretion Trap Assays Show that
ZcytoR14sR/Fc9 and IL-17F is a Receptor/Ligand Pair
[0408] A secretion trap assay was used to match the human ZcytoR14
(SEQ ID NO:2) to the human IL-17F (SEQ ID NO:16). The soluble
ZcytoR14sR/Fc9 fusion protein (Example 8) was used as a binding
reagent in a secretion assay. SV40 ori containing expression
vectors containing cDNA of human IL-17B, C, D, E, and F was
transiently transfected into COS cells. The binding of
ZcytoR14sR/Fc9 to transfected COS cells was carried out using the
secretion trap assay described below. Positive binding of
ZcytoR14sR/Fc9 was only seen to human IL-17F. These results
demonstrate the novel finding that human ZcytoR14 and IL-17F is a
receptor/ligand pair.
B) COS Cell Transfections
[0409] The COS cell transfection was performed as follows: Mix 3 ul
pooled DNA and 5 ul Lipofectamine.TM. in 92 ul serum free DMEM
media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin,
2.5 mg insulin, 1 .mu.g selenium and 5 mg fetuin in 500 ml DMEM),
incubate at room temperature for 30 minutes and then add 400 ul
serum free DMEM media. Add this 500 ul mixture onto
1.5.times.10.sup.5 COS cells/well plated on 12-well tissue culture
plate and incubate for 5 hours at 37.degree. C. Add 500 ul 20% FBS
DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg
L-glutamine in 500 ml DMEM) and incubate overnight.
C) Secretion Trap Assay
[0410] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed with TNT (0.1M
Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O), and
permeated with 0.1% Triton-X in PBS for 15 minutes, and again
washed with TNT. Cells were blocked for 1 hour with TNB (0.1M
Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance
TSA-Direct Kit) in H.sub.2O), and washed again with TNT. The cells
were incubated for 1 hour with 1 .mu.g/ml human ZcytoR14x1sR/FC9
soluble receptor fusion protein Cells were then washed with TNT.
Cells were incubated for another hour with 1:200 diluted
goat-anti-human Ig-HRP (Fc specific). Again cells were washed with
TNT.
[0411] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for
4-6 minutes, and washed with TNT. Cells were preserved with
Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted
1:5 in TNT. Cells were visualized using a FITC filter on
fluorescent microscope.
Example 18
Generation of Murine Anti-Human ZcytoR14 Monoclonal Antibodies
A. Immunization for generation of anti-ZcytoR14 Antibodies
[0412] 1. Soluble ZcytoR14-muFc
[0413] Six to twelve week old intact or ZcytoR14 knockout mice are
immunized by intraperitoneal injection with 25-50 ug of soluble
human ZcytoR14-muFc protein (Example 23) mixed 1:1 (v:v) with Ribi
adjuvant (Sigma) on a biweekly schedule. Seven to ten days
following the third immunization, blood samples were taken via
retroorbital bleed, the serum harvested and evaluated for its
ability to inhibit the binding of IL-17 or IL-17F to ZcytoR14 in
neutralization assays (e.g., described herein) and to stain
ZcytoR14 transfected versus untransfected 293 cells in a FACS
staining assay. Mice continued to be immunized and blood samples
taken and evaluated as described above until neutralization titers
reached a plateau. At that time, mice with the highest
neutralization titers were injected intravascularly with 25-50 ug
of soluble ZcytoR14-Fc protein in PBS. Three days later, the spleen
and lymph nodes from these mice were harvested and used for
hybridoma generation, for example using mouse myeloma
(P3-X63-Ag8.653.3.12.11) cells or other appropriate cell lines in
the art, using standard methods known in the art (e.g., see
Kearney, J. F. et al., J Immunol. 123:1548-50, 1979; and Lane, R.
D. J Immunol Methods 81:223-8, 1985).
[0414] 2. Soluble ZcytoR14, ZcytoR14-CEE ZcytoR14-CHIS
ZcytoR14-CFLAG
[0415] Six to twelve week old intact or ZcytoR14 knockout mice are
immunized by intraperitoneal injection with 25-50 ug of soluble
human ZcytoR14-CEE, ZcytoR14-CHIS, or ZcytoR14-CFLAG mixed 1:1
(v:v) with Ribi adjuvant (Sigma) on a biweekly schedule. Seven to
ten days following the third immunization, blood samples are taken
via retroorbital bleed, the serum harvested and evaluated for its
ability to inhibit the binding of IL-17 or IL-17F to ZcytoR14 in
neutralization assays (e.g., described herein) and to stain
ZcytoR14 transfected versus untransfected 293 cells in a FACS
staining assay. Mice are continued to be immunized and blood
samples taken and evaluated as described above until neutralization
titers reached a plateau. At that time, mice with the highest
neutralization titers are injected intravascularly with 25-50 ug of
soluble ZcytoR14, ZcytoR14-CEE, zcytor-CHIS, or ZcytoR14-CFLAG
antigen protein in PBS. Three days later, the spleen and lymph
nodes from these mice are harvested and used for hybridoma
generation, for example using mouse myeloma
(P3-X63-Ag8.653.3.12.11) cells or other appropriate cell lines in
the art, using standard methods known in the art (e.g., see
Kearney, J. F. et al., J Immunol. 123:1548-50, 1979; and Lane, R.
D. J Immunol Methods 81:223-8, 1985).
[0416] 3. P815 transfectants that express the ZcytoR14
[0417] Six to ten week old female DBA/2 mice are immunized by
intraperitoneal injection of 1.times.10.sup.5 live, transfected
P815 cells, for example P815/ZcytoR14 cells (e.g., 0.5 ml at a cell
density of 2.times.10.sup.5 cells/ml). Prior to injection, the
cells are maintained in the exponential growth phase. For injection
the cells are harvested, washed three times with PBS and then
resuspended in PBS to a density of 2.times.10.sup.5 cells/ml. In
this model, the mice develop an ascites tumor within 2-3 weeks and
progress to death by 4-6 weeks unless an immune response to the
transfected target antigen has been mounted. At three weeks mice
with no apparent abdominal swelling (indicative of ascites) are
re-immunized as above at 2-3 week intervals. Seven to ten days
following the second immunization, blood samples are taken via
retroorbital bleed, the serum harvested and evaluated for its
ability to inhibit the binding of IL-17 or IL-17F to IL-17 or
ZcytoR14 in neutralization assays (e.g., described herein) and to
stain ZcytoR14 transfected versus untransfected 293 cells in a FACS
staining assay. Mice continue to be immunized and blood samples
taken and evaluated as described above until neutralization titers
reach a plateau. At that time, the mice with the highest
neutralization titers are injected intraperitonealy with
1.times.10.sup.5 live, transfected P815 cells. Four days later, the
spleen and lymph nodes from these mice are harvested and used for
hybridoma generation, for example using mouse myeloma
(P3-X63-Ag8.653.3.12.11) cells or other appropriate cell lines in
the art, using standard methods known in the art (e.g., see
Kearney, J. F. et al., supra.; and Lane, R. D. supra.).
[0418] An alternative to the above immunization scheme with live,
transfected P815 cells involves intraperitoneal injection of
1-5.times.10.sup.6 irradiated, transfected cells every 2-3 weeks.
In this approach, no animals develop and die of ascites. Instead,
animals are monitored for a neutralizing immune response to
ZcytoR14 in their serum as outlined above, starting with a bleed
after the second immunization. Once neutralization titers have
reached a maximal level, the mice with highest titers are given a
pre-fusion, intraperitoneal injection of 5.times.10.sup.6
irradiated cells and four days later, the spleen and lymph nodes
from these mice are harvested and used for hybridoma generation,
for example using mouse myeloma (P3-X63-Ag8.653.3.12.11) cells or
other appropriate cell lines in the art, using standard methods
known in the art (e.g., see Kearney, J. F. et al., supra.; and
Lane, R. D. supra.).
B. Screening the Hybridoma Fusions for Antibodies that bind
ZcytoR14 and Inhibit the Binding of IL-17 or IL-17F to ZcytoR14
[0419] Three different primary screens are performed on the
hybridoma supernatants at 8-10 days post-fusion. For the first
assay, antibodies in supernatants were tested for their ability to
bind to plate bound soluble human ZcytoR14, ZcytoR14-muFc,
ZcytoR14-CEE, ZcytoR14-CHIS, or ZcytoR14-CFLAG protein by ELISA
using HRP-conjugated goat anti-mouse kappa and anti-lambda light
chain second step reagents to identify bound mouse antibodies. To
demonstrate specificity for the ZcytoR14 portion of the ZcytoR14
fusion proteins, positive supernatants in the initial assay were
evaluated on an irrelevant protein fused to the same murine Fc
region (mG2a), EE sequence, HIS sequence, or FLAG sequence.
Antibody in those supernatants that bound to ZcytoR14-fusion
protein and not the irrelevant muFc or other proteins containing
fusion protein sequence were deemed to be specific for ZcytoR14.
For the second assay, antibodies in all hybridoma supernatants were
evaluated by ELISA for their ability to inhibit the binding of
biotinylated human IL-17 or biotinylated human IL-17F to plate
bound ZcytoR14-muFc or ZcytoR14-fusion proteins.
[0420] All supernatants containing antibodies that bound
specifically to ZcytoR14, whether they inhibited the binding of
IL-17 or IL-17F to ZcytoR14 or not in the ELISA assay, were
subsequently tested for their ability to inhibit the binding of
IL-17 or IL-17F to ZcytoR14 transfected Baf3 or BHK cells or normal
human bronchial epithelial cells. All supernatants that were
neutralization positive in either the IL-17 or IL-17F inhibition
assays or both the IL-17 and IL-17F inhibition assays were
subsequently evaluated for their ability to stain ZcytoR14
transfected versus non-transfected Baf3 or BHK cells by FACS
analysis. This analysis was designed to confirm that inhibition of
IL-17 or IL-17F binding to ZcytoR14, was indeed due to an antibody
that specifically binds the ZcytoR14 receptor. Additionally, since
the FACS analysis was performed with an anti-IgG second step
reagent, specific FACS positive results indicate that the
neutralizing antibody was likely to be of the IgG class. By these
means, a master well was identified that bound ZcytoR14 in the
plate bound ELISA, inhibited the binding of IL-17 or IL-17F to
ZcytoR14 in the ELISA based inhibition assay, blocked the
interaction of IL-17 and IL-17F with ZcytoR14 transfected Baf3 or
BHK cells, respectively, and was strongly positive for the staining
of ZcytoR14 transfected Baf3 or BHK cells with an anti-mouse IgG
second step reagent.
[0421] The third assay consists of primary human bronchial
epithelial cells which express ZcytoR14 and can be induced to
secrete IL-8 or IL-6 in response to IL-17F treatment. The specific
monoclonal antibody is assayed by its ability to inhibit the IL-17
or IL-17F stimulated IL-8 or IL-6 production by these cells. IL-8
and IL-6 production is assayed in response to IL-17 or IL-17F as
described herein.
[0422] Alternatively, the monoclonal antibody; anti-ZcytoR14,
mediated inhibition of IL-17 or IL-17F induced luciferase
production in NIH 3T3 or other ZcytoR14 containing cells can be
used with or in place of one of the bioactivity neutralization
assays noted above. The NFkB mediated luciferase assay in NIH 3T3
cells is described herein.
C) Cloning Anti-ZcytoR14 Specific Antibody Producing Hybridomas
[0423] Hybridoma cell lines producing a specific anti-ZcytoR14 mAb
that cross-neutralized the binding of IL-17 and IL-17F to
appropriately transfected BaF3 or BHK cells are cloned by a
standard low-density dilution (less than 1 cell per well) approach.
Approximately 5-7 days after plating, the clones are screened by
ELISA on, for example, plate bound human ZcytoR14-muFc followed by
a retest of positive wells by ELISA on irrelevant muFc containing
fusion protein as described above. Selected clones, whose
supernatants bind to ZcytoR14-muFc and not the irrelevant muFc
containing fusion protein, are further confirmed for specific
antibody activity by repeating both neutralization assays as well
as the FACS analysis. All selected ZcytoR14 antibody positive
clones are cloned a minimum of two times to help insure clonality
and to assess stability of antibody production. Further rounds of
cloning are performed and screened as described until, preferably,
at least 95% of the resulting clones were positive for neutralizing
anti-ZcytoR14 antibody production.
D) Biochemical Characterization of the Molecule Recognized by
Anti-ZcytoR14 mAbs
[0424] Biochemical confirmation that the target molecule, ZcytoR14,
recognized by the putative anti-ZcytoR14 mAbs is indeed ZcytoR14
are performed by standard immunoprecipitation followed by SDS-PAGE
analysis or western blotting procedures, both employing soluble
membrane preparations from ZcytoR14 transfected versus
untransfected Baf3 or BHK cells. Moreover, soluble membrane
preparations of non-transfected cell lines that express ZcytoR14
are used show that the mAbs recognize the native receptor chain as
well as the transfected one. Alternatively, the mAbs are tested for
their ability to specifically immunoprecipitate or western blot the
soluble ZcytoR14-muFc protein.
Example 19
Neutralization of Human ZcytoR14 by Sera from Mice Injected with
P815 Cells Transfected with Human ZcytoR14
[0425] Using a cell based neutralization assay, serum from mice
injected with live human ZcytoR14 transfected P815 cells (Example
17) is added as a serial dilution at 1%, 0.5%, 0.25%, 0.13%, 0.06%,
0.03%, 0.02%, and 0%. The assay plates are incubated at
37.quadrature. C, 5% CO.sub.2 for 4 days at which time Alamar Blue
(Accumed, Chicago, Ill.) is added at 20 .mu.l/well. Plates are
again incubated at 37.quadrature. C, 5% CO.sub.2 for 16 hours.
Results showed that serum from four of the animals could neutralize
signaling of both huIL-17 and huIL-17F through human ZcytoR14.
[0426] Results such as these provide additional evidence that
effectively blocking ZcytoR14 by binding, blocking, inhibiting,
reducing, antagonizing or neutralizing IL-17 or IL-17F activity
(individually or together), for example via a neutralizing
monoclonal antibody to ZcytoR14 of the present invention, could be
advantageous in reducing the effects of IL-17 and IL-17F (alone or
together) in vivo and may reduce IL-17 and/or IL-17F-induced
inflammation, such as that seen in, for example in psoriasis, IBD,
colitis, chronic obstructive pulmonary disease, cystic fibrosis or
other inflammatory diseases induced by IL-17, and or IL-17F
including IBD, arthritis, asthma, psoriatic arthritis, colitis,
inflammatory skin conditions, and atopic dermatitis.
Example 20
Pharmacokinetics of an Anti-human ZcytoR14 Monoclonal Antibody
[0427] The test monoclonal antibody, anti-human ZcytoR14 mAb, is
provided in, for example, 3.times.3 mL aliquots at a concentration
of approximately 1 mg/mL (determined by UV Absorbance at 280 nM)
and was stored at -80.degree. C. until use. The vehicle is
1.times.PBS (50 mM NaPO4, 109 mM NaCl), pH 7.3. The mAb is thawed
at room temperature before use and aliquots 1 and 2 are used as
provided for the 100 .mu.g IV and SC dosing groups, respectively.
Half of aliquot 3 is diluted 1:2 in 1.times.PBS for the 50 .mu.g SC
dose group and the second half of aliquot 3 is diluted 1:10 in
1.times.PBS for the 10 .mu.g SC dose group. Female SCID mice (n=96)
are obtained from Charles River Labs. Animals are checked for
health on arrival and group-housed (3 animals per cage). The mice
are 12 weeks old with an average body weight of approximately 22 g
at the beginning of the study.
A) Dosing Protocol
[0428] Female SCID mice (n=24/dose group) are randomly placed into
four dosing groups (Table 5). Group 1 was administered the
anti-human ZcytoR14 mAb via IV injection of approximately 93 .mu.L
in a tail vein and Groups 2, 3, and 4 are administered the mAb via
SC injection of approximately 93 .mu.L in the scruff of the
neck.
B) Sample Collection
[0429] Prior to blood collection, mice were fully anesthetized with
halothane or isofluorane. Blood samples were collected via cardiac
stick for all time points except the 168 hr timepoint (collected
via eye bleed and the same animals were bled again at the 504 hr
timepoint via cardiac stick). Blood was collected into serum
separator tubes and allowed to clot for 15 minutes. Samples were
subsequently centrifuged for 3 minutes at 14,000 rpm. Following
centrifugation, aliquots of 125-150 uL were dispensed into labeled
eppendorf tubes and immediately stored at -80.degree. C. until
analysis. TABLE-US-00005 TABLE 5 Group # Dose (ROA) Animals PK
Timepoints 1 100 .mu.g (IV) 3 mice/timepoint* 0.25, 1, 4, 8, 24,
72, 168, 336 and 504 hr 2 100 .mu.g (SC) 3 mice/timepoint* 0.25, 1,
4, 8, 24, 72, 168, 336 and 504 hr 3 50 .mu.g (SC) 3 mice/timepoint*
0.25, 1, 4, 8, 24, 72, 168, 336 and 504 hr 4 10 .mu.g (SC) 3
mice/timepoint* 0.25, 1, 4, 8, 24, 72, 168, 336 and 504 hr *The
same animals were used for the 168 and 504 hr timepoints.
C) Quantification of Serum Anti-human ZcytoR14 mAb Concentrations
by ELISA
[0430] An Enzyme Linked Immunosorbant Assay (ELISA) is developed
and qualified to analyze mouse serum samples from animals dosed
with anti-ZcytoR14 mAb during pharmacokinetic studies. This assay
is designed to take advantage of a commercially available secondary
antibody and calorimetric detection using TMB. The dilutions used
for the standard curve were modified to improve the definition of
the linear portion of the standard curve. A standard curve in the
range of 100 ng/mL to 0.231 ng/mL with 2-fold dilutions allows for
quantitation of the mouse serum samples. QC samples are diluted to
1:100, 1:1000 and 1:10000 in 10% SCID mouse serum and back
calculated from the standard curve.
D) Pharmacokinetic Analysis
[0431] Serum concentration versus time data are downloaded into
WinNonlin Professional 4.0 software (Pharsight, Inc.; Cary, N.C.)
for pharmacokinetic analysis. Noncompartmental analysis is used to
determine pharmacokinetic parameters based on the mean data at each
time point.
Example 21
Neutralization of IL-17 and IL-17F Activity by a Anti-Human
ZcytoR14 Monoclonal Antibody
[0432] Using a cell-based neutralization assay, a purified mouse
anti-human ZcytoR14 monoclonal antibody is added as a serial
dilution, for example, at 10 .mu.g/ml, 5 .mu.g/ml, 2.5 .mu.g/ml,
1.25 .mu.g/ml, 625 ng/ml, 313 ng/ml, 156 ng/ml and 78 ng/ml. The
assay plates are incubated at 37.degree. C., 5% CO.sub.2 for 4 days
at which time Alamar Blue (Accumed, Chicago, Ill.) is added at 20
.mu.l/well. Plates are again incubated at 37.quadrature. C, 5%
CO.sub.2 for 16 hours. This assay is able to demonstrate that the
purified anti-human ZcytoR14 monoclonal antibody is able neutralize
signaling of both huIL-17 and huIL-17F through human ZcytoR14. For
highly effective antibodies, when used at approx. 10 .mu.g/ml
concentration, the antibody completely neutralizes proliferation
induced by huIL-17 or huIL-17F, with the inhibition of
proliferation decreasing in a dose dependent fashion at the lower
concentrations. An isotype-matched negative control mouse mAb,
tested at the concentrations described above, is executed to
provide no inhibition of proliferation of either cytokine. These
results are able to further demonstrate that monoclonal antibodies
to ZcytoR14 could indeed antagonize the activity of the
pro-inflammatory ligands, IL-17 and IL-17F at low
concentrations.
Example 22
IL-17A Induces Elevated Levels of IFN-gamma and TNF-alpha in Human
Peripheral Blood Mononuclear Cells
[0433] Human peripheral blood mononuclear cells (PBMC) are purified
by ficoll density gradient centrifugation and then incubated
overnight at 37.degree. C. in media alone, 50 ng/ml anti-human CD3
antibody, or the combination of 50 ng/ml anti-human CD3 antibody
plus 1 .quadrature.g/ml anti-human CD28 antibody. Replicate
cultures for each of these conditions are set up and are given no
cytokine, 25 ng/ml human IL-17A, or 25 ng/ml human IL-17F. After
24-hour incubations, supernatants from each culture are harvested
and assayed for cytokine content using B-D Bioscience's human
Th1/Th2 Cytometric Bead Array (CBA). We found that cultures that
had been stimulated with either anti-CD3 or anti-CD3 plus anti-CD28
and had been supplemented with IL-17A contained significantly
elevated levels of IFN-gamma and TNF-alpha (3-5-fold elevation of
each) over cultures with no cytokine added or those that received
IL-17F. Cultures in which no anti-CD3 stimulation was added did not
show significant changes in cytokine levels. In addition, IL-17A
addition induced no significant changes in other cytokines assayed
for with the CBA including IL-2, IL-4, IL-5, and IL-10. This data
indicates that IL-17A, but not IL-17F, can augment the production
of IFN-gamma and TNF-alpha in PBMC cultures stimulated with
anti-CD3 or anti-CD3 plus anti-CD28.
Example 23
ZcytoR14-Fc Decreases Disease Incidence and Progression in Mouse
Collagen Induced Arthritis (CIA) Model
A) Mouse Collagen Induced Arthritis (CIA) Model
[0434] Ten week old male DBA/1J mice (Jackson Labs) are divided
into 3 groups of 13 mice/group. On day-21, animals are given an
intradermal tail injection of 50-100 .mu.l of 1 mg/ml chick Type II
collagen formulated in Complete Freund's Adjuvant (prepared by
Chondrex, Redmond, Wash.), and three weeks later on Day 0 they are
given the same injection except prepared in Incomplete Freund's
Adjuvant. ZcytoR14-Fc is administered as an intraperitoneal
injection 3 times a week for 4 weeks, at different time points
ranging from Day 0, to a day in which the majority of mice exhibit
moderate symptoms of disease. Groups receive either 10 or 100 .mu.g
of ZcytoR14-Fc per animal per dose, and control groups receive the
vehicle control, PBS (Life Technologies, Rockville, Md.). Animals
begin to show symptoms of arthritis following the second collagen
injection, with most animals developing inflammation within 1.5-3
weeks. The extent of disease is evaluated in each paw by using a
caliper to measure paw thickness, and by assigning a clinical score
(0-3) to each paw: 0=Normal, 0.5=Toe(s) inflamed, 1=Mild paw
inflammation, 2=Moderate paw inflammation, and 3=Severe paw
inflammation as detailed below.
B) Monitoring Disease
[0435] Animals can begin to show signs of paw inflammation soon
after the second collagen injection, and some animals may even
begin to have signs of toe inflammation prior to the second
collagen injection. Most animals develop arthritis within 1.5-3
weeks of the boost injection, but some may require a longer period
of time. Incidence of disease in this model is typically 95-100%,
and 0-2 non-responders (determined after 6 weeks of observation)
are typically seen in a study using 40 animals. Note that as
inflammation begins, a common transient occurrence of variable
low-grade paw or toe inflammation can occur. For this reason, an
animal is not considered to have established disease until marked,
persistent paw swelling has developed.
[0436] All animals are observed daily to assess the status of the
disease in their paws, which is done by assigning a qualitative
clinical score to each of the paws. Every day, each animal has its
4 paws scored according to its state of clinical disease. To
determine the clinical score, the paw can be thought of as having 3
zones, the toes, the paw itself (manus or pes), and the wrist or
ankle joint. The extent and severity of the inflammation relative
to these zones is noted including: observation of each toe for
swelling; torn nails or redness of toes; notation of any evidence
of edema or redness in any of the paws; notation of any loss of
fine anatomic demarcation of tendons or bones; evaluation of the
wrist or ankle for any edema or redness; and notation if the
inflammation extends proximally up the leg. A paw score of 1, 2, or
3 is based first on the overall impression of severity, and second
on how many zones are involved. The scale used for clinical scoring
is shown below.
C) Clinical Score
[0437] 0=Normal
[0438] 0.5=One or more toes involved, but only the toes are
inflamed
[0439] 1=mild inflammation involving the paw (1 zone), and may
include a toe or toes
[0440] 2=moderate inflammation in the paw and may include some of
the toes and/or the wrist/ankle (2 zones)
[0441] 3=severe inflammation in the paw, wrist/ankle, and some or
all of the toes (3 zones)
[0442] Established disease is defined as a qualitative score of paw
inflammation ranking 2 or more, that persists for two days in a
row. Once established disease is present, the date is recorded and
designated as that animal's first day with "established
disease".
[0443] Blood is collected throughout the experiment to monitor
serum levels of anti-collagen antibodies, as well as serum
immunoglobulin and cytokine levels. Serum anti-collagen antibodies
correlate well with severity of disease. Animals are euthanized on
Day 21, and blood collected for serum and CBC's. From each animal,
one affected paw is collected in 110% NBF for histology and one is
frozen in liquid nitrogen and stored at -80.degree. C. for mRNA
analysis. Also, 1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph node,
one liver lobe and the left kidney are collected in RNA later for
RNA analysis, and 0.1/2 spleen, 1/2 thymus, 1/2 mesenteric lymph
node, the remaining liver, and the right kidney are collected in
10% NBF for histology. Serum is collected and frozen at -80.degree.
C. for immunoglobulin and cytokine assays.
[0444] Groups of mice receiving ZcytoR14-Fc at all time points are
characterized by a delay in the onset and/or progression of paw
inflammation. These results indicate that ZcytoR14 can reduce
inflammation, as well as disease incidence and progression
associated with this model. These results are further supported by
the observation that ZcytoR14-Fc resulted in decreased levels of
serum TNFa, IL-1b, and anti-collagen antibodies.
Example 24
Stable Over-Expression of ZcytoR14 in the Murine Assay Cell Line,
Nih3t3/kz142.8 Expressing the ap1/nfkb Transcription Factor
[0445] The murine nih3t3/kz142.8 assay cell line was transfected
with a human zcytor14x1 (SEQ ID NO:2) in an expression vector with
a methotrexate resistance gene (dihydrofolate reductase, DHFR) This
transfection was performed using a commercially available kit and
the manufacturer's recommendations. (Mirus, Madison, Wis. Cat.
#MIR218) Cells were placed in 1 .mu.M mtx amended growth medium to
select for the expression vector containing the human zcytor14X1
transgene. After selection a human zcytor14x1 transfection pool was
generated, and called nih3t3/kz142.8/hcytor14x1.
A) Luciferase Assay Using the nih3t3/kz142.8 Assay Cell Line
[0446] Since nih3t3/kz142.8 has a stable kz142 reporter, there is
no need for adenovirus infection to add this reporter. Thus the
luciferase assay protocol was shorted and done the following
way:
[0447] 1. Cell Plating
[0448] nih3t3/kz142.8 cells were plated at 5000 cells/well in solid
white, cell culture coated 96 well plates, (Cat. #3917. Costar)
using DMEM/10% FBS, containing glutamine and amended with pyruvate
and cultured overnight at 37o C. and 5% C02. On this second day,
the plating media was removed and exchanged for DMEM/1% FBS,
containing glutamine and amended with pyruvate and cultured
overnight at 37o C. and 5% C02.
[0449] 2. Luciferase Assay Measuring IL-17A and F Activation of the
Stable kz142 Reporter
[0450] Following the overnight incubation in the 1% fbs, DMEM
media, human IL-17A, and IL-17F ligand dilutions were made in serum
free media, amended with BSA to a 0.28% level. After adding the
ligand dilutions, cells were incubated at 37o C. and 5% C02 for 4
hours, after which the media was removed, cells lysed for 15
minutes and mean fluorescence intensity (MFI) measured using the
luciferase assay system and reagents, (Cat.#e1531 Promega. Madison,
Wis.) and a Microplate luminometer. Activity was detected for both
ligands at concentrations ranging from 0.1-1000 ng/ml. The
nih3t3/kz142.8/hcytor14x1 transfection pool showed similar activity
for the murine IL-17A ligand as did the parental cell line.
(example 14) However, the cytor14x1 transfectant pool showed an
elevated responsiveness to human IL-17A and F treatments, even when
these ligand concentrations were as low as 20 femptograms. The fact
that the mIL-17A signaling is comparable to that in the parental
cell line (example 14) suggests that there isn't a general,
non-specific problem with human ZcytoR14-expressing cells and that
the murine IL-17A is probably signaling through the endogenous
murine nih3t3 cell IL-17R or ZcytoR14 receptor. Thus, the fact that
human IL-17A and IL-17F cause an elevation of MFI at such low
ligand concentrations may indicate a specific hyper-responsiveness
of the cells to those ligands, which is mediated through the
over-expressed human ZcytoR14 receptor.
[0451] This result has significant clinical and biological
ramifications and utility. For example, physiological situations
could cause local up-regulation of the ZcytoR14 receptors which
could then make these areas hyper-responsive to IL-17A and IL-17F,
resulting in biological activation at much lower ligand
concentrations than those suggested without ZcytoR14
over-expression. Thus, far lower soluble receptor levels may be
sufficient to antagonize these hypothetically lower ligand
concentrations, than previously thought or recognized by those in
the field.
Example 25
Antagonists to IL-17F and IL-17A activity Decrease Disease
Incidence and Progression in an Inflammatory Bowel Disease (IBD)
Model
[0452] This model is designed to show that cultured intestinal
tissue from patients with IBD produce higher levels of inflammatory
mediators compared to tissue from healthy controls. This enhanced
production of inflammatory mediators (including but not limited to
IL-1b, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A and F,
IL-18, IL-23, TNF-a, IFN-g, MIP family members, MCP-1, G- and
GM-CSF, etc.) contributes to the symptoms and pathology associated
with IBDs such as Crohn's disease (CD) and ulcerative colitis (UC)
by way of their effect(s) on activating inflammatory pathways and
downstream effector cells. These pathways and components then lead
to tissue and cell damage/destruction observed in vivo. Therefore,
this model can simulate this enhanced inflammatory mediator aspect
of IBD. Furthermore, when intestinal tissue from healthy controls
or from human intestinal epithelial cell (IEC) lines is cultured in
the presence of these inflammatory components, inflammatory pathway
signaling can be observed, as well as evidence of tissue and cell
damage.
[0453] Therapeutics that would be efficacious in human IBD in vivo
would work in the above ex vivo or IEC models by inhibiting and/or
neutralizing the production and/or presence of inflammatory
mediators.
[0454] In this model, human intestinal tissue is collected from
patients with IBD or from healthy controls undergoing intestinal
biopsy, re-sectioning or from post-mortem tissue collection, and
processed using a modification of Alexakis et al (Gut 53:85-90;
2004). Under aseptic conditions, samples are gently cleaned with
copious amounts of PBS, followed by culturing of minced sections of
tissue, in the presence of complete tissue culture media (plus
antibiotics to prevent bacterial overgrowth). Samples from the same
pool of minced tissue are treated with one of the following:
vehicle (PBS); recombinant human (rh) IL-17A; rhIL-17F; or
rhIL-17A+rhIL-17F. In addition, these are treated with or without
an antagonist of either IL-17A or IL-17F, alone or in combination
(such as a soluble ZcytoR14). This experimental protocol is
followed for studies with human IEC lines, with the exception that
cells are passaged from existing stocks. After varying times in
culture (from 1 h to several days), supernatants are collected and
analyzed for levels of inflammatory mediators, including those
listed above. In samples from patients with IBD or in samples
treated with rhIL-17A and/or F, levels of inflammatory cytokines
and chemokines are elevated compared to untreated healthy control
tissue samples. The addition of antagonists to IL-17F and/or IL-17A
activity, such as ZcytoR14 soluble receptors and antibodies thereto
including the anti-human-ZcytoR14 monoclonal and neutralizing
antibodies of the present invention markedly reduces the production
of inflammatory mediators, and thus, would expect to be efficacious
in human IBD.
Example 26
Antagonists to IL-17F and IL-17A activity Decrease Disease
Incidence and Progression in a Multiple Sclerosis (MS) Model
[0455] Multiple sclerosis (MS) is a complex disease that is thought
to be mediated by a number of factors, including the presence of
lymphocytic and mononuclear cell inflammatory infiltrates and
demyelination throughout the CNS. Microglia are macrophage-like
cells that populate the central nervous system (CNS) and become
activated upon injury or infection. Microglia have been implicated
as playing critical roles in various CNS diseases including MS, and
may be used to study mechanism(s) of initiation, progression, and
therapy of the disease (Nagai et al. Neurobiol Dis 8:1057-1068;
2001; Olson et al. J Neurosci Methods 128:33-43; 2003).
Immortalized human microglial cell lines and/or established human
astroglia cell lines can, therefore, be used to study some of the
effects of inflammatory mediators on these cell types and their
potential for neutralization. Inflammatory mediators (including but
not limited to IL-1b, IL-6, IL-8, IL-12, IL-13, IL-15, IL-17 A and
F, IL-18, IL-23, TNF-a, IFN-g, MIP family members, RANTES, IP-10,
MCP-1, G- and GM-CSF, etc.) can contribute to the symptoms and
pathology associated with MS by way of their effect(s) on
activating inflammatory pathways and downstream effector cells.
[0456] In order to evaluate the pro-inflammatory actions of IL-17A
and IL-17F, and the ability of an antagonist to IL-17F and/or
IL-17A activity, such as ZcytoR14 soluble receptors and antibodies
thereto including the anti-human-ZcytoR14 monoclonal and
neutralizing antibodies of the present invention to neutralize or
decrease these effects, cultured glial cells are treated with one
of the following: vehicle; rhIL-17A; rhIL-17F; rhIL-17A+IL-17F. In
addition, these are treated with or without an antagonist of either
IL-17A or IL-17F, alone or in combination (such as a soluble
ZcytoR14). After varying times in culture (from 1 h to several
days), supernatants and cells are collected and analyzed for levels
and/or expression of inflammatory mediators, including those listed
above. Levels of inflammatory cytokines and chemokines are elevated
in the presence of rhIL-17A and/or IL-17F compared to cultures
treated with vehicle alone. The addition of antagonists to IL-17F
and/or IL-17A activity, such as ZcytoR14 soluble receptors and
antibodies thereto including the anti-human-ZcytoR14 monoclonal and
neutralizing antibodies of the present invention markedly reduces
the production and expression of inflammatory mediators, and thus,
would expect to be efficacious in inflammatory aspects associated
with human MS.
Example 27
Antagonists to IL-17F and IL-17A activity Decrease Disease
Incidence and Progression in a Rheumatoid Arthritis (RA) and
Osteoarthritis (OA) Model
[0457] This model is designed to show that human synovial cultures
(including synovial macrophages, synovial fibroblasts, and
articular chondrocytes) and explants from patients with RA and OA
produce higher levels of inflammatory mediators compared to
cultures/explants from healthy controls. This enhanced production
of inflammatory mediators (including but not limited to oncostatin
M, IL-1b, IL-6, IL-8, IL-12, IL-15, IL-17 A and F, IL-18, IL-23,
TNF-a, IFN-g, IP-10, RANTES, RANKL, MIP family members, MCP-1, G-
and GM-CSF, nitric oxide, etc.) contributes to the symptoms and
pathology associated with RA and OA by way of their effect(s) on
activating inflammatory pathways and downstream effector cells.
These pathways and components then lead to inflammatory
infiltrates, cartilage and matrix loss/destruction, bone loss, and
upregulation of prostaglandins and cyclooxygenases. Therefore, this
model can simulate the destructive inflammatory aspects of RA and
OA in in vitro and ex vivo experiments. Furthermore, when explants
and synovial cultures from healthy controls are cultured in the
presence of several of these inflammatory components (e.g.
oncostatin M, TNF-a, IL-1b, IL-6, IL-17A and F, IL-15, etc.),
inflammatory pathway signaling can be observed. Therapeutics that
would be efficacious in human RA in vivo would work in the above in
vitro and ex vivo models by inhibiting and/or neutralizing the
production and/or presence of inflammatory mediators.
[0458] In this model, human synovial explants are collected from
patients with RA, OA, or from healthy controls undergoing joint
replacement or from post-mortem tissue collection, and processed
using a modification of Wooley and Tetlow (Arthritis Res 2: 65-70;
2000) and van't Hof et al (Rheumatology 39:1004-1008; 2000).
Cultures of synovial fibroblasts, synovial macrophages and
articular chondrocytes are also studied. Replicate samples are
treated with one of the following: vehicle (PBS); recombinant human
(rh) IL-17A; rhIL-17F; or rhIL-17A+rhIL-17F, and some samples
contain various combinations of oncostatin M, TNF-a, IL-1b, IL-6,
IL-17A, IL-17F, and IL-15. In addition, these are treated with or
without an antagonist to IL-17F and/or IL-17A activity, such as
ZcytoR14 soluble receptors and antibodies thereto including the
anti-human-ZcytoR14 monoclonal and neutralizing antibodies of the
present invention. After varying time of culture (from 1 h to
several days), supernatants are collected and analyzed for levels
of inflammatory mediators, including those listed above. In samples
from patients with RA or OA, or in samples treated with rhIL-17A
and/or F (either alone or in combination with other inflammatory
cytokines), levels of inflammatory cytokines and chemokines are
elevated compared to untreated healthy control explants or in
untreated cell cultures. The addition of antagonists to IL-17F
and/or IL-17A activity, such as ZcytoR14 soluble receptors and
antibodies thereto including the anti-human-ZcytoR14 monoclonal and
neutralizing antibodies of the present invention markedly reduces
the production of inflammatory mediators, and thus, would expect to
be efficacious in human RA and OA.
Example 28
IL-17A and IL-17F Functional Responses
[0459] NIH-3T3/KZ142 cells were stably transfected with human
zcytoR14x1 (SEQ ID NO:1) and mouse zcytoR14x1 (SEQ ID NO:25). As
described above, each line was treated for 7 and 15 minutes with a
dose response of IL-17A, IL-17F, murine IL-17F, and appropriate
controls. Both IL-17A and IL-17F gave a dose dependent response in
phosphorylated I.kappa.B-.alpha. and p38 MAPK transcription factors
when ZcytoR14x1 (SEQ ID NO:1) was transfected, approximately 30%
greater then the inherent signaling from the control line. IL-17A
and IL-17F gave no increase in signaling when the murine ZcytoR14x1
(SEQ ID NO:25) was transfected. Murine IL-17F gave no increase in
signaling for either human or murine ZcytoR14x1.
Example 29
IL-17A, IL-17F, IL-17R and ZcytoR14 Expression in Murine Disease
Models
[0460] Four murine models of disease (asthma, DSS colitis, atopic
dermatitis and experimental allergic encephalomyelitis) were
analyzed using know techniques for the expression of IL-17A,
IL-17F, IL-17R and ZcytoR14.
[0461] In the asthma model, IL-17A and IL-17F are expressed at very
low to undetectable levels in lung, spleen, lung draining lymph
nodes and lung infiltrating cells in diseased and non-diseased
mice. Zcytor14 message was found to be more highly expressed in
lung compared to spleen and lymph node but was not regulated with
disease. IL-17R was more highly expressed in spleen and lung
draining lymph node compared to lung but was also not regulated
with disease.
[0462] Contrary to the asthma model, IL-17A and IL-17F were highly
up-regulated in diseased but not normal mice in the DSS-colitis
model in both proximal and distal colon. Neither cytokine was
significantly up-regulated in the mesenteric lymph node. Further,
it was found that up-regulation of both cytokines in the context of
acute DSS-induced colitis and not in chronic DSS-induced colitis.
IL-17R was found to be prominently expressed in mesenteric lymph
nodes as compared to proximal and distal colon, but was not
regulated with disease. In contrast, ZcytoR14 was more highly
expressed in proximal distal colon tissue compared to mesenteric
lymph nodes. ZcytoR14 expression was also not regulated with
disease.
[0463] In atopic dermatitis, IL-17A mRNA was not detectable. IL-17F
was found to be expressed in both skin and skin-draining lymph
nodes but did not appear to be significantly regulated with
disease. IL-17R mRNA was more highly expressed in skin-draining
lymph nodes as compared to skin but was not regulated with disease.
Zcytor14 was more highly expressed in skin compared to
skin-draining lymph nodes but was also not regulated with
disease.
[0464] In experimental allergic encephalomyelitis, both IL-17A and
IL-17F appeared to up-regulated in spinal chord in diseased but not
healthy mice. IL-17F may have been more highly expressed in lymph
nodes compared to spinal cord but expression in the lymph nodes was
not regulated with disease. However, overall levels of expression
in these tissues was quite low. IL-17R was more highly expressed in
lymph node tissue compared to brain and spinal cord. Zcytor14 was
not tested.
[0465] In short, IL-17A and IL-17F expression appears to be
regulated with disease in the context of the DSS-induced colitis
and experimental allergic encephalomyelitis models but apparently
not for asthma or atopic dermatitis. IL-17R and zcytor14 expression
does not appear to be regulated with disease but IL-17R expression
appears to be enriched in lymphoid tissues while zcytor14
expression appears to be enriched in non-lymphoid tissues.
Example 30
ZcytoR14 is a Mediator of Activation to Both IL-17A and IL-17F
[0466] The murine nih3t3/kz142.8 assay cell line was transfected
with a human ZcytoR14X1 (SEQ ID NO:2) in an expression vector with
a methotrexate resistance gene. (dihydrofolate reductase, DHFR)
Human IL-17RA (SEQ ID NO:21) was similarly transfected into this
cell line. Transfections were performed using a commercially
available kit and the manufacturer's recommendations. (Mirus,
Madison, Wis. Cat. #MIR218) Cells were placed in 1 .mu.M mtx
amended growth medium to select for the expression vector
containing the expression constructs. After selection transfection
pools were generated, and called nih3t3/kz142.8/hcytor14X1 and
nih3t3/kz142.8/IL-17R.
A) Luciferase Assay Using the nih3t3/kz142.8-Based Cell Lines.
[0467] Since nih3t3/kz142.8 based cell lines have stable ap1/nfkb
reporters (kz142), there is no need for adenovirus infection to add
this reporter. Thus the luciferase assay protocol was shorted and
done the following way:
[0468] 1. Cell Plating
[0469] Cells were plated at 5000 cells/well in solid white, cell
culture coated 96 well plates, (Cat. #3917. Costar) using DMEM/10%
FBS, containing glutamine and amended with pyruvate and cultured
overnight at 37o C. and 5% C02. On this second day, the plating
media was removed and exchanged for DMEM/1% FBS, containing
glutamine and amended with pyruvate and cultured overnight at 37o
C. and 5% C02.
[0470] 2. Luciferase Assay Measuring IL-17A and F Activation of the
Stable kz142 Reporter
[0471] Following the overnight incubation in the 1% fbs, DMEM
media, human IL-17A, and IL-17F ligand dilutions were made in serum
free media, amended with BSA to a 0.28% level. After adding the
ligand dilutions, cells were incubated at 37o C. and 5% C02 for 4
hours, after which the media was removed, cells lysed for 15
minutes and mean fluorescence intensity (MFI) measured using the
luciferase assay system and reagents, (Cat.#e1531 Promega. Madison,
Wis.) and a Microplate luminometer. Activity was detected for both
ligands at concentrations ranging from 1-100 ng/ml.
[0472] The EC.sub.50s discussed below are averages of at least 4
experiments. The nih3t3/kz142.8/hcytor14x1 transfection pool showed
similar activity for the murine IL-17A ligand as did the parental
cell line, with an EC.sub.50 of about 4 ng/ml. (example 14) The
fact that the mIL-17A signaling in the hcytor14x1 recombinant line
is comparable to that in the parental cell line (example 14)
suggests that murine IL-17A is probably signaling through the
endogenous murine nih3t3 cell IL-17RA or zcytor14 receptors and
does not activate the cells through hcytor14X1. However, the
hzcytor14X1 transfectant pool showed an elevated responsiveness to
human IL-17A treatment, with an EC.sub.50 of 0.41 ng/ml Vs 2.8
ng/ml (averages of 4 experiments) in the parental line (a 6.8 fold
more potent EC.sub.50 in the recombinant line) In addition, the
hIL-17RCX1 recombinant line had an enhanced responsiveness to
hIL-17F, with an EC.sub.50 of 0.61 ng/ml in the recombinant line Vs
10 ng/ml in the parental line. (a 17 fold more potent EC.sub.50 in
the recombinant line). The increased potency to hIL-17A and F in
the hIL-17RCX1 line is consistent with human zcytor14X1 being a
high affinity receptor for both human IL-17A and IL-17F. In
contrast, the hIL-17RA recombinant line had enhanced sensitivity
only to hIL-17A, with an EC.sub.50 of 0.6 ng/ml vs 2.8 ng/ml for
the parental line. There was not an enhancement of the hIL-17F
E.sub.50 in the hIL-17RA recombinant line, with an IL-17F E.sub.50
of 12.4 ng/ml vs 8.9 ng/ml in the parental line.
[0473] This result is significant because it specifically
implicates hzcytor14X1 as a mediator of activation to both hIL-17A
and hIL-17F and suggests that hIL-17RA mediates signaling only to
hIL-17A activation and not hIL-17F.
Example 31
Intravenous Administration of IL-17A and IL-17F
[0474] To determine the effect of i.v. delivery of murine or human
IL-17A or IL-17F on complete blood counts (CBC) and serum
cytokines/chemokines in BALB/c mice at various time points.
[0475] I.V. administration of 1 ug mIL-17A resulted in an
approximate 2-fold increase in circulating neutrophils (by CBC) and
approximate 10-fold increase in serum KC and MCP-1 (by Luminex) 1-2
h following administration; similar results in these chemokines
were observed with 5 ug hIL-17A. Blood monocyte levels were also
significantly increased in mice treated with 1 ug mIL-17A (showed
the greatest increase), 5 ug hIL-17A or 5 ug hIL-17F at the 2 h
timepoint. I.V. administration of m and hIL-17F resulted in marked
increases in serum IL-15 (by Luminex) at the 1 and 2 h time points,
and small increases in serum KC and MCP-1 at these same
timepoints.
Example 32
Neutralization of i.v. Administered IL-17A and IL-17F
[0476] To neutralize the i.v. IL-17A and IL-17F-mediated increases
in cytokines and chemokines with i.p. soluble receptors
(mIL-17RA:Fc for murine ligands; soluble human ZcytoR14 for human
ligands). Female BALB/c mice were administered by i.p. injection
either PBS, 100 ug mIL-17RA:Fc, or 100 ug soluble human ZcytoR14
three hours prior to receiving by i.v. tail injection: PBS; 2 ug of
either mIL-17A, mIL-17F, or 2 ug of both mIL-17A and F (for mice
that received mIL-17RA:Fc); or 2 ug of either hIL-17A, hIL-17F, or
2 ug of both hIL-17A and F (for mice that received soluble human
ZcytoR14). Serum was collected 1 h following ligand administration
and analyzed for a small number of serum cytokines and
chemokines.
[0477] Mice pretreated with i.p. soluble receptor had marked
reductions in IL-17A-mediated increases in serum concentrations of
IL-17A and KC compared to mice treated with PBS +IL-17A.
[0478] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
66 1 2255 DNA Homo sapiens CDS (154)...(2229) 1 aactacccag
cacagccccc tccgccccct ctggaggctg aagagggatt ccagcccctg 60
ccacccacag acacgggctg actggggtgt ctgcccccct tggggggggg cagcacaggg
120 cctcaggcct gggtgccacc tggcacctag aag atg cct gtg ccc tgg ttc
ttg 174 Met Pro Val Pro Trp Phe Leu 1 5 ctg tcc ttg gca ctg ggc cga
agc cca gtg gtc ctt tct ctg gag agg 222 Leu Ser Leu Ala Leu Gly Arg
Ser Pro Val Val Leu Ser Leu Glu Arg 10 15 20 ctt gtg ggg cct cag
gac gct acc cac tgc tct ccg ggc ctc tcc tgc 270 Leu Val Gly Pro Gln
Asp Ala Thr His Cys Ser Pro Gly Leu Ser Cys 25 30 35 cgc ctc tgg
gac agt gac ata ctc tgc ctg cct ggg gac atc gtg cct 318 Arg Leu Trp
Asp Ser Asp Ile Leu Cys Leu Pro Gly Asp Ile Val Pro 40 45 50 55 gct
ccg ggc ccc gtg ctg gcg cct acg cac ctg cag aca gag ctg gtg 366 Ala
Pro Gly Pro Val Leu Ala Pro Thr His Leu Gln Thr Glu Leu Val 60 65
70 ctg agg tgc cag aag gag acc gac tgt gac ctc tgt ctg cgt gtg gct
414 Leu Arg Cys Gln Lys Glu Thr Asp Cys Asp Leu Cys Leu Arg Val Ala
75 80 85 gtc cac ttg gcc gtg cat ggg cac tgg gaa gag cct gaa gat
gag gaa 462 Val His Leu Ala Val His Gly His Trp Glu Glu Pro Glu Asp
Glu Glu 90 95 100 aag ttt gga gga gca gct gac tca ggg gtg gag gag
cct agg aat gcc 510 Lys Phe Gly Gly Ala Ala Asp Ser Gly Val Glu Glu
Pro Arg Asn Ala 105 110 115 tct ctc cag gcc caa gtc gtg ctc tcc ttc
cag gcc tac cct act gcc 558 Ser Leu Gln Ala Gln Val Val Leu Ser Phe
Gln Ala Tyr Pro Thr Ala 120 125 130 135 cgc tgc gtc ctg ctg gag gtg
caa gtg cct gct gcc ctt gtg cag ttt 606 Arg Cys Val Leu Leu Glu Val
Gln Val Pro Ala Ala Leu Val Gln Phe 140 145 150 ggt cag tct gtg ggc
tct gtg gta tat gac tgc ttc gag gct gcc cta 654 Gly Gln Ser Val Gly
Ser Val Val Tyr Asp Cys Phe Glu Ala Ala Leu 155 160 165 ggg agt gag
gta cga atc tgg tcc tat act cag ccc agg tac gag aag 702 Gly Ser Glu
Val Arg Ile Trp Ser Tyr Thr Gln Pro Arg Tyr Glu Lys 170 175 180 gaa
ctc aac cac aca cag cag ctg cct gcc ctg ccc tgg ctc aac gtg 750 Glu
Leu Asn His Thr Gln Gln Leu Pro Ala Leu Pro Trp Leu Asn Val 185 190
195 tca gca gat ggt gac aac gtg cat ctg gtt ctg aat gtc tct gag gag
798 Ser Ala Asp Gly Asp Asn Val His Leu Val Leu Asn Val Ser Glu Glu
200 205 210 215 cag cac ttc ggc ctc tcc ctg tac tgg aat cag gtc cag
ggc ccc cca 846 Gln His Phe Gly Leu Ser Leu Tyr Trp Asn Gln Val Gln
Gly Pro Pro 220 225 230 aaa ccc cgg tgg cac aaa aac ctg act gga ccg
cag atc att acc ttg 894 Lys Pro Arg Trp His Lys Asn Leu Thr Gly Pro
Gln Ile Ile Thr Leu 235 240 245 aac cac aca gac ctg gtt ccc tgc ctc
tgt att cag gtg tgg cct ctg 942 Asn His Thr Asp Leu Val Pro Cys Leu
Cys Ile Gln Val Trp Pro Leu 250 255 260 gaa cct gac tcc gtt agg acg
aac atc tgc ccc ttc agg gag gac ccc 990 Glu Pro Asp Ser Val Arg Thr
Asn Ile Cys Pro Phe Arg Glu Asp Pro 265 270 275 cgc gca cac cag aac
ctc tgg caa gcc gcc cga ctg cga ctg ctg acc 1038 Arg Ala His Gln
Asn Leu Trp Gln Ala Ala Arg Leu Arg Leu Leu Thr 280 285 290 295 ctg
cag agc tgg ctg ctg gac gca ccg tgc tcg ctg ccc gca gaa gcg 1086
Leu Gln Ser Trp Leu Leu Asp Ala Pro Cys Ser Leu Pro Ala Glu Ala 300
305 310 gca ctg tgc tgg cgg gct ccg ggt ggg gac ccc tgc cag cca ctg
gtc 1134 Ala Leu Cys Trp Arg Ala Pro Gly Gly Asp Pro Cys Gln Pro
Leu Val 315 320 325 cca ccg ctt tcc tgg gag aac gtc act gtg gac aag
gtt ctc gag ttc 1182 Pro Pro Leu Ser Trp Glu Asn Val Thr Val Asp
Lys Val Leu Glu Phe 330 335 340 cca ttg ctg aaa ggc cac cct aac ctc
tgt gtt cag gtg aac agc tcg 1230 Pro Leu Leu Lys Gly His Pro Asn
Leu Cys Val Gln Val Asn Ser Ser 345 350 355 gag aag ctg cag ctg cag
gag tgc ttg tgg gct gac tcc ctg ggg cct 1278 Glu Lys Leu Gln Leu
Gln Glu Cys Leu Trp Ala Asp Ser Leu Gly Pro 360 365 370 375 ctc aaa
gac gat gtg cta ctg ttg gag aca cga ggc ccc cag gac aac 1326 Leu
Lys Asp Asp Val Leu Leu Leu Glu Thr Arg Gly Pro Gln Asp Asn 380 385
390 aga tcc ctc tgt gcc ttg gaa ccc agt ggc tgt act tca cta ccc agc
1374 Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly Cys Thr Ser Leu Pro
Ser 395 400 405 aaa gcc tcc acg agg gca gct cgc ctt gga gag tac tta
cta caa gac 1422 Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu Tyr
Leu Leu Gln Asp 410 415 420 ctg cag tca ggc cag tgt ctg cag cta tgg
gac gat gac ttg gga gcg 1470 Leu Gln Ser Gly Gln Cys Leu Gln Leu
Trp Asp Asp Asp Leu Gly Ala 425 430 435 cta tgg gcc tgc ccc atg gac
aaa tac atc cac aag cgc tgg gcc ctc 1518 Leu Trp Ala Cys Pro Met
Asp Lys Tyr Ile His Lys Arg Trp Ala Leu 440 445 450 455 gtg tgg ctg
gcc tgc cta ctc ttt gcc gct gcg ctt tcc ctc atc ctc 1566 Val Trp
Leu Ala Cys Leu Leu Phe Ala Ala Ala Leu Ser Leu Ile Leu 460 465 470
ctt ctc aaa aag gat cac gcg aaa gcg gcc gcc agg ggc cgc gcg gct
1614 Leu Leu Lys Lys Asp His Ala Lys Ala Ala Ala Arg Gly Arg Ala
Ala 475 480 485 ctg ctc ctc tac tca gcc gat gac tcg ggt ttc gag cgc
ctg gtg ggc 1662 Leu Leu Leu Tyr Ser Ala Asp Asp Ser Gly Phe Glu
Arg Leu Val Gly 490 495 500 gcc ctg gcg tcg gcc ctg tgc cag ctg ccg
ctg cgc gtg gcc gta gac 1710 Ala Leu Ala Ser Ala Leu Cys Gln Leu
Pro Leu Arg Val Ala Val Asp 505 510 515 ctg tgg agc cgt cgt gaa ctg
agc gcg cag ggg ccc gtg gct tgg ttt 1758 Leu Trp Ser Arg Arg Glu
Leu Ser Ala Gln Gly Pro Val Ala Trp Phe 520 525 530 535 cac gcg cag
cgg cgc cag acc ctg cag gag ggc ggc gtg gtg gtc ttg 1806 His Ala
Gln Arg Arg Gln Thr Leu Gln Glu Gly Gly Val Val Val Leu 540 545 550
ctc ttc tct ccc ggt gcg gtg gcg ctg tgc agc gag tgg cta cag gat
1854 Leu Phe Ser Pro Gly Ala Val Ala Leu Cys Ser Glu Trp Leu Gln
Asp 555 560 565 ggg gtg tcc ggg ccc ggg gcg cac ggc ccg cac gac gcc
ttc cgc gcc 1902 Gly Val Ser Gly Pro Gly Ala His Gly Pro His Asp
Ala Phe Arg Ala 570 575 580 tcg ctc agc tgc gtg ctg ccc gac ttc ttg
cag ggc cgg gcg ccc ggc 1950 Ser Leu Ser Cys Val Leu Pro Asp Phe
Leu Gln Gly Arg Ala Pro Gly 585 590 595 agc tac gtg ggg gcc tgc ttc
gac agg ctg ctc cac ccg gac gcc gta 1998 Ser Tyr Val Gly Ala Cys
Phe Asp Arg Leu Leu His Pro Asp Ala Val 600 605 610 615 ccc gcc ctt
ttc cgc acc gtg ccc gtc ttc aca ctg ccc tcc caa ctg 2046 Pro Ala
Leu Phe Arg Thr Val Pro Val Phe Thr Leu Pro Ser Gln Leu 620 625 630
cca gac ttc ctg ggg gcc ctg cag cag cct cgc gcc ccg cgt tcc ggg
2094 Pro Asp Phe Leu Gly Ala Leu Gln Gln Pro Arg Ala Pro Arg Ser
Gly 635 640 645 cgg ctc caa gag aga gcg gag caa gtg tcc cgg gcc ctt
cag cca gcc 2142 Arg Leu Gln Glu Arg Ala Glu Gln Val Ser Arg Ala
Leu Gln Pro Ala 650 655 660 ctg gat agc tac ttc cat ccc ccg ggg act
ccc gcg ccg gga cgc ggg 2190 Leu Asp Ser Tyr Phe His Pro Pro Gly
Thr Pro Ala Pro Gly Arg Gly 665 670 675 gtg gga cca ggg gcg gga cct
ggg gcg ggg gac ggg act taaataaagg 2239 Val Gly Pro Gly Ala Gly Pro
Gly Ala Gly Asp Gly Thr 680 685 690 cagacgctgt ttttct 2255 2 692
PRT Homo sapiens 2 Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu
Gly Arg Ser Pro 1 5 10 15 Val Val Leu Ser Leu Glu Arg Leu Val Gly
Pro Gln Asp Ala Thr His 20 25 30 Cys Ser Pro Gly Leu Ser Cys Arg
Leu Trp Asp Ser Asp Ile Leu Cys 35 40 45 Leu Pro Gly Asp Ile Val
Pro Ala Pro Gly Pro Val Leu Ala Pro Thr 50 55 60 His Leu Gln Thr
Glu Leu Val Leu Arg Cys Gln Lys Glu Thr Asp Cys 65 70 75 80 Asp Leu
Cys Leu Arg Val Ala Val His Leu Ala Val His Gly His Trp 85 90 95
Glu Glu Pro Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly 100
105 110 Val Glu Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu
Ser 115 120 125 Phe Gln Ala Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu
Val Gln Val 130 135 140 Pro Ala Ala Leu Val Gln Phe Gly Gln Ser Val
Gly Ser Val Val Tyr 145 150 155 160 Asp Cys Phe Glu Ala Ala Leu Gly
Ser Glu Val Arg Ile Trp Ser Tyr 165 170 175 Thr Gln Pro Arg Tyr Glu
Lys Glu Leu Asn His Thr Gln Gln Leu Pro 180 185 190 Ala Leu Pro Trp
Leu Asn Val Ser Ala Asp Gly Asp Asn Val His Leu 195 200 205 Val Leu
Asn Val Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp 210 215 220
Asn Gln Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr 225
230 235 240 Gly Pro Gln Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro
Cys Leu 245 250 255 Cys Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val
Arg Thr Asn Ile 260 265 270 Cys Pro Phe Arg Glu Asp Pro Arg Ala His
Gln Asn Leu Trp Gln Ala 275 280 285 Ala Arg Leu Arg Leu Leu Thr Leu
Gln Ser Trp Leu Leu Asp Ala Pro 290 295 300 Cys Ser Leu Pro Ala Glu
Ala Ala Leu Cys Trp Arg Ala Pro Gly Gly 305 310 315 320 Asp Pro Cys
Gln Pro Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr 325 330 335 Val
Asp Lys Val Leu Glu Phe Pro Leu Leu Lys Gly His Pro Asn Leu 340 345
350 Cys Val Gln Val Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu
355 360 365 Trp Ala Asp Ser Leu Gly Pro Leu Lys Asp Asp Val Leu Leu
Leu Glu 370 375 380 Thr Arg Gly Pro Gln Asp Asn Arg Ser Leu Cys Ala
Leu Glu Pro Ser 385 390 395 400 Gly Cys Thr Ser Leu Pro Ser Lys Ala
Ser Thr Arg Ala Ala Arg Leu 405 410 415 Gly Glu Tyr Leu Leu Gln Asp
Leu Gln Ser Gly Gln Cys Leu Gln Leu 420 425 430 Trp Asp Asp Asp Leu
Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr 435 440 445 Ile His Lys
Arg Trp Ala Leu Val Trp Leu Ala Cys Leu Leu Phe Ala 450 455 460 Ala
Ala Leu Ser Leu Ile Leu Leu Leu Lys Lys Asp His Ala Lys Ala 465 470
475 480 Ala Ala Arg Gly Arg Ala Ala Leu Leu Leu Tyr Ser Ala Asp Asp
Ser 485 490 495 Gly Phe Glu Arg Leu Val Gly Ala Leu Ala Ser Ala Leu
Cys Gln Leu 500 505 510 Pro Leu Arg Val Ala Val Asp Leu Trp Ser Arg
Arg Glu Leu Ser Ala 515 520 525 Gln Gly Pro Val Ala Trp Phe His Ala
Gln Arg Arg Gln Thr Leu Gln 530 535 540 Glu Gly Gly Val Val Val Leu
Leu Phe Ser Pro Gly Ala Val Ala Leu 545 550 555 560 Cys Ser Glu Trp
Leu Gln Asp Gly Val Ser Gly Pro Gly Ala His Gly 565 570 575 Pro His
Asp Ala Phe Arg Ala Ser Leu Ser Cys Val Leu Pro Asp Phe 580 585 590
Leu Gln Gly Arg Ala Pro Gly Ser Tyr Val Gly Ala Cys Phe Asp Arg 595
600 605 Leu Leu His Pro Asp Ala Val Pro Ala Leu Phe Arg Thr Val Pro
Val 610 615 620 Phe Thr Leu Pro Ser Gln Leu Pro Asp Phe Leu Gly Ala
Leu Gln Gln 625 630 635 640 Pro Arg Ala Pro Arg Ser Gly Arg Leu Gln
Glu Arg Ala Glu Gln Val 645 650 655 Ser Arg Ala Leu Gln Pro Ala Leu
Asp Ser Tyr Phe His Pro Pro Gly 660 665 670 Thr Pro Ala Pro Gly Arg
Gly Val Gly Pro Gly Ala Gly Pro Gly Ala 675 680 685 Gly Asp Gly Thr
690 3 432 PRT Homo sapiens 3 Leu Glu Arg Leu Val Gly Pro Gln Asp
Ala Thr His Cys Ser Pro Gly 1 5 10 15 Leu Ser Cys Arg Leu Trp Asp
Ser Asp Ile Leu Cys Leu Pro Gly Asp 20 25 30 Ile Val Pro Ala Pro
Gly Pro Val Leu Ala Pro Thr His Leu Gln Thr 35 40 45 Glu Leu Val
Leu Arg Cys Gln Lys Glu Thr Asp Cys Asp Leu Cys Leu 50 55 60 Arg
Val Ala Val His Leu Ala Val His Gly His Trp Glu Glu Pro Glu 65 70
75 80 Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly Val Glu Glu
Pro 85 90 95 Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser Phe
Gln Ala Tyr 100 105 110 Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln
Val Pro Ala Ala Leu 115 120 125 Val Gln Phe Gly Gln Ser Val Gly Ser
Val Val Tyr Asp Cys Phe Glu 130 135 140 Ala Ala Leu Gly Ser Glu Val
Arg Ile Trp Ser Tyr Thr Gln Pro Arg 145 150 155 160 Tyr Glu Lys Glu
Leu Asn His Thr Gln Gln Leu Pro Ala Leu Pro Trp 165 170 175 Leu Asn
Val Ser Ala Asp Gly Asp Asn Val His Leu Val Leu Asn Val 180 185 190
Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp Asn Gln Val Gln 195
200 205 Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr Gly Pro Gln
Ile 210 215 220 Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys
Ile Gln Val 225 230 235 240 Trp Pro Leu Glu Pro Asp Ser Val Arg Thr
Asn Ile Cys Pro Phe Arg 245 250 255 Glu Asp Pro Arg Ala His Gln Asn
Leu Trp Gln Ala Ala Arg Leu Arg 260 265 270 Leu Leu Thr Leu Gln Ser
Trp Leu Leu Asp Ala Pro Cys Ser Leu Pro 275 280 285 Ala Glu Ala Ala
Leu Cys Trp Arg Ala Pro Gly Gly Asp Pro Cys Gln 290 295 300 Pro Leu
Val Pro Pro Leu Ser Trp Glu Asn Val Thr Val Asp Lys Val 305 310 315
320 Leu Glu Phe Pro Leu Leu Lys Gly His Pro Asn Leu Cys Val Gln Val
325 330 335 Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp Ala
Asp Ser 340 345 350 Leu Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu
Thr Arg Gly Pro 355 360 365 Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu
Pro Ser Gly Cys Thr Ser 370 375 380 Leu Pro Ser Lys Ala Ser Thr Arg
Ala Ala Arg Leu Gly Glu Tyr Leu 385 390 395 400 Leu Gln Asp Leu Gln
Ser Gly Gln Cys Leu Gln Leu Trp Asp Asp Asp 405 410 415 Leu Gly Ala
Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile His Lys Arg 420 425 430 4
1753 DNA Homo sapiens CDS (2)...(1726) 4 g gag gag cct agg aat gcc
tct ctc cag gcc caa gtc gtg ctc tcc ttc 49 Glu Glu Pro Arg Asn Ala
Ser Leu Gln Ala Gln Val Val Leu Ser Phe 1 5 10 15 cag gcc tac cct
act gcc cgc tgc gtc ctg ctg gag gtg caa gtg cct 97 Gln Ala Tyr Pro
Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val Pro 20 25 30 gct gcc
ctt gtg cag ttt ggt cag tct gtg ggc tct gtg gta tat gac 145 Ala Ala
Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr Asp 35 40 45
tgc ttc gag gct gcc cta ggg agt gag gta cga atc tgg tcc tat act 193
Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr Thr 50
55 60 cag ccc agg tac gag aag gaa ctc aac cac aca cag cag ctg cct
gcc 241 Gln Pro Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln Leu Pro
Ala 65 70 75 80 ctg ccc tgg ctc aac gtg tca gca gat ggt gac aac gtg
cat ctg gtt 289 Leu Pro Trp Leu Asn Val Ser Ala Asp Gly Asp Asn Val
His Leu Val
85 90 95 ctg aat gtc tct gag gag cag cac ttc ggc ctc tcc ctg tac
tgg aat 337 Leu Asn Val Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr
Trp Asn 100 105 110 cag gtc cag ggc ccc cca aaa ccc cgg tgg cac aaa
aac ctg act gga 385 Gln Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys
Asn Leu Thr Gly 115 120 125 ccg cag atc att acc ttg aac cac aca gac
ctg gtt ccc tgc ctc tgt 433 Pro Gln Ile Ile Thr Leu Asn His Thr Asp
Leu Val Pro Cys Leu Cys 130 135 140 att cag gtg tgg cct ctg gaa cct
gac tcc gtt agg acg aac atc tgc 481 Ile Gln Val Trp Pro Leu Glu Pro
Asp Ser Val Arg Thr Asn Ile Cys 145 150 155 160 ccc ttc agg gag gac
ccc cgc gca cac cag aac ctc tgg caa gcc gcc 529 Pro Phe Arg Glu Asp
Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala 165 170 175 cga ctg cga
ctg ctg acc ctg cag agc tgg ctg ctg gac gca ccg tgc 577 Arg Leu Arg
Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro Cys 180 185 190 tcg
ctg ccc gca gaa gcg gca ctg tgc tgg cgg gct ccg ggt ggg gac 625 Ser
Leu Pro Ala Glu Ala Ala Leu Cys Trp Arg Ala Pro Gly Gly Asp 195 200
205 ccc tgc cag cca ctg gtc cca ccg ctt tcc tgg gag aac gtc act gtg
673 Pro Cys Gln Pro Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr Val
210 215 220 gac gtg aac agc tcg gag aag ctg cag ctg cag gag tgc ttg
tgg gct 721 Asp Val Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu
Trp Ala 225 230 235 240 gac tcc ctg ggg cct ctc aaa gac gat gtg cta
ctg ttg gag aca cga 769 Asp Ser Leu Gly Pro Leu Lys Asp Asp Val Leu
Leu Leu Glu Thr Arg 245 250 255 ggc ccc cag gac aac aga tcc ctc tgt
gcc ttg gaa ccc agt ggc tgt 817 Gly Pro Gln Asp Asn Arg Ser Leu Cys
Ala Leu Glu Pro Ser Gly Cys 260 265 270 act tca cta ccc agc aaa gcc
tcc acg agg gca gct cgc ctt gga gag 865 Thr Ser Leu Pro Ser Lys Ala
Ser Thr Arg Ala Ala Arg Leu Gly Glu 275 280 285 tac tta cta caa gac
ctg cag tca ggc cag tgt ctg cag cta tgg gac 913 Tyr Leu Leu Gln Asp
Leu Gln Ser Gly Gln Cys Leu Gln Leu Trp Asp 290 295 300 gat gac ttg
gga gcg cta tgg gcc tgc ccc atg gac aaa tac atc cac 961 Asp Asp Leu
Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile His 305 310 315 320
aag cgc tgg gcc ctc gtg tgg ctg gcc tgc cta ctc ttt gcc gct gcg
1009 Lys Arg Trp Ala Leu Val Trp Leu Ala Cys Leu Leu Phe Ala Ala
Ala 325 330 335 ctt tcc ctc atc ctc ctt ctc aaa aag gat cac gcg aaa
ggg tgg ctg 1057 Leu Ser Leu Ile Leu Leu Leu Lys Lys Asp His Ala
Lys Gly Trp Leu 340 345 350 agg ctc ttg aaa cag gac gtc cgc tcg ggg
gcg gcc gcc agg ggc cgc 1105 Arg Leu Leu Lys Gln Asp Val Arg Ser
Gly Ala Ala Ala Arg Gly Arg 355 360 365 gcg gct ctg ctc ctc tac tca
gcc gat gac tcg ggt ttc gag cgc ctg 1153 Ala Ala Leu Leu Leu Tyr
Ser Ala Asp Asp Ser Gly Phe Glu Arg Leu 370 375 380 gtg ggc gcc ctg
gcg tcg gcc ctg tgc cag ctg ccg ctg cgc gtg gcc 1201 Val Gly Ala
Leu Ala Ser Ala Leu Cys Gln Leu Pro Leu Arg Val Ala 385 390 395 400
gta gac ctg tgg agc cgt cgt gaa ctg agc gcg cag ggg ccc gtg gct
1249 Val Asp Leu Trp Ser Arg Arg Glu Leu Ser Ala Gln Gly Pro Val
Ala 405 410 415 tgg ttt cac gcg cag cgg cgc cag acc ctg cag gag ggc
ggc gtg gtg 1297 Trp Phe His Ala Gln Arg Arg Gln Thr Leu Gln Glu
Gly Gly Val Val 420 425 430 gtc ttg ctc ttc tct ccc ggt gcg gtg gcg
ctg tgc agc gag tgg cta 1345 Val Leu Leu Phe Ser Pro Gly Ala Val
Ala Leu Cys Ser Glu Trp Leu 435 440 445 cag gat ggg gtg tcc ggg ccc
ggg gcg cac ggc ccg cac gac gcc ttc 1393 Gln Asp Gly Val Ser Gly
Pro Gly Ala His Gly Pro His Asp Ala Phe 450 455 460 cgc gcc tcg ctc
agc tgc gtg ctg ccc gac ttc ttg cag ggc cgg gcg 1441 Arg Ala Ser
Leu Ser Cys Val Leu Pro Asp Phe Leu Gln Gly Arg Ala 465 470 475 480
ccc ggc agc tac gtg ggg gcc tgc ttc gac agg ctg ctc cac ccg gac
1489 Pro Gly Ser Tyr Val Gly Ala Cys Phe Asp Arg Leu Leu His Pro
Asp 485 490 495 gcc gta ccc gcc ctt ttc cgc acc gtg ccc gtc ttc aca
ctg ccc tcc 1537 Ala Val Pro Ala Leu Phe Arg Thr Val Pro Val Phe
Thr Leu Pro Ser 500 505 510 caa ctg cca gac ttc ctg ggg gcc ctg cag
cag cct cgc gcc ccg cgt 1585 Gln Leu Pro Asp Phe Leu Gly Ala Leu
Gln Gln Pro Arg Ala Pro Arg 515 520 525 tcc ggg cgg ctc caa gag aga
gcg gag caa gtg tcc cgg gcc ctt cag 1633 Ser Gly Arg Leu Gln Glu
Arg Ala Glu Gln Val Ser Arg Ala Leu Gln 530 535 540 cca gcc ctg gat
agc tac ttc cat ccc ccg ggg act ccc gcg ccg gga 1681 Pro Ala Leu
Asp Ser Tyr Phe His Pro Pro Gly Thr Pro Ala Pro Gly 545 550 555 560
cgc ggg gtg gga cca ggg gcg gga cct ggg gcg ggg gac ggg act 1726
Arg Gly Val Gly Pro Gly Ala Gly Pro Gly Ala Gly Asp Gly Thr 565 570
575 taaataaagg cagacgctgt ttttcta 1753 5 575 PRT Homo sapiens 5 Glu
Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser Phe 1 5 10
15 Gln Ala Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val Pro
20 25 30 Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val
Tyr Asp 35 40 45 Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile
Trp Ser Tyr Thr 50 55 60 Gln Pro Arg Tyr Glu Lys Glu Leu Asn His
Thr Gln Gln Leu Pro Ala 65 70 75 80 Leu Pro Trp Leu Asn Val Ser Ala
Asp Gly Asp Asn Val His Leu Val 85 90 95 Leu Asn Val Ser Glu Glu
Gln His Phe Gly Leu Ser Leu Tyr Trp Asn 100 105 110 Gln Val Gln Gly
Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr Gly 115 120 125 Pro Gln
Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys 130 135 140
Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys 145
150 155 160 Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln
Ala Ala 165 170 175 Arg Leu Arg Leu Leu Thr Leu Gln Ser Trp Leu Leu
Asp Ala Pro Cys 180 185 190 Ser Leu Pro Ala Glu Ala Ala Leu Cys Trp
Arg Ala Pro Gly Gly Asp 195 200 205 Pro Cys Gln Pro Leu Val Pro Pro
Leu Ser Trp Glu Asn Val Thr Val 210 215 220 Asp Val Asn Ser Ser Glu
Lys Leu Gln Leu Gln Glu Cys Leu Trp Ala 225 230 235 240 Asp Ser Leu
Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr Arg 245 250 255 Gly
Pro Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly Cys 260 265
270 Thr Ser Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu
275 280 285 Tyr Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln Leu
Trp Asp 290 295 300 Asp Asp Leu Gly Ala Leu Trp Ala Cys Pro Met Asp
Lys Tyr Ile His 305 310 315 320 Lys Arg Trp Ala Leu Val Trp Leu Ala
Cys Leu Leu Phe Ala Ala Ala 325 330 335 Leu Ser Leu Ile Leu Leu Leu
Lys Lys Asp His Ala Lys Gly Trp Leu 340 345 350 Arg Leu Leu Lys Gln
Asp Val Arg Ser Gly Ala Ala Ala Arg Gly Arg 355 360 365 Ala Ala Leu
Leu Leu Tyr Ser Ala Asp Asp Ser Gly Phe Glu Arg Leu 370 375 380 Val
Gly Ala Leu Ala Ser Ala Leu Cys Gln Leu Pro Leu Arg Val Ala 385 390
395 400 Val Asp Leu Trp Ser Arg Arg Glu Leu Ser Ala Gln Gly Pro Val
Ala 405 410 415 Trp Phe His Ala Gln Arg Arg Gln Thr Leu Gln Glu Gly
Gly Val Val 420 425 430 Val Leu Leu Phe Ser Pro Gly Ala Val Ala Leu
Cys Ser Glu Trp Leu 435 440 445 Gln Asp Gly Val Ser Gly Pro Gly Ala
His Gly Pro His Asp Ala Phe 450 455 460 Arg Ala Ser Leu Ser Cys Val
Leu Pro Asp Phe Leu Gln Gly Arg Ala 465 470 475 480 Pro Gly Ser Tyr
Val Gly Ala Cys Phe Asp Arg Leu Leu His Pro Asp 485 490 495 Ala Val
Pro Ala Leu Phe Arg Thr Val Pro Val Phe Thr Leu Pro Ser 500 505 510
Gln Leu Pro Asp Phe Leu Gly Ala Leu Gln Gln Pro Arg Ala Pro Arg 515
520 525 Ser Gly Arg Leu Gln Glu Arg Ala Glu Gln Val Ser Arg Ala Leu
Gln 530 535 540 Pro Ala Leu Asp Ser Tyr Phe His Pro Pro Gly Thr Pro
Ala Pro Gly 545 550 555 560 Arg Gly Val Gly Pro Gly Ala Gly Pro Gly
Ala Gly Asp Gly Thr 565 570 575 6 1725 DNA Artificial Sequence
degenerate sequence misc_feature (1)...(1725) n = A,T,C or G 6
gargarccnm gnaaygcnws nytncargcn cargtngtny tnwsnttyca rgcntayccn
60 acngcnmgnt gygtnytnyt ngargtncar gtnccngcng cnytngtnca
rttyggncar 120 wsngtnggnw sngtngtnta ygaytgytty gargcngcny
tnggnwsnga rgtnmgnath 180 tggwsntaya cncarccnmg ntaygaraar
garytnaayc ayacncarca rytnccngcn 240 ytnccntggy tnaaygtnws
ngcngayggn gayaaygtnc ayytngtnyt naaygtnwsn 300 gargarcarc
ayttyggnyt nwsnytntay tggaaycarg tncarggncc nccnaarccn 360
mgntggcaya araayytnac nggnccncar athathacny tnaaycayac ngayytngtn
420 ccntgyytnt gyathcargt ntggccnytn garccngayw sngtnmgnac
naayathtgy 480 ccnttymgng argayccnmg ngcncaycar aayytntggc
argcngcnmg nytnmgnytn 540 ytnacnytnc arwsntggyt nytngaygcn
ccntgywsny tnccngcnga rgcngcnytn 600 tgytggmgng cnccnggngg
ngayccntgy carccnytng tnccnccnyt nwsntgggar 660 aaygtnacng
tngaygtnaa ywsnwsngar aarytncary tncargartg yytntgggcn 720
gaywsnytng gnccnytnaa rgaygaygtn ytnytnytng aracnmgngg nccncargay
780 aaymgnwsny tntgygcnyt ngarccnwsn ggntgyacnw snytnccnws
naargcnwsn 840 acnmgngcng cnmgnytngg ngartayytn ytncargayy
tncarwsngg ncartgyytn 900 carytntggg aygaygayyt nggngcnytn
tgggcntgyc cnatggayaa rtayathcay 960 aarmgntggg cnytngtntg
gytngcntgy ytnytnttyg cngcngcnyt nwsnytnath 1020 ytnytnytna
araargayca ygcnaarggn tggytnmgny tnytnaarca rgaygtnmgn 1080
wsnggngcng cngcnmgngg nmgngcngcn ytnytnytnt aywsngcnga ygaywsnggn
1140 ttygarmgny tngtnggngc nytngcnwsn gcnytntgyc arytnccnyt
nmgngtngcn 1200 gtngayytnt ggwsnmgnmg ngarytnwsn gcncarggnc
cngtngcntg gttycaygcn 1260 carmgnmgnc aracnytnca rgarggnggn
gtngtngtny tnytnttyws nccnggngcn 1320 gtngcnytnt gywsngartg
gytncargay ggngtnwsng gnccnggngc ncayggnccn 1380 caygaygcnt
tymgngcnws nytnwsntgy gtnytnccng ayttyytnca rggnmgngcn 1440
ccnggnwsnt aygtnggngc ntgyttygay mgnytnytnc ayccngaygc ngtnccngcn
1500 ytnttymgna cngtnccngt nttyacnytn ccnwsncary tnccngaytt
yytnggngcn 1560 ytncarcarc cnmgngcncc nmgnwsnggn mgnytncarg
armgngcnga rcargtnwsn 1620 mgngcnytnc arccngcnyt ngaywsntay
ttycayccnc cnggnacncc ngcnccnggn 1680 mgnggngtng gnccnggngc
nggnccnggn gcnggngayg gnacn 1725 7 2076 DNA Artificial Sequence
degenerate sequence misc_feature (1)...(2076) n = A,T,C or G 7
atgccngtnc cntggttyyt nytnwsnytn gcnytnggnm gnwsnccngt ngtnytnwsn
60 ytngarmgny tngtnggncc ncargaygcn acncaytgyw snccnggnyt
nwsntgymgn 120 ytntgggayw sngayathyt ntgyytnccn ggngayathg
tnccngcncc nggnccngtn 180 ytngcnccna cncayytnca racngarytn
gtnytnmgnt gycaraarga racngaytgy 240 gayytntgyy tnmgngtngc
ngtncayytn gcngtncayg gncaytggga rgarccngar 300 gaygargara
arttyggngg ngcngcngay wsnggngtng argarccnmg naaygcnwsn 360
ytncargcnc argtngtnyt nwsnttycar gcntayccna cngcnmgntg ygtnytnytn
420 gargtncarg tnccngcngc nytngtncar ttyggncarw sngtnggnws
ngtngtntay 480 gaytgyttyg argcngcnyt nggnwsngar gtnmgnatht
ggwsntayac ncarccnmgn 540 taygaraarg arytnaayca yacncarcar
ytnccngcny tnccntggyt naaygtnwsn 600 gcngayggng ayaaygtnca
yytngtnytn aaygtnwsng argarcarca yttyggnytn 660 wsnytntayt
ggaaycargt ncarggnccn ccnaarccnm gntggcayaa raayytnacn 720
ggnccncara thathacnyt naaycayacn gayytngtnc cntgyytntg yathcargtn
780 tggccnytng arccngayws ngtnmgnacn aayathtgyc cnttymgnga
rgayccnmgn 840 gcncaycara ayytntggca rgcngcnmgn ytnmgnytny
tnacnytnca rwsntggytn 900 ytngaygcnc cntgywsnyt nccngcngar
gcngcnytnt gytggmgngc nccnggnggn 960 gayccntgyc arccnytngt
nccnccnytn wsntgggara aygtnacngt ngayaargtn 1020 ytngarttyc
cnytnytnaa rggncayccn aayytntgyg tncargtnaa ywsnwsngar 1080
aarytncary tncargartg yytntgggcn gaywsnytng gnccnytnaa rgaygaygtn
1140 ytnytnytng aracnmgngg nccncargay aaymgnwsny tntgygcnyt
ngarccnwsn 1200 ggntgyacnw snytnccnws naargcnwsn acnmgngcng
cnmgnytngg ngartayytn 1260 ytncargayy tncarwsngg ncartgyytn
carytntggg aygaygayyt nggngcnytn 1320 tgggcntgyc cnatggayaa
rtayathcay aarmgntggg cnytngtntg gytngcntgy 1380 ytnytnttyg
cngcngcnyt nwsnytnath ytnytnytna araargayca ygcnaargcn 1440
gcngcnmgng gnmgngcngc nytnytnytn taywsngcng aygaywsngg nttygarmgn
1500 ytngtnggng cnytngcnws ngcnytntgy carytnccny tnmgngtngc
ngtngayytn 1560 tggwsnmgnm gngarytnws ngcncarggn ccngtngcnt
ggttycaygc ncarmgnmgn 1620 caracnytnc argarggngg ngtngtngtn
ytnytnttyw snccnggngc ngtngcnytn 1680 tgywsngart ggytncarga
yggngtnwsn ggnccnggng cncayggncc ncaygaygcn 1740 ttymgngcnw
snytnwsntg ygtnytnccn gayttyytnc arggnmgngc nccnggnwsn 1800
taygtnggng cntgyttyga ymgnytnytn cayccngayg cngtnccngc nytnttymgn
1860 acngtnccng tnttyacnyt nccnwsncar ytnccngayt tyytnggngc
nytncarcar 1920 ccnmgngcnc cnmgnwsngg nmgnytncar garmgngcng
arcargtnws nmgngcnytn 1980 carccngcny tngaywsnta yttycayccn
ccnggnacnc cngcnccngg nmgnggngtn 2040 ggnccnggng cnggnccngg
ngcnggngay ggnacn 2076 8 21 DNA Artificial Sequence PCR primer 8
cggcgtggtg gtcttgctct t 21 9 16 PRT Artificial Sequence peptide
linker 9 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser 1 5 10 15 10 688 PRT Artificial Sequence Chimeric Zcytor14
protein 10 Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg
Ser Pro 1 5 10 15 Val Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln
Asp Ala Thr His 20 25 30 Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp
Asp Ser Asp Ile Leu Cys 35 40 45 Leu Pro Gly Asp Ile Val Pro Ala
Pro Gly Pro Val Leu Ala Pro Thr 50 55 60 His Leu Gln Thr Glu Leu
Val Leu Arg Cys Gln Lys Glu Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu
Arg Val Ala Val His Leu Ala Val His Gly His Trp 85 90 95 Glu Glu
Pro Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly 100 105 110
Val Glu Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser 115
120 125 Phe Gln Ala Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln
Val 130 135 140 Pro Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser
Val Val Tyr 145 150 155 160 Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu
Val Arg Ile Trp Ser Tyr 165 170 175 Thr Gln Pro Arg Tyr Glu Lys Glu
Leu Asn His Thr Gln Gln Leu Pro 180 185 190 Ala Leu Pro Trp Leu Asn
Val Ser Ala Asp Gly Asp Asn Val His Leu 195 200 205 Val Leu Asn Val
Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp 210 215 220 Asn Gln
Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr 225 230 235
240 Gly Pro Gln Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu
245 250 255 Cys Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr
Asn Ile 260 265 270 Cys Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn
Leu Trp Gln Ala 275 280 285 Ala Arg Leu Arg Leu Leu Thr Leu Gln Ser
Trp Leu Leu Asp Ala Pro 290 295 300 Cys Ser Leu Pro Ala Glu Ala Ala
Leu Cys Trp Arg Ala Pro Gly Gly 305 310 315 320 Asp Pro Cys Gln Pro
Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr 325 330 335 Val Asp Val
Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp 340 345 350 Ala
Asp Ser Leu Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr 355 360
365 Arg Gly Pro Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly
370 375 380 Cys Thr Ser Leu Pro
Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly 385 390 395 400 Glu Tyr
Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln Leu Trp 405 410 415
Asp Asp Asp Leu Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile 420
425 430 His Lys Arg Trp Ala Leu Val Trp Leu Ala Cys Leu Leu Phe Ala
Ala 435 440 445 Ala Leu Ser Leu Ile Leu Leu Leu Lys Lys Asp His Ala
Lys Gly Trp 450 455 460 Leu Arg Leu Leu Lys Gln Asp Val Arg Ser Gly
Ala Ala Ala Arg Gly 465 470 475 480 Arg Ala Ala Leu Leu Leu Tyr Ser
Ala Asp Asp Ser Gly Phe Glu Arg 485 490 495 Leu Val Gly Ala Leu Ala
Ser Ala Leu Cys Gln Leu Pro Leu Arg Val 500 505 510 Ala Val Asp Leu
Trp Ser Arg Arg Glu Leu Ser Ala Gln Gly Pro Val 515 520 525 Ala Trp
Phe His Ala Gln Arg Arg Gln Thr Leu Gln Glu Gly Gly Val 530 535 540
Val Val Leu Leu Phe Ser Pro Gly Ala Val Ala Leu Cys Ser Glu Trp 545
550 555 560 Leu Gln Asp Gly Val Ser Gly Pro Gly Ala His Gly Pro His
Asp Ala 565 570 575 Phe Arg Ala Ser Leu Ser Cys Val Leu Pro Asp Phe
Leu Gln Gly Arg 580 585 590 Ala Pro Gly Ser Tyr Val Gly Ala Cys Phe
Asp Arg Leu Leu His Pro 595 600 605 Asp Ala Val Pro Ala Leu Phe Arg
Thr Val Pro Val Phe Thr Leu Pro 610 615 620 Ser Gln Leu Pro Asp Phe
Leu Gly Ala Leu Gln Gln Pro Arg Ala Pro 625 630 635 640 Arg Ser Gly
Arg Leu Gln Glu Arg Ala Glu Gln Val Ser Arg Ala Leu 645 650 655 Gln
Pro Ala Leu Asp Ser Tyr Phe His Pro Pro Gly Thr Pro Ala Pro 660 665
670 Gly Arg Gly Val Gly Pro Gly Ala Gly Pro Gly Ala Gly Asp Gly Thr
675 680 685 11 705 PRT Artificial Sequence Chimeric Zcytor14
protein 11 Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg
Ser Pro 1 5 10 15 Val Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln
Asp Ala Thr His 20 25 30 Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp
Asp Ser Asp Ile Leu Cys 35 40 45 Leu Pro Gly Asp Ile Val Pro Ala
Pro Gly Pro Val Leu Ala Pro Thr 50 55 60 His Leu Gln Thr Glu Leu
Val Leu Arg Cys Gln Lys Glu Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu
Arg Val Ala Val His Leu Ala Val His Gly His Trp 85 90 95 Glu Glu
Pro Glu Asp Glu Glu Lys Phe Gly Gly Ala Ala Asp Ser Gly 100 105 110
Val Glu Glu Pro Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu Ser 115
120 125 Phe Gln Ala Tyr Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln
Val 130 135 140 Pro Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser
Val Val Tyr 145 150 155 160 Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu
Val Arg Ile Trp Ser Tyr 165 170 175 Thr Gln Pro Arg Tyr Glu Lys Glu
Leu Asn His Thr Gln Gln Leu Pro 180 185 190 Ala Leu Pro Trp Leu Asn
Val Ser Ala Asp Gly Asp Asn Val His Leu 195 200 205 Val Leu Asn Val
Ser Glu Glu Gln His Phe Gly Leu Ser Leu Tyr Trp 210 215 220 Asn Gln
Val Gln Gly Pro Pro Lys Pro Arg Trp His Lys Asn Leu Thr 225 230 235
240 Gly Pro Gln Ile Ile Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu
245 250 255 Cys Ile Gln Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr
Asn Ile 260 265 270 Cys Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn
Leu Trp Gln Ala 275 280 285 Ala Arg Leu Arg Leu Leu Thr Leu Gln Ser
Trp Leu Leu Asp Ala Pro 290 295 300 Cys Ser Leu Pro Ala Glu Ala Ala
Leu Cys Trp Arg Ala Pro Gly Gly 305 310 315 320 Asp Pro Cys Gln Pro
Leu Val Pro Pro Leu Ser Trp Glu Asn Val Thr 325 330 335 Val Asp Lys
Val Leu Glu Phe Pro Leu Leu Lys Gly His Pro Asn Leu 340 345 350 Cys
Val Gln Val Asn Ser Ser Glu Lys Leu Gln Leu Gln Glu Cys Leu 355 360
365 Trp Ala Asp Ser Leu Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu
370 375 380 Thr Arg Gly Pro Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu
Pro Ser 385 390 395 400 Gly Cys Thr Ser Leu Pro Ser Lys Ala Ser Thr
Arg Ala Ala Arg Leu 405 410 415 Gly Glu Tyr Leu Leu Gln Asp Leu Gln
Ser Gly Gln Cys Leu Gln Leu 420 425 430 Trp Asp Asp Asp Leu Gly Ala
Leu Trp Ala Cys Pro Met Asp Lys Tyr 435 440 445 Ile His Lys Arg Trp
Ala Leu Val Trp Leu Ala Cys Leu Leu Phe Ala 450 455 460 Ala Ala Leu
Ser Leu Ile Leu Leu Leu Lys Lys Asp His Ala Lys Gly 465 470 475 480
Trp Leu Arg Leu Leu Lys Gln Asp Val Arg Ser Gly Ala Ala Ala Arg 485
490 495 Gly Arg Ala Ala Leu Leu Leu Tyr Ser Ala Asp Asp Ser Gly Phe
Glu 500 505 510 Arg Leu Val Gly Ala Leu Ala Ser Ala Leu Cys Gln Leu
Pro Leu Arg 515 520 525 Val Ala Val Asp Leu Trp Ser Arg Arg Glu Leu
Ser Ala Gln Gly Pro 530 535 540 Val Ala Trp Phe His Ala Gln Arg Arg
Gln Thr Leu Gln Glu Gly Gly 545 550 555 560 Val Val Val Leu Leu Phe
Ser Pro Gly Ala Val Ala Leu Cys Ser Glu 565 570 575 Trp Leu Gln Asp
Gly Val Ser Gly Pro Gly Ala His Gly Pro His Asp 580 585 590 Ala Phe
Arg Ala Ser Leu Ser Cys Val Leu Pro Asp Phe Leu Gln Gly 595 600 605
Arg Ala Pro Gly Ser Tyr Val Gly Ala Cys Phe Asp Arg Leu Leu His 610
615 620 Pro Asp Ala Val Pro Ala Leu Phe Arg Thr Val Pro Val Phe Thr
Leu 625 630 635 640 Pro Ser Gln Leu Pro Asp Phe Leu Gly Ala Leu Gln
Gln Pro Arg Ala 645 650 655 Pro Arg Ser Gly Arg Leu Gln Glu Arg Ala
Glu Gln Val Ser Arg Ala 660 665 670 Leu Gln Pro Ala Leu Asp Ser Tyr
Phe His Pro Pro Gly Thr Pro Ala 675 680 685 Pro Gly Arg Gly Val Gly
Pro Gly Ala Gly Pro Gly Ala Gly Asp Gly 690 695 700 Thr 705 12 675
PRT Artificial Sequence Chimeric Zcytor14 protein 12 Met Pro Val
Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg Ser Pro 1 5 10 15 Val
Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His 20 25
30 Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys
35 40 45 Leu Pro Gly Asp Ile Val Pro Ala Pro Gly Pro Val Leu Ala
Pro Thr 50 55 60 His Leu Gln Thr Glu Leu Val Leu Arg Cys Gln Lys
Glu Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu Arg Val Ala Val His Leu
Ala Val His Gly His Trp 85 90 95 Glu Glu Pro Glu Asp Glu Glu Lys
Phe Gly Gly Ala Ala Asp Ser Gly 100 105 110 Val Glu Glu Pro Arg Asn
Ala Ser Leu Gln Ala Gln Val Val Leu Ser 115 120 125 Phe Gln Ala Tyr
Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val 130 135 140 Pro Ala
Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr 145 150 155
160 Asp Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr
165 170 175 Thr Gln Pro Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln
Leu Pro 180 185 190 Ala Leu Pro Trp Leu Asn Val Ser Ala Asp Gly Asp
Asn Val His Leu 195 200 205 Val Leu Asn Val Ser Glu Glu Gln His Phe
Gly Leu Ser Leu Tyr Trp 210 215 220 Asn Gln Val Gln Gly Pro Pro Lys
Pro Arg Trp His Lys Asn Leu Thr 225 230 235 240 Gly Pro Gln Ile Ile
Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu 245 250 255 Cys Ile Gln
Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile 260 265 270 Cys
Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala 275 280
285 Ala Arg Leu Arg Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro
290 295 300 Cys Ser Leu Pro Ala Glu Ala Ala Leu Cys Trp Arg Ala Pro
Gly Gly 305 310 315 320 Asp Pro Cys Gln Pro Leu Val Pro Pro Leu Ser
Trp Glu Asn Val Thr 325 330 335 Val Asp Val Asn Ser Ser Glu Lys Leu
Gln Leu Gln Glu Cys Leu Trp 340 345 350 Ala Asp Ser Leu Gly Pro Leu
Lys Asp Asp Val Leu Leu Leu Glu Thr 355 360 365 Arg Gly Pro Gln Asp
Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly 370 375 380 Cys Thr Ser
Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly 385 390 395 400
Glu Tyr Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln Leu Trp 405
410 415 Asp Asp Asp Leu Gly Ala Leu Trp Ala Cys Pro Met Asp Lys Tyr
Ile 420 425 430 His Lys Arg Trp Ala Leu Val Trp Leu Ala Cys Leu Leu
Phe Ala Ala 435 440 445 Ala Leu Ser Leu Ile Leu Leu Leu Lys Lys Asp
His Ala Lys Ala Ala 450 455 460 Ala Arg Gly Arg Ala Ala Leu Leu Leu
Tyr Ser Ala Asp Asp Ser Gly 465 470 475 480 Phe Glu Arg Leu Val Gly
Ala Leu Ala Ser Ala Leu Cys Gln Leu Pro 485 490 495 Leu Arg Val Ala
Val Asp Leu Trp Ser Arg Arg Glu Leu Ser Ala Gln 500 505 510 Gly Pro
Val Ala Trp Phe His Ala Gln Arg Arg Gln Thr Leu Gln Glu 515 520 525
Gly Gly Val Val Val Leu Leu Phe Ser Pro Gly Ala Val Ala Leu Cys 530
535 540 Ser Glu Trp Leu Gln Asp Gly Val Ser Gly Pro Gly Ala His Gly
Pro 545 550 555 560 His Asp Ala Phe Arg Ala Ser Leu Ser Cys Val Leu
Pro Asp Phe Leu 565 570 575 Gln Gly Arg Ala Pro Gly Ser Tyr Val Gly
Ala Cys Phe Asp Arg Leu 580 585 590 Leu His Pro Asp Ala Val Pro Ala
Leu Phe Arg Thr Val Pro Val Phe 595 600 605 Thr Leu Pro Ser Gln Leu
Pro Asp Phe Leu Gly Ala Leu Gln Gln Pro 610 615 620 Arg Ala Pro Arg
Ser Gly Arg Leu Gln Glu Arg Ala Glu Gln Val Ser 625 630 635 640 Arg
Ala Leu Gln Pro Ala Leu Asp Ser Tyr Phe His Pro Pro Gly Thr 645 650
655 Pro Ala Pro Gly Arg Gly Val Gly Pro Gly Ala Gly Pro Gly Ala Gly
660 665 670 Asp Gly Thr 675 13 1874 DNA Homo sapiens 13 gaattccggc
aggcacaaac tcatccatcc ccagttgatt ggaagaaaca acgatgactc 60
ctgggaagac ctcattggtg tcactgctac tgctgctgag cctggaggcc atagtgaagg
120 caggaatcac aatcccacga aatccaggat gcccaaattc tgaggacaag
aacttccccc 180 ggactgtgat ggtcaacctg aacatccata accggaatac
caataccaat cccaaaaggt 240 cctcagatta ctacaaccga tccacctcac
cttggaatct ccaccgcaat gaggaccctg 300 agagatatcc ctctgtgatc
tgggaggcaa agtgccgcca cttgggctgc atcaacgctg 360 atgggaacgt
ggactaccac atgaactctg tccccatcca gcaagagatc ctggtcctgc 420
gcagggagcc tccacactgc cccaactcct tccggctgga gaagatactg gtgtccgtgg
480 gctgcacctg tgtcaccccg attgtccacc atgtggccta agagctctgg
ggagcccaca 540 ctccccaaag cagttagact atggagagcc gacccagccc
ctcaggaacc ctcatccttc 600 aaagacagcc tcatttcgga ctaaactcat
tagagttctt aaggcagttt gtccaattaa 660 agcttcagag gtaacacttg
gccaagatat gagatctgaa ttacctttcc ctctttccaa 720 gaaggaaggt
ttgactgagt accaatttgc ttcttgttta cttttttaag ggctttaagt 780
tatttatgta tttaatatgc cctgagataa ctttggggta taagattcca ttttaatgaa
840 ttacctactt tattttgttt gtctttttaa agaagataag attctgggct
tgggaatttt 900 attatttaaa aggtaaaacc tgtatttatt tgagctattt
aaggatctat ttatgtttaa 960 gtatttagaa aaaggtgaaa aagcactatt
atcagttctg cctaggtaaa tgtaagatag 1020 aattaaatgg cagtgcaaaa
tttctgagtc tttacaacat acggatatag tatttcctcc 1080 tctttgtttt
taaaagttat aacatggctg aaaagaaaga ttaaacctac tttcatatgt 1140
attaatttaa attttgcaat ttgttgaggt tttacaagag atacagcaag tctaactctc
1200 tgttccatta aacccttata ataaaatcct tctgtaataa taaagtttca
aaagaaaatg 1260 tttatttgtt ctcattaaat gtattttagc aaactcagct
cttccctatt gggaagagtt 1320 atgcaaattc tcctataagc aaaacaaagc
atgtctttga gtaacaatga cctggaaata 1380 cccaaaattc caagttctcg
atttcacatg ccttcaagac tgaacaccga ctaaggtttt 1440 catactatta
gccaatgctg tagacagaag cattttgata ggaatagagc aaataagata 1500
atggccctga ggaatggcat gtcattatta aagatcatat ggggaaaatg aaaccctccc
1560 caaaatacaa gaagttctgg gaggagacat tgtcttcaga ctacaatgtc
cagtttctcc 1620 cctagactca ggcttccttt ggagattaag gcccctcaga
gatcaacaga ccaacatttt 1680 tctcttcctc aagcaacact cctagggcct
ggcttctgtc tgatcaaggc accacacaac 1740 ccagaaagga gctgatgggg
cagaatgaac tttaagtatg agaaaagttc agcccaagta 1800 aaataaaaac
tcaatcacat tcaattccag agtagtttca agtttcacat cgtaaccatt 1860
ttcgcccgga attc 1874 14 155 PRT Homo sapiens 14 Met Thr Pro Gly Lys
Thr Ser Leu Val Ser Leu Leu Leu Leu Leu Ser 1 5 10 15 Leu Glu Ala
Ile Val Lys Ala Gly Ile Thr Ile Pro Arg Asn Pro Gly 20 25 30 Cys
Pro Asn Ser Glu Asp Lys Asn Phe Pro Arg Thr Val Met Val Asn 35 40
45 Leu Asn Ile His Asn Arg Asn Thr Asn Thr Asn Pro Lys Arg Ser Ser
50 55 60 Asp Tyr Tyr Asn Arg Ser Thr Ser Pro Trp Asn Leu His Arg
Asn Glu 65 70 75 80 Asp Pro Glu Arg Tyr Pro Ser Val Ile Trp Glu Ala
Lys Cys Arg His 85 90 95 Leu Gly Cys Ile Asn Ala Asp Gly Asn Val
Asp Tyr His Met Asn Ser 100 105 110 Val Pro Ile Gln Gln Glu Ile Leu
Val Leu Arg Arg Glu Pro Pro His 115 120 125 Cys Pro Asn Ser Phe Arg
Leu Glu Lys Ile Leu Val Ser Val Gly Cys 130 135 140 Thr Cys Val Thr
Pro Ile Val His His Val Ala 145 150 155 15 923 DNA Homo sapiens 15
ggcttcagtt actagctagg ctactgagtt tagttctcag tttggcacct tgataccttt
60 aggtgtgagt gttcccattt ccaggtgagg aactgaggtg caaagagaag
ccctgatccc 120 ataaaaggac aggaatgctg agttccgcca gaccatgcat
ctcttgctag taggtgaggc 180 gagtctctaa ctgattgcag cgtcttctat
tttccaggtc aagtacttgc tgctgtcgat 240 attggggctt gcctttctga
gtgaggcggc agctcggaaa atccccaaag taggacatac 300 ttttttccaa
aagcctgaga gttgcccgcc tgtgccagga ggtagtatga agcttgacat 360
tggcatcatc aatgaaaacc agcgcgtttc catgtcacgt aacatcgaga gccgctccac
420 ctccccctgg aattacactg tcacttggga ccccaaccgg tacccctcgg
aagttgtaca 480 ggcccagtgt aggaacttgg gctgcatcaa tgctcaagga
aaggaagaca tctccatgaa 540 ttccgttccc atccagcaag agaccctggt
cgtccggagg aagcaccaag gctgctctgt 600 ttctttccag ttggagaagg
tgctggtgac tgttggctgc acctgcgtca cccctgtcat 660 ccaccatgtg
cagtaagagg tgcatatcca ctcagctgaa gaagctgtag aaatgccact 720
ccttacccag tgctctgcaa caagtcctgt ctgaccccca attccctcca cttcacagga
780 ctcttaataa gacctgcacg gatggaaaca taaaatattc acaatgtatg
tgtgtatgta 840 ctacacttta tatttgatat ctaaaatgtt aggagaaaaa
ttaatatatt cagtgctaat 900 ataataaagt attaataatg tta 923 16 153 PRT
Homo sapiens 16 Met Val Lys Tyr Leu Leu Leu Ser Ile Leu Gly Leu Ala
Phe Leu Ser 1 5 10 15 Glu Ala Ala Ala Arg Lys Ile Pro Lys Val Gly
His Thr Phe Phe Gln 20 25 30 Lys Pro Glu Ser Cys Pro Pro Val Pro
Gly Gly Ser Met Lys Leu Asp 35 40 45 Ile Gly Ile Ile Asn Glu Asn
Gln Arg Val Ser Met Ser Arg Asn Ile 50 55 60 Glu Ser Arg Ser Thr
Ser Pro Trp Asn Tyr Thr Val Thr Trp Asp Pro 65 70 75 80 Asn Arg Tyr
Pro Ser Glu Val Val Gln Ala Gln Cys Arg Asn Leu Gly 85 90 95 Cys
Ile Asn Ala Gln Gly Lys Glu Asp Ile Ser Met Asn Ser Val Pro 100 105
110 Ile Gln Gln
Glu Thr Leu Val Val Arg Arg Lys His Gln Gly Cys Ser 115 120 125 Val
Ser Phe Gln Leu Glu Lys Val Leu Val Thr Val Gly Cys Thr Cys 130 135
140 Val Thr Pro Val Ile His His Val Gln 145 150 17 1172 DNA Mus
musculus 17 gatccacctc acacgaggca caagtgcacc cagcaccagc tgatcaggac
gcgcaaacat 60 gagtccaggg agagcttcat ctgtgtctct gatgctgttg
ctgctgctga gcctggcggc 120 tacagtgaag gcagcagcga tcatccctca
aagctcagcg tgtccaaaca ctgaggccaa 180 ggacttcctc cagaatgtga
aggtcaacct caaagtcttt aactcccttg gcgcaaaagt 240 gagctccaga
aggccctcag actacctcaa ccgttccacg tcaccctgga ctctccaccg 300
caatgaagac cctgatagat atccctctgt gatctgggaa gctcagtgcc gccaccagcg
360 ctgtgtcaat gcggagggaa agctggacca ccacatgaat tctgttctca
tccagcaaga 420 gatcctggtc ctgaagaggg agcctgagag ctgccccttc
actttcaggg tcgagaagat 480 gctggtgggt gtgggctgca cctgcgtggc
ctcgattgtc cgccaggcag cctaaacaga 540 gacccgcggc tgacccctaa
gaaaccccca cgtttctcag caaacttact tgcattttta 600 aaacagttcg
tgctattgat tttcagcaag gaatgtggat tcagaggcag attcagaatt 660
gtctgccctc cacaatgaaa agaaggtgta aaggggtccc aaactgcttc gtgtttgttt
720 ttctgtggac tttaaattat ttgtgtattt acaatatccc aagatagctt
tgaagcgtaa 780 cttattttaa tgaagtatct acattattat tatgtttctt
tctgaagaag acaaaattca 840 agactcagaa attttattat ttaaaaggta
aagcctatat ttatatgagc tatttatgaa 900 tctatttatt tttcttcagt
atttgaagta ttaagaacat gattttcaga tctacctagg 960 gaagtcctaa
gtaagattaa atattaatgg aaatttcagc tttactattt gtttatttaa 1020
ggttctctcc tctgaatggg gtgaaaacca aacttagttt tatgtttaat aactttttaa
1080 attattgaag attcaaaaaa ttggataatt tagctcccta ctctgtttta
aaaaaaaatt 1140 gtaacaatat cactgtaata ataaagtttt gg 1172 18 158 PRT
Mus musculus 18 Met Ser Pro Gly Arg Ala Ser Ser Val Ser Leu Met Leu
Leu Leu Leu 1 5 10 15 Leu Ser Leu Ala Ala Thr Val Lys Ala Ala Ala
Ile Ile Pro Gln Ser 20 25 30 Ser Ala Cys Pro Asn Thr Glu Ala Lys
Asp Phe Leu Gln Asn Val Lys 35 40 45 Val Asn Leu Lys Val Phe Asn
Ser Leu Gly Ala Lys Val Ser Ser Arg 50 55 60 Arg Pro Ser Asp Tyr
Leu Asn Arg Ser Thr Ser Pro Trp Thr Leu His 65 70 75 80 Arg Asn Glu
Asp Pro Asp Arg Tyr Pro Ser Val Ile Trp Glu Ala Gln 85 90 95 Cys
Arg His Gln Arg Cys Val Asn Ala Glu Gly Lys Leu Asp His His 100 105
110 Met Asn Ser Val Leu Ile Gln Gln Glu Ile Leu Val Leu Lys Arg Glu
115 120 125 Pro Glu Ser Cys Pro Phe Thr Phe Arg Val Glu Lys Met Leu
Val Gly 130 135 140 Val Gly Cys Thr Cys Val Ala Ser Ile Val Arg Gln
Ala Ala 145 150 155 19 462 DNA Mus musculus 19 atggtcaagt
ctttgctact gttgatgttg ggacttgcca ttctgaggga ggtagcagct 60
cggaagaacc ccaaagcagg ggttcctgcc ttgcagaagg ctgggaactg tcctcccctg
120 gaggataaca ctgtgagagt tgacattcga atcttcaacc aaaaccaggg
catttctgtc 180 ccacgtgaat tccagaaccg ctccagttcc ccatgggatt
acaacatcac tcgagacccc 240 caccggttcc cctcagagat cgctgaggcc
cagtgcagac actcaggctg catcaatgcc 300 cagggtcagg aagacagcac
catgaactcc gtcgccattc agcaagaaat cctggtcctt 360 cggagggagc
cccagggctg ttctaattcc ttcaggttgg agaagatgct cctaaaagtt 420
ggctgcacct gtgtcaagcc cattgtccac caagcggcct ga 462 20 153 PRT Mus
musculus 20 Met Val Lys Ser Leu Leu Leu Leu Met Leu Gly Leu Ala Ile
Leu Arg 1 5 10 15 Glu Val Ala Ala Arg Lys Asn Pro Lys Ala Gly Val
Pro Ala Leu Gln 20 25 30 Lys Ala Gly Asn Cys Pro Pro Leu Glu Asp
Asn Thr Val Arg Val Asp 35 40 45 Ile Arg Ile Phe Asn Gln Asn Gln
Gly Ile Ser Val Pro Arg Glu Phe 50 55 60 Gln Asn Arg Ser Ser Ser
Pro Trp Asp Tyr Asn Ile Thr Arg Asp Pro 65 70 75 80 His Arg Phe Pro
Ser Glu Ile Ala Glu Ala Gln Cys Arg His Ser Gly 85 90 95 Cys Ile
Asn Ala Gln Gly Gln Glu Asp Ser Thr Met Asn Ser Val Ala 100 105 110
Ile Gln Gln Glu Ile Leu Val Leu Arg Arg Glu Pro Gln Gly Cys Ser 115
120 125 Asn Ser Phe Arg Leu Glu Lys Met Leu Leu Lys Val Gly Cys Thr
Cys 130 135 140 Val Lys Pro Ile Val His Gln Ala Ala 145 150 21 320
PRT Homo sapien 21 Met Gly Ala Ala Arg Ser Pro Pro Ser Ala Val Pro
Gly Pro Leu Leu 1 5 10 15 Gly Leu Leu Leu Leu Leu Leu Gly Val Leu
Ala Pro Gly Gly Ala Ser 20 25 30 Leu Arg Leu Leu Asp His Arg Ala
Leu Val Cys Ser Gln Pro Gly Leu 35 40 45 Asn Cys Thr Val Lys Asn
Ser Thr Cys Leu Asp Asp Ser Trp Ile His 50 55 60 Pro Arg Asn Leu
Thr Pro Ser Ser Pro Lys Asp Leu Gln Ile Gln Leu 65 70 75 80 His Phe
Ala His Thr Gln Gln Gly Asp Leu Phe Pro Val Ala His Ile 85 90 95
Glu Trp Thr Leu Gln Thr Asp Ala Ser Ile Leu Tyr Leu Glu Gly Ala 100
105 110 Glu Leu Ser Val Leu Gln Leu Asn Thr Asn Glu Arg Leu Cys Val
Arg 115 120 125 Phe Glu Phe Leu Ser Lys Leu Arg His His His Arg Arg
Trp Arg Phe 130 135 140 Thr Phe Ser His Phe Val Val Asp Pro Asp Gln
Glu Tyr Glu Val Thr 145 150 155 160 Val His His Leu Pro Lys Pro Ile
Pro Asp Gly Asp Pro Asn His Gln 165 170 175 Ser Lys Asn Phe Leu Val
Pro Asp Cys Glu His Ala Arg Met Lys Val 180 185 190 Thr Thr Pro Cys
Met Ser Ser Gly Ser Leu Trp Asp Pro Asn Ile Thr 195 200 205 Val Glu
Thr Leu Glu Ala His Gln Leu Arg Val Ser Phe Thr Leu Trp 210 215 220
Asn Glu Ser Thr His Tyr Gln Ile Leu Leu Thr Ser Phe Pro His Met 225
230 235 240 Glu Asn His Ser Cys Phe Glu His Met His His Ile Pro Ala
Pro Arg 245 250 255 Pro Glu Glu Phe His Gln Arg Ser Asn Val Thr Leu
Thr Leu Arg Asn 260 265 270 Leu Lys Gly Cys Cys Arg His Gln Val Gln
Ile Gln Pro Phe Phe Ser 275 280 285 Ser Cys Leu Asn Asp Cys Leu Arg
His Ser Ala Thr Val Ser Cys Pro 290 295 300 Glu Met Pro Asp Thr Pro
Glu Pro Ile Pro Asp Tyr Met Pro Leu Trp 305 310 315 320 22 221 PRT
Homo sapien 22 Lys Pro Arg Trp His Lys Asn Leu Thr Gly Pro Gln Ile
Ile Thr Leu 1 5 10 15 Asn His Thr Asp Leu Val Pro Cys Leu Cys Ile
Gln Val Trp Pro Leu 20 25 30 Glu Pro Asp Ser Val Arg Thr Asn Ile
Cys Pro Phe Arg Glu Asp Pro 35 40 45 Arg Ala His Gln Asn Leu Trp
Gln Ala Ala Arg Leu Arg Leu Leu Thr 50 55 60 Leu Gln Ser Trp Leu
Leu Asp Ala Pro Cys Ser Leu Pro Ala Glu Ala 65 70 75 80 Ala Leu Cys
Trp Arg Ala Pro Gly Gly Asp Pro Cys Gln Pro Leu Val 85 90 95 Pro
Pro Leu Ser Trp Glu Asn Val Thr Val Asp Lys Val Leu Glu Phe 100 105
110 Pro Leu Leu Lys Gly His Pro Asn Leu Cys Val Gln Val Asn Ser Ser
115 120 125 Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp Ala Asp Ser Leu
Gly Pro 130 135 140 Leu Lys Asp Asp Val Leu Leu Leu Glu Thr Arg Gly
Pro Gln Asp Asn 145 150 155 160 Arg Ser Leu Cys Ala Leu Glu Pro Ser
Gly Cys Thr Ser Leu Pro Ser 165 170 175 Lys Ala Ser Thr Arg Ala Ala
Arg Leu Gly Glu Tyr Leu Leu Gln Asp 180 185 190 Leu Gln Ser Gly Gln
Cys Leu Gln Leu Trp Asp Asp Asp Leu Gly Ala 195 200 205 Leu Trp Ala
Cys Pro Met Asp Lys Tyr Ile His Lys Arg 210 215 220 23 2180 DNA
Homo sapien 23 aactacccag cacagccccc tccgccccct ctggaggctg
aagagggatt ccagcccctg 60 ccacccacag acacgggctg actggggtgt
ctgcccccct tggggggggg cagcacaggg 120 cctcaggcct gggtgccacc
tggcacctag aagatgcctg tgccctggtt cttgctgtcc 180 ttggcactgg
gccgaagccc agtggtcctt tctctggaga ggcttgtggg gcctcaggac 240
gctacccact gctctccggg cctctcctgc cgcctctggg acagtgacat actctgcctg
300 cctggggaca tcgtgcctgc tccgggcccc gtgctggcgc ctacgcacct
gcagacagag 360 ctggtgctga ggtgccagaa ggagaccgac tgtgacctct
gtctgcgtgt ggctgtccac 420 ttggccgtgc atgcctctct ccaggcccaa
gtcgtgctct ccttccaggc ctaccctact 480 gcccgctgcg tcctgctgga
ggtgcaagtg cctgctgccc ttgtgcagtt tggtcagtct 540 gtgggctctg
tggtatatga ctgcttcgag gctgccctag ggagtgaggt acgaatctgg 600
tcctatactc agcccaggta cgagaaggaa ctcaaccaca cacagcagct gcctgccctg
660 ccctggctca acgtgtcagc agatggtgac aacgtgcatc tggttctgaa
tgtctctgag 720 gagcagcact tcggcctctc cctgtactgg aatcaggtcc
agggcccccc aaaaccccgg 780 tggcacaaaa acctgactgg accgcagatc
attaccttga accacacaga cctggttccc 840 tgcctctgta ttcaggtgtg
gcctctggaa cctgactccg ttaggacgaa catctgcccc 900 ttcagggagg
acccccgcgc acaccagaac ctctggcaag ccgcccgact gcgactgctg 960
accctgcaga gctggctgct ggacgcaccg tgctcgctgc ccgcagaagc ggcactgtgc
1020 tggcgggctc cgggtgggga cccctgccag ccactggtcc caccgctttc
ctgggagaac 1080 gtcactgtgg acaaggttct cgagttccca ttgctgaaag
gccaccctaa cctctgtgtt 1140 caggtgaaca gctcggagaa gctgcagctg
caggagtgct tgtgggctga ctccctgggg 1200 cctctcaaag acgatgtgct
actgttggag acacgaggcc cccaggacaa cagatccctc 1260 tgtgccttgg
aacccagtgg ctgtacttca ctacccagca aagcctccac gagggcagct 1320
cgccttggag agtacttact acaagacctg cagtcaggcc agtgtctgca gctatgggac
1380 gatgacttgg gagcgctatg ggcctgcccc atggacaaat acatccacaa
gcgctgggcc 1440 ctcgtgtggc tggcctgcct actctttgcc gctgcgcttt
ccctcatcct ccttctcaaa 1500 aaggatcacg cgaaagcggc cgccaggggc
cgcgcggctc tgctcctcta ctcagccgat 1560 gactcgggtt tcgagcgcct
ggtgggcgcc ctggcgtcgg ccctgtgcca gctgccgctg 1620 cgcgtggccg
tagacctgtg gagccgtcgt gaactgagcg cgcaggggcc cgtggcttgg 1680
tttcacgcgc agcggcgcca gaccctgcag gagggcggcg tggtggtctt gctcttctct
1740 cccggtgcgg tggcgctgtg cagcgagtgg ctacaggatg gggtgtccgg
gcccggggcg 1800 cacggcccgc acgacgcctt ccgcgcctcg ctcagctgcg
tgctgcccga cttcttgcag 1860 ggccgggcgc ccggcagcta cgtgggggcc
tgcttcgaca ggctgctcca cccggacgcc 1920 gtacccgccc ttttccgcac
cgtgcccgtc ttcacactgc cctcccaact gccagacttc 1980 ctgggggccc
tgcagcagcc tcgcgccccg cgttccgggc ggctccaaga gagagcggag 2040
caagtgtccc gggcccttca gccagccctg gatagctact tccatccccc ggggactccc
2100 gcgccgggac gcggggtggg accaggggcg ggacctgggg cgggggacgg
gacttaaata 2160 aaggcagacg ctgtttttct 2180 24 667 PRT Homo sapien
24 Met Pro Val Pro Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg Ser Pro
1 5 10 15 Val Val Leu Ser Leu Glu Arg Leu Val Gly Pro Gln Asp Ala
Thr His 20 25 30 Cys Ser Pro Gly Leu Ser Cys Arg Leu Trp Asp Ser
Asp Ile Leu Cys 35 40 45 Leu Pro Gly Asp Ile Val Pro Ala Pro Gly
Pro Val Leu Ala Pro Thr 50 55 60 His Leu Gln Thr Glu Leu Val Leu
Arg Cys Gln Lys Glu Thr Asp Cys 65 70 75 80 Asp Leu Cys Leu Arg Val
Ala Val His Leu Ala Val His Ala Ser Leu 85 90 95 Gln Ala Gln Val
Val Leu Ser Phe Gln Ala Tyr Pro Thr Ala Arg Cys 100 105 110 Val Leu
Leu Glu Val Gln Val Pro Ala Ala Leu Val Gln Phe Gly Gln 115 120 125
Ser Val Gly Ser Val Val Tyr Asp Cys Phe Glu Ala Ala Leu Gly Ser 130
135 140 Glu Val Arg Ile Trp Ser Tyr Thr Gln Pro Arg Tyr Glu Lys Glu
Leu 145 150 155 160 Asn His Thr Gln Gln Leu Pro Ala Leu Pro Trp Leu
Asn Val Ser Ala 165 170 175 Asp Gly Asp Asn Val His Leu Val Leu Asn
Val Ser Glu Glu Gln His 180 185 190 Phe Gly Leu Ser Leu Tyr Trp Asn
Gln Val Gln Gly Pro Pro Lys Pro 195 200 205 Arg Trp His Lys Asn Leu
Thr Gly Pro Gln Ile Ile Thr Leu Asn His 210 215 220 Thr Asp Leu Val
Pro Cys Leu Cys Ile Gln Val Trp Pro Leu Glu Pro 225 230 235 240 Asp
Ser Val Arg Thr Asn Ile Cys Pro Phe Arg Glu Asp Pro Arg Ala 245 250
255 His Gln Asn Leu Trp Gln Ala Ala Arg Leu Arg Leu Leu Thr Leu Gln
260 265 270 Ser Trp Leu Leu Asp Ala Pro Cys Ser Leu Pro Ala Glu Ala
Ala Leu 275 280 285 Cys Trp Arg Ala Pro Gly Gly Asp Pro Cys Gln Pro
Leu Val Pro Pro 290 295 300 Leu Ser Trp Glu Asn Val Thr Val Asp Lys
Val Leu Glu Phe Pro Leu 305 310 315 320 Leu Lys Gly His Pro Asn Leu
Cys Val Gln Val Asn Ser Ser Glu Lys 325 330 335 Leu Gln Leu Gln Glu
Cys Leu Trp Ala Asp Ser Leu Gly Pro Leu Lys 340 345 350 Asp Asp Val
Leu Leu Leu Glu Thr Arg Gly Pro Gln Asp Asn Arg Ser 355 360 365 Leu
Cys Ala Leu Glu Pro Ser Gly Cys Thr Ser Leu Pro Ser Lys Ala 370 375
380 Ser Thr Arg Ala Ala Arg Leu Gly Glu Tyr Leu Leu Gln Asp Leu Gln
385 390 395 400 Ser Gly Gln Cys Leu Gln Leu Trp Asp Asp Asp Leu Gly
Ala Leu Trp 405 410 415 Ala Cys Pro Met Asp Lys Tyr Ile His Lys Arg
Trp Ala Leu Val Trp 420 425 430 Leu Ala Cys Leu Leu Phe Ala Ala Ala
Leu Ser Leu Ile Leu Leu Leu 435 440 445 Lys Lys Asp His Ala Lys Ala
Ala Ala Arg Gly Arg Ala Ala Leu Leu 450 455 460 Leu Tyr Ser Ala Asp
Asp Ser Gly Phe Glu Arg Leu Val Gly Ala Leu 465 470 475 480 Ala Ser
Ala Leu Cys Gln Leu Pro Leu Arg Val Ala Val Asp Leu Trp 485 490 495
Ser Arg Arg Glu Leu Ser Ala Gln Gly Pro Val Ala Trp Phe His Ala 500
505 510 Gln Arg Arg Gln Thr Leu Gln Glu Gly Gly Val Val Val Leu Leu
Phe 515 520 525 Ser Pro Gly Ala Val Ala Leu Cys Ser Glu Trp Leu Gln
Asp Gly Val 530 535 540 Ser Gly Pro Gly Ala His Gly Pro His Asp Ala
Phe Arg Ala Ser Leu 545 550 555 560 Ser Cys Val Leu Pro Asp Phe Leu
Gln Gly Arg Ala Pro Gly Ser Tyr 565 570 575 Val Gly Ala Cys Phe Asp
Arg Leu Leu His Pro Asp Ala Val Pro Ala 580 585 590 Leu Phe Arg Thr
Val Pro Val Phe Thr Leu Pro Ser Gln Leu Pro Asp 595 600 605 Phe Leu
Gly Ala Leu Gln Gln Pro Arg Ala Pro Arg Ser Gly Arg Leu 610 615 620
Gln Glu Arg Ala Glu Gln Val Ser Arg Ala Leu Gln Pro Ala Leu Asp 625
630 635 640 Ser Tyr Phe His Pro Pro Gly Thr Pro Ala Pro Gly Arg Gly
Val Gly 645 650 655 Pro Gly Ala Gly Pro Gly Ala Gly Asp Gly Thr 660
665 25 2269 DNA Mus musculus 25 aaatcgaaag cactccagct gaaactgggc
ctggagtcca ggctcactgg agtggggaag 60 catggctgga gaggaattct
agcccttgct ctctcccagg gacacggggc tgattgtcag 120 caggggcgag
gggtctgccc ccccttgggg gggcaggacg gggcctcagg cctgggtgct 180
gtccggcacc tggaagatgc ctgtgtcctg gttcctgctg tccttggcac tgggccgaaa
240 ccctgtggtc gtctctctgg agagactgat ggagcctcag gacactgcac
gctgctctct 300 aggcctctcc tgccacctct gggatggtga cgtgctctgc
ctgcctggaa gcctccagtc 360 tgccccaggc cctgtgctag tgcctacccg
cctgcagacg gagctggtgc tgaggtgtcc 420 acagaagaca gattgcgccc
tctgtgtccg tgtggtggtc cacttggccg tgcatgggca 480 ctgggcagag
cctgaagaag ctggaaagtc tgattcagaa ctccaggagt ctaggaacgc 540
ctctctccag gcccaggtgg tgctctcctt ccaggcctac cccatcgccc gctgtgccct
600 gctggaggtc caggtgcccg ctgacctggt gcagcctggt cagtccgtgg
gttctgcggt 660 atttgactgt ttcgaggcta gtcttggggc tgaggtacag
atctggtcct acacgaagcc 720 caggtaccag aaagagctca acctcacaca
gcagctgcct gtcctgccct ggctcaatgt 780 gtctacagat ggtgacaatg
tccttctgac actggatgtc tctgaggagc aggactttag 840 cttcttactg
tacctgcgtc cagtcccgga tgctctcaaa tccttgtggt acaaaaacct 900
gactggacct cagaacatta ctttaaacca cacagacctg gttccctgcc tctgcattca
960 ggtgtggtcg ctagagccag actctgagag ggtcgaattc tgccccttcc
gggaagatcc 1020 cggtgcacac aggaacctct ggcacatagc caggctgcgg
gtactgtccc caggggtatg 1080 gcagctagat gcgccttgct gtctgccggg
caaggtaaca ctgtgctggc aggcaccaga 1140 ccagagtccc tgccagccac
ttgtgccacc agtgccccag aagaacgcca ctgtgaatga 1200 gccacaagat
ttccagttgg tggcaggcca ccccaacctc tgtgtccagg tgagcacctg 1260
ggagaaggtt cagctgcaag cgtgcttgtg ggctgactcc ttggggccct tcaaggatga
1320 tatgctgtta gtggagatga aaaccggcct caacaacaca tcagtctgtg
ccttggaacc 1380 cagtggctgt acaccactgc ccagcatggc ctccacgaga
gctgctcgcc tgggagagga 1440 gttgctgcaa gacttccgat cacaccagtg
tatgcagctg tggaacgatg acaacatggg 1500 atcgctatgg gcctgcccca
tggacaagta catccacagg cgctgggtcc tagtatggct 1560 ggcctgccta
ctcttggctg cggcgctttt cttcttcctc cttctaaaaa aggaccgcag 1620
gaaagcggcc cgtggctccc gcacggcctt gctcctccac tccgccgacg gagcgggcta
1680 cgagcgtctg gtgggagcac tggcgtccgc gttgagccag atgccactgc
gcgtggccgt 1740 ggacctgtgg agccgccgcg agctgagcgc gcacggagcc
ctagcctggt tccaccacca 1800 gcgacgccgt atcctgcagg agggtggcgt
ggtaatcctt ctcttctcgc ccgcggccgt 1860 ggcgcagtgt cagcagtggc
tgcagctcca gacagtggag cccgggccgc atgacgccct 1920 cgccgcctgg
ctcagctgcg tgctacccga tttcctgcaa ggccgggcga ccggccgcta 1980
cgtcggggtc tacttcgacg ggctgctgca cccagactct gtgccctccc cgttccgcgt
2040 cgccccgctc ttctccctgc cctcgcagct gccggctttc ctggatgcac
tgcagggagg 2100 ctgctccact tccgcggggc gacccgcgga ccgggtggaa
cgagtgaccc aggcgctgcg 2160 gtccgccctg gacagctgta cttctagctc
ggaagcccca ggctgctgcg aggaatggga 2220 cctgggaccc tgcactacac
tagaataaaa gccgatacag tattcctaa 2269 26 683 PRT Mus musculus 26 Met
Pro Val Ser Trp Phe Leu Leu Ser Leu Ala Leu Gly Arg Asn Pro 1 5 10
15 Val Val Val Ser Leu Glu Arg Leu Met Glu Pro Gln Asp Thr Ala Arg
20 25 30 Cys Ser Leu Gly Leu Ser Cys His Leu Trp Asp Gly Asp Val
Leu Cys 35 40 45 Leu Pro Gly Ser Leu Gln Ser Ala Pro Gly Pro Val
Leu Val Pro Thr 50 55 60 Arg Leu Gln Thr Glu Leu Val Leu Arg Cys
Pro Gln Lys Thr Asp Cys 65 70 75 80 Ala Leu Cys Val Arg Val Val Val
His Leu Ala Val His Gly His Trp 85 90 95 Ala Glu Pro Glu Glu Ala
Gly Lys Ser Asp Ser Glu Leu Gln Glu Ser 100 105 110 Arg Asn Ala Ser
Leu Gln Ala Gln Val Val Leu Ser Phe Gln Ala Tyr 115 120 125 Pro Ile
Ala Arg Cys Ala Leu Leu Glu Val Gln Val Pro Ala Asp Leu 130 135 140
Val Gln Pro Gly Gln Ser Val Gly Ser Ala Val Phe Asp Cys Phe Glu 145
150 155 160 Ala Ser Leu Gly Ala Glu Val Gln Ile Trp Ser Tyr Thr Lys
Pro Arg 165 170 175 Tyr Gln Lys Glu Leu Asn Leu Thr Gln Gln Leu Pro
Val Leu Pro Trp 180 185 190 Leu Asn Val Ser Thr Asp Gly Asp Asn Val
Leu Leu Thr Leu Asp Val 195 200 205 Ser Glu Glu Gln Asp Phe Ser Phe
Leu Leu Tyr Leu Arg Pro Val Pro 210 215 220 Asp Ala Leu Lys Ser Leu
Trp Tyr Lys Asn Leu Thr Gly Pro Gln Asn 225 230 235 240 Ile Thr Leu
Asn His Thr Asp Leu Val Pro Cys Leu Cys Ile Gln Val 245 250 255 Trp
Ser Leu Glu Pro Asp Ser Glu Arg Val Glu Phe Cys Pro Phe Arg 260 265
270 Glu Asp Pro Gly Ala His Arg Asn Leu Trp His Ile Ala Arg Leu Arg
275 280 285 Val Leu Ser Pro Gly Val Trp Gln Leu Asp Ala Pro Cys Cys
Leu Pro 290 295 300 Gly Lys Val Thr Leu Cys Trp Gln Ala Pro Asp Gln
Ser Pro Cys Gln 305 310 315 320 Pro Leu Val Pro Pro Val Pro Gln Lys
Asn Ala Thr Val Asn Glu Pro 325 330 335 Gln Asp Phe Gln Leu Val Ala
Gly His Pro Asn Leu Cys Val Gln Val 340 345 350 Ser Thr Trp Glu Lys
Val Gln Leu Gln Ala Cys Leu Trp Ala Asp Ser 355 360 365 Leu Gly Pro
Phe Lys Asp Asp Met Leu Leu Val Glu Met Lys Thr Gly 370 375 380 Leu
Asn Asn Thr Ser Val Cys Ala Leu Glu Pro Ser Gly Cys Thr Pro 385 390
395 400 Leu Pro Ser Met Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu Glu
Leu 405 410 415 Leu Gln Asp Phe Arg Ser His Gln Cys Met Gln Leu Trp
Asn Asp Asp 420 425 430 Asn Met Gly Ser Leu Trp Ala Cys Pro Met Asp
Lys Tyr Ile His Arg 435 440 445 Arg Trp Val Leu Val Trp Leu Ala Cys
Leu Leu Leu Ala Ala Ala Leu 450 455 460 Phe Phe Phe Leu Leu Leu Lys
Lys Asp Arg Arg Lys Ala Ala Arg Gly 465 470 475 480 Ser Arg Thr Ala
Leu Leu Leu His Ser Ala Asp Gly Ala Gly Tyr Glu 485 490 495 Arg Leu
Val Gly Ala Leu Ala Ser Ala Leu Ser Gln Met Pro Leu Arg 500 505 510
Val Ala Val Asp Leu Trp Ser Arg Arg Glu Leu Ser Ala His Gly Ala 515
520 525 Leu Ala Trp Phe His His Gln Arg Arg Arg Ile Leu Gln Glu Gly
Gly 530 535 540 Val Val Ile Leu Leu Phe Ser Pro Ala Ala Val Ala Gln
Cys Gln Gln 545 550 555 560 Trp Leu Gln Leu Gln Thr Val Glu Pro Gly
Pro His Asp Ala Leu Ala 565 570 575 Ala Trp Leu Ser Cys Val Leu Pro
Asp Phe Leu Gln Gly Arg Ala Thr 580 585 590 Gly Arg Tyr Val Gly Val
Tyr Phe Asp Gly Leu Leu His Pro Asp Ser 595 600 605 Val Pro Ser Pro
Phe Arg Val Ala Pro Leu Phe Ser Leu Pro Ser Gln 610 615 620 Leu Pro
Ala Phe Leu Asp Ala Leu Gln Gly Gly Cys Ser Thr Ser Ala 625 630 635
640 Gly Arg Pro Ala Asp Arg Val Glu Arg Val Thr Gln Ala Leu Arg Ser
645 650 655 Ala Leu Asp Ser Cys Thr Ser Ser Ser Glu Ala Pro Gly Cys
Cys Glu 660 665 670 Glu Trp Asp Leu Gly Pro Cys Thr Thr Leu Glu 675
680 27 449 PRT Mus musculus 27 Met Pro Val Ser Trp Phe Leu Leu Ser
Leu Ala Leu Gly Arg Asn Pro 1 5 10 15 Val Val Val Ser Leu Glu Arg
Leu Met Glu Pro Gln Asp Thr Ala Arg 20 25 30 Cys Ser Leu Gly Leu
Ser Cys His Leu Trp Asp Gly Asp Val Leu Cys 35 40 45 Leu Pro Gly
Ser Leu Gln Ser Ala Pro Gly Pro Val Leu Val Pro Thr 50 55 60 Arg
Leu Gln Thr Glu Leu Val Leu Arg Cys Pro Gln Lys Thr Asp Cys 65 70
75 80 Ala Leu Cys Val Arg Val Val Val His Leu Ala Val His Gly His
Trp 85 90 95 Ala Glu Pro Glu Glu Ala Gly Lys Ser Asp Ser Glu Leu
Gln Glu Ser 100 105 110 Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu
Ser Phe Gln Ala Tyr 115 120 125 Pro Ile Ala Arg Cys Ala Leu Leu Glu
Val Gln Val Pro Ala Asp Leu 130 135 140 Val Gln Pro Gly Gln Ser Val
Gly Ser Ala Val Phe Asp Cys Phe Glu 145 150 155 160 Ala Ser Leu Gly
Ala Glu Val Gln Ile Trp Ser Tyr Thr Lys Pro Arg 165 170 175 Tyr Gln
Lys Glu Leu Asn Leu Thr Gln Gln Leu Pro Val Leu Pro Trp 180 185 190
Leu Asn Val Ser Thr Asp Gly Asp Asn Val Leu Leu Thr Leu Asp Val 195
200 205 Ser Glu Glu Gln Asp Phe Ser Phe Leu Leu Tyr Leu Arg Pro Val
Pro 210 215 220 Asp Ala Leu Lys Ser Leu Trp Tyr Lys Asn Leu Thr Gly
Pro Gln Asn 225 230 235 240 Ile Thr Leu Asn His Thr Asp Leu Val Pro
Cys Leu Cys Ile Gln Val 245 250 255 Trp Ser Leu Glu Pro Asp Ser Glu
Arg Val Glu Phe Cys Pro Phe Arg 260 265 270 Glu Asp Pro Gly Ala His
Arg Asn Leu Trp His Ile Ala Arg Leu Arg 275 280 285 Val Leu Ser Pro
Gly Val Trp Gln Leu Asp Ala Pro Cys Cys Leu Pro 290 295 300 Gly Lys
Val Thr Leu Cys Trp Gln Ala Pro Asp Gln Ser Pro Cys Gln 305 310 315
320 Pro Leu Val Pro Pro Val Pro Gln Lys Asn Ala Thr Val Asn Glu Pro
325 330 335 Gln Asp Phe Gln Leu Val Ala Gly His Pro Asn Leu Cys Val
Gln Val 340 345 350 Ser Thr Trp Glu Lys Val Gln Leu Gln Ala Cys Leu
Trp Ala Asp Ser 355 360 365 Leu Gly Pro Phe Lys Asp Asp Met Leu Leu
Val Glu Met Lys Thr Gly 370 375 380 Leu Asn Asn Thr Ser Val Cys Ala
Leu Glu Pro Ser Gly Cys Thr Pro 385 390 395 400 Leu Pro Ser Met Ala
Ser Thr Arg Ala Ala Arg Leu Gly Glu Glu Leu 405 410 415 Leu Gln Asp
Phe Arg Ser His Gln Cys Met Gln Leu Trp Asn Asp Asp 420 425 430 Asn
Met Gly Ser Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile His Arg 435 440
445 Arg 28 222 PRT Mus musculus 28 Lys Ser Leu Trp Tyr Lys Asn Leu
Thr Gly Pro Gln Asn Ile Thr Leu 1 5 10 15 Asn His Thr Asp Leu Val
Pro Cys Leu Cys Ile Gln Val Trp Ser Leu 20 25 30 Glu Pro Asp Ser
Glu Arg Val Glu Phe Cys Pro Phe Arg Glu Asp Pro 35 40 45 Gly Ala
His Arg Asn Leu Trp His Ile Ala Arg Leu Arg Val Leu Ser 50 55 60
Pro Gly Val Trp Gln Leu Asp Ala Pro Cys Cys Leu Pro Gly Lys Val 65
70 75 80 Thr Leu Cys Trp Gln Ala Pro Asp Gln Ser Pro Cys Gln Pro
Leu Val 85 90 95 Pro Pro Val Pro Gln Lys Asn Ala Thr Val Asn Glu
Pro Gln Asp Phe 100 105 110 Gln Leu Val Ala Gly His Pro Asn Leu Cys
Val Gln Val Ser Thr Trp 115 120 125 Glu Lys Val Gln Leu Gln Ala Cys
Leu Trp Ala Asp Ser Leu Gly Pro 130 135 140 Phe Lys Asp Asp Met Leu
Leu Val Glu Met Lys Thr Gly Leu Asn Asn 145 150 155 160 Thr Ser Val
Cys Ala Leu Glu Pro Ser Gly Cys Thr Pro Leu Pro Ser 165 170 175 Met
Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu Glu Leu Leu Gln Asp 180 185
190 Phe Arg Ser His Gln Cys Met Gln Leu Trp Asn Asp Asp Asn Met Gly
195 200 205 Ser Leu Trp Ala Cys Pro Met Asp Lys Tyr Ile His Arg Arg
210 215 220 29 2287 DNA Mus musculus 29 aaatcgaaag cactccagct
gaaactgggc ctggagtcca ggctcactgg agtggggaag 60 catggctgga
gaggaattct agcccttgct ctctcccagg gacacggggc tgattgtcag 120
caggggcgag gggtctgccc ccccttgggg gggcaggacg gggcctcagg cctgggtgct
180 gtccggcacc tggaagatgc ctgtgtcctg gttcctgctg tccttggcac
tgggccgaaa 240 ccctgtggtc gtctctctgg agagactgat ggagcctcag
gacactgcac gctgctctct 300 aggcctctcc tgccacctct gggatggtga
cgtgctctgc ctgcctggaa gcctccagtc 360 tgccccaggc cctgtgctag
tgcctacccg cctgcagacg gagctggtgc tgaggtgtcc 420 acagaagaca
gattgcgccc tctgtgtccg tgtggtggtc cacttggccg tgcatgggca 480
ctgggcagag cctgaagaag ctggaaagtc tgattcagaa ctccaggagt ctaggaacgc
540 ctctctccag gcccaggtgg tgctctcctt ccaggcctac cccatcgccc
gctgtgccct 600 gctggaggtc caggtgcccg ctgacctggt gcagcctggt
cagtccgtgg gttctgcggt 660 atttgactgt ttcgaggcta gtcttggggc
tgaggtacag atctggtcct acacgaagcc 720 caggtaccag aaagagctca
acctcacaca gcagctgcct gactgcaggg gtcttgaagt 780 ccgggacagc
atccagagct gctgggatgg tgacaatgtc cttctgacac tggatgtctc 840
tgaggagcag gactttagct tcttactgta cctgcgtcca gtcccggatg ctctcaaatc
900 cttgtggtac aaaaacctga ctggacctca gaacattact ttaaaccaca
cagacctggt 960 tccctgcctc tgcattcagg tgtggtcgct agagccagac
tctgagaggg tcgaattctg 1020 ccccttccgg gaagatcccg gtgcacacag
gaacctctgg cacatagcca ggctgcgggt 1080 actgtcccca ggggtatggc
agctagatgc gccttgctgt ctgccgggca aggtaacact 1140 gtgctggcag
gcaccagacc agagtccctg ccagccactt gtgccaccag tgccccagaa 1200
gaacgccact gtgaatgagc cacaagattt ccagttggtg gcaggccacc ccaacctctg
1260 tgtccaggtg agcacctggg agaaggttca gctgcaagcg tgcttgtggg
ctgactcctt 1320 ggggcccttc aaggatgata tgctgttagt ggagatgaaa
accggcctca acaacacatc 1380 agtctgtgcc ttggaaccca gtggctgtac
accactgccc agcatggcct ccacgagagc 1440 tgctcgcctg ggagaggagt
tgctgcaaga cttccgatca caccagtgta tgcagctgtg 1500 gaacgatgac
aacatgggat cgctatgggc ctgccccatg gacaagtaca tccacaggcg 1560
ctgggtccta gtatggctgg cctgcctact cttggctgcg gcgcttttct tcttcctcct
1620 tctaaaaaag gaccgcagga aagcggcccg tggctcccgc acggccttgc
tcctccactc 1680 cgccgacgga gcgggctacg agcgtctggt gggagcactg
gcgtccgcgt tgagccagat 1740 gccactgcgc gtggccgtgg acctgtggag
ccgccgcgag ctgagcgcgc acggagccct 1800 agcctggttc caccaccagc
gacgccgtat cctgcaggag ggtggcgtgg taatccttct 1860 cttctcgccc
gcggccgtgg cgcagtgtca gcagtggctg cagctccaga cagtggagcc 1920
cgggccgcat gacgccctcg ccgcctggct cagctgcgtg ctacccgatt tcctgcaagg
1980 ccgggcgacc ggccgctacg tcggggtcta cttcgacggg ctgctgcacc
cagactctgt 2040 gccctccccg ttccgcgtcg ccccgctctt ctccctgccc
tcgcagctgc cggctttcct 2100 ggatgcactg cagggaggct gctccacttc
cgcggggcga cccgcggacc gggtggaacg 2160 agtgacccag gcgctgcggt
ccgccctgga cagctgtact tctagctcgg aagccccagg 2220 ctgctgcgag
gaatgggacc tgggaccctg cactacacta gaataaaagc cgatacagta 2280 ttcctaa
2287 30 689 PRT Mus musculus 30 Met Pro Val Ser Trp Phe Leu Leu Ser
Leu Ala Leu Gly Arg Asn Pro 1 5 10 15 Val Val Val Ser Leu Glu Arg
Leu Met Glu Pro Gln Asp Thr Ala Arg 20 25 30 Cys Ser Leu Gly Leu
Ser Cys His Leu Trp Asp Gly Asp Val Leu Cys 35 40 45 Leu Pro Gly
Ser Leu Gln Ser Ala Pro Gly Pro Val Leu Val Pro Thr 50 55 60 Arg
Leu Gln Thr Glu Leu Val Leu Arg Cys Pro Gln Lys Thr Asp Cys 65 70
75 80 Ala Leu Cys Val Arg Val Val Val His Leu Ala Val His Gly His
Trp 85 90 95 Ala Glu Pro Glu Glu Ala Gly Lys Ser Asp Ser Glu Leu
Gln Glu Ser 100 105 110 Arg Asn Ala Ser Leu Gln Ala Gln Val Val Leu
Ser Phe Gln Ala Tyr 115 120 125 Pro Ile Ala Arg Cys Ala Leu Leu Glu
Val Gln Val Pro Ala Asp Leu 130 135 140 Val Gln Pro Gly Gln Ser Val
Gly Ser Ala Val Phe Asp Cys Phe Glu 145 150 155 160 Ala Ser Leu Gly
Ala Glu Val Gln Ile Trp Ser Tyr Thr Lys Pro Arg 165 170 175 Tyr Gln
Lys Glu Leu Asn Leu Thr Gln Gln Leu Pro Asp Cys Arg Gly 180 185 190
Leu Glu Val Arg Asp Ser Ile Gln Ser Cys Trp Asp Gly Asp Asn Val 195
200 205 Leu Leu Thr Leu Asp Val Ser Glu Glu Gln Asp Phe Ser Phe Leu
Leu 210 215 220 Tyr Leu Arg Pro Val Pro Asp Ala Leu Lys Ser Leu Trp
Tyr Lys Asn 225 230 235 240 Leu Thr Gly Pro Gln Asn Ile Thr Leu Asn
His Thr Asp Leu Val Pro 245 250 255 Cys Leu Cys Ile Gln Val Trp Ser
Leu Glu Pro Asp Ser Glu Arg Val 260 265 270 Glu Phe Cys Pro Phe Arg
Glu Asp Pro Gly Ala His Arg Asn Leu Trp 275 280 285 His Ile Ala Arg
Leu Arg Val Leu Ser Pro Gly Val Trp Gln Leu Asp 290 295 300 Ala Pro
Cys Cys Leu Pro Gly Lys Val Thr Leu Cys Trp Gln Ala Pro 305 310 315
320 Asp Gln Ser Pro Cys Gln Pro Leu Val Pro Pro Val Pro Gln Lys Asn
325 330 335 Ala Thr Val Asn Glu Pro Gln Asp Phe Gln Leu Val Ala Gly
His Pro 340 345 350 Asn Leu Cys Val Gln Val Ser Thr Trp Glu Lys Val
Gln Leu Gln Ala 355 360 365 Cys Leu Trp Ala Asp Ser Leu Gly Pro Phe
Lys Asp Asp Met Leu Leu 370 375 380 Val Glu Met Lys Thr Gly Leu Asn
Asn Thr Ser Val Cys Ala Leu Glu 385 390 395 400 Pro Ser Gly Cys Thr
Pro Leu Pro Ser Met Ala Ser Thr Arg Ala Ala 405 410 415 Arg Leu Gly
Glu Glu Leu Leu Gln Asp Phe Arg Ser His Gln Cys Met 420 425 430 Gln
Leu Trp Asn Asp Asp Asn Met Gly Ser Leu Trp Ala Cys Pro Met 435 440
445 Asp Lys Tyr Ile His Arg Arg Trp Val Leu Val Trp Leu Ala Cys Leu
450 455 460 Leu Leu Ala Ala Ala Leu Phe Phe Phe Leu Leu Leu Lys Lys
Asp Arg 465 470 475 480 Arg Lys Ala Ala Arg Gly Ser Arg Thr Ala Leu
Leu Leu His Ser Ala 485 490 495 Asp Gly Ala Gly Tyr Glu Arg Leu Val
Gly Ala Leu Ala Ser Ala Leu 500 505 510 Ser Gln Met Pro Leu Arg Val
Ala Val Asp Leu Trp Ser Arg Arg Glu 515 520 525 Leu Ser Ala His Gly
Ala Leu Ala Trp Phe His His Gln Arg Arg Arg 530 535 540 Ile Leu Gln
Glu Gly Gly Val Val Ile Leu Leu Phe
Ser Pro Ala Ala 545 550 555 560 Val Ala Gln Cys Gln Gln Trp Leu Gln
Leu Gln Thr Val Glu Pro Gly 565 570 575 Pro His Asp Ala Leu Ala Ala
Trp Leu Ser Cys Val Leu Pro Asp Phe 580 585 590 Leu Gln Gly Arg Ala
Thr Gly Arg Tyr Val Gly Val Tyr Phe Asp Gly 595 600 605 Leu Leu His
Pro Asp Ser Val Pro Ser Pro Phe Arg Val Ala Pro Leu 610 615 620 Phe
Ser Leu Pro Ser Gln Leu Pro Ala Phe Leu Asp Ala Leu Gln Gly 625 630
635 640 Gly Cys Ser Thr Ser Ala Gly Arg Pro Ala Asp Arg Val Glu Arg
Val 645 650 655 Thr Gln Ala Leu Arg Ser Ala Leu Asp Ser Cys Thr Ser
Ser Ser Glu 660 665 670 Ala Pro Gly Cys Cys Glu Glu Trp Asp Leu Gly
Pro Cys Thr Thr Leu 675 680 685 Glu 31 21 DNA Artificial Sequence
oligonucleotide primer 31 tcccgtcccc cgccccaggt c 21 32 25 DNA
Artificial Sequence oligonucleotide primer 32 ctctccatcc ttatctttca
tcaac 25 33 24 DNA Artificial Sequence oligonucleotide primer 33
ctctctgctg gctaaacaaa acac 24 34 26 DNA Artificial Sequence
oligonucleotide primer 34 ctcatattgc tcaactgtgt gaaaag 26 35 25 DNA
Artificial Sequence oligonucleotide primer 35 tagaagccac ctgaacacaa
atctg 25 36 28 DNA Artificial Sequence oligonucleotide primer 36
atcttgcgtt gtatgttgaa aatcaatt 28 37 25 DNA Artificial Sequence
oligonucleotide primer 37 ttctccacca ggtaaacaag tctac 25 38 24 DNA
Artificial Sequence oligonucleotide primer 38 ctctccaggc ccaagtcgtg
ctct 24 39 24 DNA Artificial Sequence oligonucleotide primer 39
ttgtcctggg ggcctcgtgt ctcc 24 40 24 DNA Artificial Sequence
oligonucleotide primer 40 acgaagccca ggtaccagaa agag 24 41 24 DNA
Artificial Sequence oligonucleotide primer 41 aaaagcgccg cagccaagag
tagg 24 42 1293 DNA Homo sapiens 42 ctggagaggc ttgtggggcc
tcaggacgct acccactgct ctccgggcct ctcctgccgc 60 ctctgggaca
gtgacatact ctgcctgcct ggggacatcg tgcctgctcc gggccccgtg 120
ctggcgccta cgcacctgca gacagagctg gtgctgaggt gccagaagga gaccgactgt
180 gacctctgtc tgcgtgtggc tgtccacttg gccgtgcatg ggcactggga
agagcctgaa 240 gatgaggaaa agtttggagg agcagctgac tcaggggtgg
aggagcctag gaatgcctct 300 ctccaggccc aagtcgtgct ctccttccag
gcctacccta ctgcccgctg cgtcctgctg 360 gaggtgcaag tgcctgctgc
ccttgtgcag tttggtcagt ctgtgggctc tgtggtatat 420 gactgcttcg
aggctgccct agggagtgag gtacgaatct ggtcctatac tcagcccagg 480
tacgagaagg aactcaacca cacacagcag ctgcctgccc tgccctggct caacgtgtca
540 gcagatggtg acaacgtgca tctggttctg aatgtctctg aggagcagca
cttcggcctc 600 tccctgtact ggaatcaggt ccagggcccc ccaaaacccc
ggtggcacaa aaacctgact 660 ggaccgcaga tcattacctt gaaccacaca
gacctggttc cctgcctctg tattcaggtg 720 tggcctctgg aacctgactc
cgttaggacg aacatctgcc ccttcaggga ggacccccgc 780 gcacaccaga
acctctggca agccgcccga ctgcgactgc tgaccctgca gagctggctg 840
ctggacgcac cgtgctcgct gcccgcagaa gcggcactgt gctggcgggc tccgggtggg
900 gacccctgcc agccactggt cccaccgctt tcctgggaga acgtcactgt
ggacaaggtt 960 ctcgagttcc cattgctgaa aggccaccct aacctctgtg
ttcaggtgaa cagctcggag 1020 aagctgcagc tgcaggagtg cttgtgggct
gactccctgg ggcctctcaa agacgatgtg 1080 ctactgttgg agacacgagg
cccccaggac aacagatccc tctgtgcctt ggaacccagt 1140 ggctgtactt
cactacccag caaagcctcc acgagggcag ctcgccttgg agagtactta 1200
ctacaagacc tgcagtcagg ccagtgtctg cagctatggg acgatgactt gggagcgcta
1260 tgggcctgcc ccatggacaa atacatccac aag 1293 43 431 PRT Homo
sapiens 43 Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His Cys Ser
Pro Gly 1 5 10 15 Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys
Leu Pro Gly Asp 20 25 30 Ile Val Pro Ala Pro Gly Pro Val Leu Ala
Pro Thr His Leu Gln Thr 35 40 45 Glu Leu Val Leu Arg Cys Gln Lys
Glu Thr Asp Cys Asp Leu Cys Leu 50 55 60 Arg Val Ala Val His Leu
Ala Val His Gly His Trp Glu Glu Pro Glu 65 70 75 80 Asp Glu Glu Lys
Phe Gly Gly Ala Ala Asp Ser Gly Val Glu Glu Pro 85 90 95 Arg Asn
Ala Ser Leu Gln Ala Gln Val Val Leu Ser Phe Gln Ala Tyr 100 105 110
Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val Pro Ala Ala Leu 115
120 125 Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr Asp Cys Phe
Glu 130 135 140 Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr Thr
Gln Pro Arg 145 150 155 160 Tyr Glu Lys Glu Leu Asn His Thr Gln Gln
Leu Pro Ala Leu Pro Trp 165 170 175 Leu Asn Val Ser Ala Asp Gly Asp
Asn Val His Leu Val Leu Asn Val 180 185 190 Ser Glu Glu Gln His Phe
Gly Leu Ser Leu Tyr Trp Asn Gln Val Gln 195 200 205 Gly Pro Pro Lys
Pro Arg Trp His Lys Asn Leu Thr Gly Pro Gln Ile 210 215 220 Ile Thr
Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys Ile Gln Val 225 230 235
240 Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys Pro Phe Arg
245 250 255 Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala Arg
Leu Arg 260 265 270 Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro
Cys Ser Leu Pro 275 280 285 Ala Glu Ala Ala Leu Cys Trp Arg Ala Pro
Gly Gly Asp Pro Cys Gln 290 295 300 Pro Leu Val Pro Pro Leu Ser Trp
Glu Asn Val Thr Val Asp Lys Val 305 310 315 320 Leu Glu Phe Pro Leu
Leu Lys Gly His Pro Asn Leu Cys Val Gln Val 325 330 335 Asn Ser Ser
Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp Ala Asp Ser 340 345 350 Leu
Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr Arg Gly Pro 355 360
365 Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly Cys Thr Ser
370 375 380 Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg Leu Gly Glu
Tyr Leu 385 390 395 400 Leu Gln Asp Leu Gln Ser Gly Gln Cys Leu Gln
Leu Trp Asp Asp Asp 405 410 415 Leu Gly Ala Leu Trp Ala Cys Pro Met
Asp Lys Tyr Ile His Lys 420 425 430 44 699 DNA Homo sapiens 44
gagcccagag ggcccacaat caagccctgt cctccatgca aatgcccagc acctaacctc
60 ttgggtggac catccgtctt catcttccct ccaaagatca aggatgtact
catgatctcc 120 ctgagcccca tagtcacatg tgtggtggtg gatgtgagcg
aggatgaccc agatgtccag 180 atcagctggt ttgtgaacaa cgtggaagta
cacacagctc agacacaaac ccatagagag 240 gattacaaca gtactctccg
ggtggtcagt gccctcccca tccagcacca ggactggatg 300 agtggcaagg
agttcaaatg caaggtcaac aacaaagacc tcccagcgcc catcgagaga 360
accatctcaa aacccaaagg gtcagtaaga gctccacagg tatatgtctt gcctccacca
420 gaagaagaga tgactaagaa acaggtcact ctgacctgca tggtcacaga
cttcatgcct 480 gaagacattt acgtggagtg gaccaacaac gggaaaacag
agctaaacta caagaacact 540 gaaccagtcc tggactctga tggttcttac
ttcatgtaca gcaagctgag agtggaaaag 600 aagaactggg tggaaagaaa
tagctactcc tgttcagtgg tccacgaggg tctgcacaat 660 caccacacga
ctaagagctt ctcccggact ccgggtaaa 699 45 233 PRT Homo sapiens 45 Glu
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro 1 5 10
15 Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
20 25 30 Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr
Cys Val 35 40 45 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln
Ile Ser Trp Phe 50 55 60 Val Asn Asn Val Glu Val His Thr Ala Gln
Thr Gln Thr His Arg Glu 65 70 75 80 Asp Tyr Asn Ser Thr Leu Arg Val
Val Ser Ala Leu Pro Ile Gln His 85 90 95 Gln Asp Trp Met Ser Gly
Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105 110 Asp Leu Pro Ala
Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser 115 120 125 Val Arg
Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130 135 140
Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro 145
150 155 160 Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu
Leu Asn 165 170 175 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly
Ser Tyr Phe Met 180 185 190 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn
Trp Val Glu Arg Asn Ser 195 200 205 Tyr Ser Cys Ser Val Val His Glu
Gly Leu His Asn His His Thr Thr 210 215 220 Lys Ser Phe Ser Arg Thr
Pro Gly Lys 225 230 46 69 DNA Artificial Sequence oligonucleotide
primer 46 gtttcgctca gccaggaaat ccatgccgag ttgagacgct tccgtagact
ggagaggctt 60 gtggggcct 69 47 36 DNA Artificial Sequence
oligonucleotide primer 47 tgtgggccct ctgggctcct tgtggatgta tttgtc
36 48 36 DNA Artificial Sequence oligonucleotide primer 48
gacaaataca tccacaagga gcccagaggg cccaca 36 49 55 DNA Artificial
Sequence oligonucleotide primer 49 caaccccaga gctgttttaa ggcgcgcctc
tagattattt acccggagtc cggga 55 50 76 DNA Artificial Sequence
oligonucleotide primer 50 caaccccaga gctgttttaa ggcgcgcctc
tagattattc catgggcatg tattcttcct 60 tgtggatgta tttgtc 76 51 10 PRT
Artificial Sequence C-terminal his tag 51 Gly Ser Gly Gly His His
His His His His 1 5 10 52 10 PRT Artificial Sequence C-terminal
FLAG tag 52 Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 10 53 7 PRT
Artificial Sequence Glu-Glu tag 53 Glu Glu Tyr Met Pro Met Glu 1 5
54 85 DNA Artificial Sequence oligonucleotide primer 54 caaccccaga
gctgttttaa ggcgcgcctc tagattagtg atggtgatgg tgatgtccac 60
cagatccctt gtggatgtat ttgtc 85 55 85 DNA Artificial Sequence
oligonucleotide primer 55 caaccccaga gctgttttaa ggcgcgcctc
tagattactt atcatcatca tccttataat 60 cggatccctt gtggatgtat ttgtc 85
56 24 DNA Artificial Sequence oligonucleotide primer 56 acgaagccca
ggtaccagaa agag 24 57 24 DNA Artificial Sequence oligonucleotide
primer 57 aaaagcgccg cagccaagag tagg 24 58 20 DNA Artificial
Sequence oligonucleotide primer 58 cgtaagcggt ggcggttttc 20 59 20
DNA Artificial Sequence oligonucleotide primer 59 tgggcagggc
acagtcacag 20 60 24 DNA Artificial Sequence oligonucleotide primer
60 acttgccatt ctgagggagg tagc 24 61 24 DNA Artificial Sequence
oligonucleotide primer 61 cacaggtgca gccaactttt agga 24 62 20 DNA
Artificial Sequence oligonucleotide primer 62 gtgggccgct ctaggcacca
20 63 25 DNA Artificial Sequence oligonucleotide primer 63
cggttggcct tagggttcag ggggg 25 64 2127 DNA Homo sapiens 64
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggcgc cgtcttcgtt
60 tcgctcagcc aggaaatcca tgccgagttg agacgcttcc gtagactgga
gaggcttgtg 120 gggcctcagg acgctaccca ctgctctccg ggcctctcct
gccgcctctg ggacagtgac 180 atactctgcc tgcctgggga catcgtgcct
gctccgggcc ccgtgctggc gcctacgcac 240 ctgcagacag agctggtgct
gaggtgccag aaggagaccg actgtgacct ctgtctgcgt 300 gtggctgtcc
acttggccgt gcatgggcac tgggaagagc ctgaagatga ggaaaagttt 360
ggaggagcag ctgactcagg ggtggaggag cctaggaatg cctctctcca ggcccaagtc
420 gtgctctcct tccaggccta ccctactgcc cgctgcgtcc tgctggaggt
gcaagtgcct 480 gctgcccttg tgcagtttgg tcagtctgtg ggctctgtgg
tatatgactg cttcgaggct 540 gccctaggga gtgaggtacg aatctggtcc
tatactcagc ccaggtacga gaaggaactc 600 aaccacacac agcagctgcc
tgccctgccc tggctcaacg tgtcagcaga tggtgacaac 660 gtgcatctgg
ttctgaatgt ctctgaggag cagcacttcg gcctctccct gtactggaat 720
caggtccagg gccccccaaa accccggtgg cacaaaaacc tgactggacc gcagatcatt
780 accttgaacc acacagacct ggttccctgc ctctgtattc aggtgtggcc
tctggaacct 840 gactccgtta ggacgaacat ctgccccttc agggaggacc
cccgcgcaca ccagaacctc 900 tggcaagccg cccgactgcg actgctgacc
ctgcagagct ggctgctgga cgcaccgtgc 960 tcgctgcccg cagaagcggc
actgtgctgg cgggctccgg gtggggaccc ctgccagcca 1020 ctggtcccac
cgctttcctg ggagaacgtc actgtggaca aggttctcga gttcccattg 1080
ctgaaaggcc accctaacct ctgtgttcag gtgaacagct cggagaagct gcagctgcag
1140 gagtgcttgt gggctgactc cctggggcct ctcaaagacg atgtgctact
gttggagaca 1200 cgaggccccc aggacaacag atccctctgt gccttggaac
ccagtggctg tacttcacta 1260 cccagcaaag cctccacgag ggcagctcgc
cttggagagt acttactaca agacctgcag 1320 tcaggccagt gtctgcagct
atgggacgat gacttgggag cgctatgggc ctgccccatg 1380 gacaaataca
tccacaaggg aggaagtggc ggaggaacag gaagtttggt ccctcgtgga 1440
agcgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca
1500 gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 1560 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 1620 gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 1680 taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 1740 aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1800
aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc
1860 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga
catcgccgtg 1920 gagtgggaga gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac 1980 tccgacggct ccttcttcct ctacagcaag
ctcaccgtgg acaagagcag gtggcagcag 2040 gggaacgtct tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacgcagaag 2100 agcctctccc
tgtctccggg taaataa 2127 65 708 PRT Homo sapiens 65 Met Asp Ala Met
Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val
Phe Val Ser Leu Ser Gln Glu Ile His Ala Glu Leu Arg Arg 20 25 30
Phe Arg Arg Leu Glu Arg Leu Val Gly Pro Gln Asp Ala Thr His Cys 35
40 45 Ser Pro Gly Leu Ser Cys Arg Leu Trp Asp Ser Asp Ile Leu Cys
Leu 50 55 60 Pro Gly Asp Ile Val Pro Ala Pro Gly Pro Val Leu Ala
Pro Thr His 65 70 75 80 Leu Gln Thr Glu Leu Val Leu Arg Cys Gln Lys
Glu Thr Asp Cys Asp 85 90 95 Leu Cys Leu Arg Val Ala Val His Leu
Ala Val His Gly His Trp Glu 100 105 110 Glu Pro Glu Asp Glu Glu Lys
Phe Gly Gly Ala Ala Asp Ser Gly Val 115 120 125 Glu Glu Pro Arg Asn
Ala Ser Leu Gln Ala Gln Val Val Leu Ser Phe 130 135 140 Gln Ala Tyr
Pro Thr Ala Arg Cys Val Leu Leu Glu Val Gln Val Pro 145 150 155 160
Ala Ala Leu Val Gln Phe Gly Gln Ser Val Gly Ser Val Val Tyr Asp 165
170 175 Cys Phe Glu Ala Ala Leu Gly Ser Glu Val Arg Ile Trp Ser Tyr
Thr 180 185 190 Gln Pro Arg Tyr Glu Lys Glu Leu Asn His Thr Gln Gln
Leu Pro Ala 195 200 205 Leu Pro Trp Leu Asn Val Ser Ala Asp Gly Asp
Asn Val His Leu Val 210 215 220 Leu Asn Val Ser Glu Glu Gln His Phe
Gly Leu Ser Leu Tyr Trp Asn 225 230 235 240 Gln Val Gln Gly Pro Pro
Lys Pro Arg Trp His Lys Asn Leu Thr Gly 245 250 255 Pro Gln Ile Ile
Thr Leu Asn His Thr Asp Leu Val Pro Cys Leu Cys 260 265 270 Ile Gln
Val Trp Pro Leu Glu Pro Asp Ser Val Arg Thr Asn Ile Cys 275 280 285
Pro Phe Arg Glu Asp Pro Arg Ala His Gln Asn Leu Trp Gln Ala Ala 290
295 300 Arg Leu Arg Leu Leu Thr Leu Gln Ser Trp Leu Leu Asp Ala Pro
Cys 305 310 315 320 Ser Leu Pro Ala Glu Ala Ala Leu Cys Trp Arg Ala
Pro Gly Gly Asp 325 330 335 Pro Cys Gln Pro Leu Val Pro Pro Leu Ser
Trp Glu Asn Val Thr Val 340 345 350 Asp Lys Val Leu Glu Phe Pro Leu
Leu Lys Gly His Pro Asn Leu Cys 355 360 365 Val Gln Val Asn Ser Ser
Glu Lys Leu Gln Leu Gln Glu Cys Leu Trp 370 375 380 Ala Asp
Ser Leu Gly Pro Leu Lys Asp Asp Val Leu Leu Leu Glu Thr 385 390 395
400 Arg Gly Pro Gln Asp Asn Arg Ser Leu Cys Ala Leu Glu Pro Ser Gly
405 410 415 Cys Thr Ser Leu Pro Ser Lys Ala Ser Thr Arg Ala Ala Arg
Leu Gly 420 425 430 Glu Tyr Leu Leu Gln Asp Leu Gln Ser Gly Gln Cys
Leu Gln Leu Trp 435 440 445 Asp Asp Asp Leu Gly Ala Leu Trp Ala Cys
Pro Met Asp Lys Tyr Ile 450 455 460 His Lys Gly Gly Ser Gly Gly Gly
Thr Gly Ser Leu Val Pro Arg Gly 465 470 475 480 Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 485 490 495 Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 500 505 510 Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 515 520
525 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
530 535 540 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr 545 550 555 560 Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn 565 570 575 Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 580 585 590 Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 595 600 605 Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 610 615 620 Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 625 630 635 640
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 645
650 655 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 660 665 670 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val 675 680 685 Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 690 695 700 Ser Pro Gly Lys 705 66 1416 DNA
Artificial Sequence synthetic insert 66 agccaggaaa tccatgccga
gttgagacgc ttccgtagac tggagaggct tgtggggcct 60 caggacgcta
cccactgctc tccgggcctc tcctgccgcc tctgggacag tgacatactc 120
tgcctgcctg gggacatcgt gcctgctccg ggccccgtgc tggcgcctac gcacctgcag
180 acagagctgg tgctgaggtg ccagaaggag accgactgtg acctctgtct
gcgtgtggct 240 gtccacttgg ccgtgcatgg gcactgggaa gagcctgaag
atgaggaaaa gtttggagga 300 gcagctgact caggggtgga ggagcctagg
aatgcctctc tccaggccca agtcgtgctc 360 tccttccagg cctaccctac
tgcccgctgc gtcctgctgg aggtgcaagt gcctgctgcc 420 cttgtgcagt
ttggtcagtc tgtgggctct gtggtatatg actgcttcga ggctgcccta 480
gggagtgagg tacgaatctg gtcctatact cagcccaggt acgagaagga actcaaccac
540 acacagcagc tgcctgccct gccctggctc aacgtgtcag cagatggtga
caacgtgcat 600 ctggttctga atgtctctga ggagcagcac ttcggcctct
ccctgtactg gaatcaggtc 660 cagggccccc caaaaccccg gtggcacaaa
aacctgactg gaccgcagat cattaccttg 720 aaccacacag acctggttcc
ctgcctctgt attcaggtgt ggcctctgga acctgactcc 780 gttaggacga
acatctgccc cttcagggag gacccccgcg cacaccagaa cctctggcaa 840
gccgcccgac tgcgactgct gaccctgcag agctggctgc tggacgcacc gtgctcgctg
900 cccgcagaag cggcactgtg ctggcgggct ccgggtgggg acccctgcca
gccactggtc 960 ccaccgcttt cctgggagaa cgtcactgtg gacaaggttc
tcgagttccc attgctgaaa 1020 ggccacccta acctctgtgt tcaggtgaac
agctcggaga agctgcagct gcaggagtgc 1080 ttgtgggctg actccctggg
gcctctcaaa gacgatgtgc tactgttgga gacacgaggc 1140 ccccaggaca
acagatccct ctgtgccttg gaacccagtg gctgtacttc actacccagc 1200
aaagcctcca cgagggcagc tcgccttgga gagtacttac tacaagacct gcagtcaggc
1260 cagtgtctgc agctatggga cgatgacttg ggagcgctat gggcctgccc
catggacaaa 1320 tacatccaca agggaggaag tggcggagga acaggaagtt
tggtccctcg tggaagcgac 1380 aaaactcaca catgcccacc gtgcccagca cctgaa
1416
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