U.S. patent application number 14/330909 was filed with the patent office on 2014-11-13 for mammalian cytokines; related reagents and methods.
The applicant listed for this patent is Merck Sharp & Dohme Corp.. Invention is credited to Rene de Waal Malefyt, Robert A. Kastelein, Sergio A. Lira, Satwant K. Narula, Birgit Oppmann, Donna M. Rennick, Maria T. Wiekowski.
Application Number | 20140335094 14/330909 |
Document ID | / |
Family ID | 27761345 |
Filed Date | 2014-11-13 |
United States Patent
Application |
20140335094 |
Kind Code |
A1 |
Oppmann; Birgit ; et
al. |
November 13, 2014 |
MAMMALIAN CYTOKINES; RELATED REAGENTS AND METHODS
Abstract
Purified genes encoding cytokine from a mammal, reagents related
thereto including purified proteins, specific antibodies, and
nucleic acids encoding this molecule are provided. Methods of using
said reagents and diagnostic kits are also provided.
Inventors: |
Oppmann; Birgit; (Berlin,
DE) ; de Waal Malefyt; Rene; (Sunnyvale, CA) ;
Rennick; Donna M.; (Los Altos, CA) ; Kastelein;
Robert A.; (Portola Valley, CA) ; Wiekowski; Maria
T.; (Wayne, NJ) ; Lira; Sergio A.; (Chatham,
NJ) ; Narula; Satwant K.; (West Caldwell,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp. |
Rahway |
NJ |
US |
|
|
Family ID: |
27761345 |
Appl. No.: |
14/330909 |
Filed: |
July 14, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12984523 |
Jan 4, 2011 |
|
|
|
14330909 |
|
|
|
|
11503324 |
Aug 10, 2006 |
7883695 |
|
|
12984523 |
|
|
|
|
09658699 |
Sep 8, 2000 |
7090847 |
|
|
11503324 |
|
|
|
|
60164616 |
Nov 10, 1999 |
|
|
|
60153281 |
Sep 9, 1999 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
530/387.3; 530/387.9 |
Current CPC
Class: |
C07K 14/5434 20130101;
C07K 2319/00 20130101; A01K 2217/05 20130101; C07K 2319/02
20130101; C07K 16/244 20130101; C07K 14/54 20130101; A61P 29/00
20180101; Y10S 530/827 20130101; A61K 38/00 20130101; C07K 2317/32
20130101; A61P 31/12 20180101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-50. (canceled)
51. An isolated antibody, or antigen binding fragment thereof, that
specifically binds to a complex of an IL-12 p40 polypeptide and an
IL-B30 polypeptide, wherein: a) the IL-12 p40 polypeptide comprises
the mature sequence of SEQ ID NO:20; and b) the IL-B30 polypeptide
comprises the mature sequence of SEQ ID NO:2; wherein the antibody,
or antigen binding fragment thereof, binds to sequences within the
mature sequence of SEQ ID NO:20 and the mature sequence of SEQ ID
NO:2; and further wherein the antibody, or antigen binding fragment
thereof does not bind to either the mature human IL-12p40
polypeptide alone or the mature human IL-B30 polypeptide alone, and
yet further wherein the complex of IL-12p40 and IL-B30 is generated
by co-expressing nucleic acids encoding the respective polypeptide
chains in a mammalian cell and collecting the IL-12p40/IL-B30
complex secreted from the cell.
52. The antibody or antigen binding fragment of claim 51 comprising
a monoclonal antibody or antigen binding fragment thereof.
53. The antibody or antigen binding fragment of claim 51 comprising
a humanized antibody or antigen binding fragment thereof.
54. The antigen binding fragment of claim 51 comprising an Fv
fragment, an Fab fragment, or an F(ab')2 fragment.
55. A pharmaceutical formulation comprising: a) the antibody or
antigen binding fragment of claim 51; and b) a pharmaceutically
acceptable carrier.
56. An isolated antibody, or antigen binding fragment thereof, that
specifically binds to a complex of an IL-12 p40 polypeptide and an
IL-B30 polypeptide, wherein: a) the IL-12 p40 polypeptide consists
of the mature sequence of SEQ ID NO:20; and b) the IL-B30
polypeptide consists of the mature sequence of SEQ ID NO:2; wherein
the antibody, or antigen binding fragment thereof, does not bind to
either the mature human IL-12p40 polypeptide alone or the mature
human IL-B30 polypeptide alone, and further wherein the complex of
IL-12p40 and IL-B30 is generated by co-expressing genes encoding
the respective polypeptide chains in a mammalian cell and
collecting the IL-12p40/IL-B30 complex secreted from the cell.
57. The antibody or antigen binding fragment of claim 56 comprising
a monoclonal antibody or antigen binding fragment thereof.
58. The antibody or antigen binding fragment of claim 56 comprising
a humanized antibody or antigen binding fragment thereof.
59. The antigen binding fragment of claim 56 comprising an Fv
fragment, an Fab fragment, or an F(ab)2 fragment.
60. A pharmaceutical formulation comprising: a) the antibody or
antigen binding fragment of claim 56; and b) a pharmaceutically
acceptable carrier.
Description
[0001] This application claims the benefit of U.S. patent
application Ser. No. 09/393,090 filed Sep. 9, 1999 and U.S. Patent
Provisional Application No. 60/164,616 filed Nov. 10, 1999. This
filing is a U.S. Utility patent application.
FIELD OF THE INVENTION
[0002] The present invention pertains to compositions and methods
related to proteins which function in controlling biology and
physiology of mammalian cells, e.g., cells of a mammalian immune
system. In particular, it provides purified genes, proteins,
antibodies, related reagents, and methods useful, e.g., to regulate
activation, development, differentiation, and function of various
cell types, including hematopoietic cells.
BACKGROUND OF THE INVENTION
[0003] Recombinant DNA technology refers generally to the technique
of integrating genetic information from a donor source into vectors
for subsequent processing, such as through introduction into a
host, whereby the transferred genetic information is copied and/or
expressed in the new environment. Commonly, the genetic information
exists in the form of complementary DNA (cDNA) derived from
messenger RNA (mRNA) coding for a desired protein product. The
carrier is frequently a plasmid having the capacity to incorporate
cDNA for later replication in a host and, in some cases, actually
to control expression of the cDNA and thereby direct synthesis of
the encoded product in the host.
[0004] For some time, it has been known that the mammalian immune
response is based on a series of complex cellular interactions,
called the "immune network". See, e.g., Paul, (1998) Fundamental
Immunology (4th ed.) Raven Press, NY. Recent research has provided
new insights into the inner workings of this network. While it
remains clear that much of the response does, in fact, revolve
around the network-like interactions of lymphocytes, macrophages,
granulocytes, and other cells, immunologists now generally hold the
opinion that soluble proteins, known as lymphokines, cytokines, or
monokines, play a critical role in controlling these cellular
interactions. Thus, there is considerable interest in the
isolation, characterization, and mechanisms of action of cell
modulatory factors, an understanding of which will lead to
significant advancements in the diagnosis and therapy of numerous
medical abnormalities, e.g., immune system disorders. Some of these
factors are hematopoietic growth factors, e.g., granulocyte colony
stimulating factor (G-CSF). See, e.g., Thomson, (ed. 1998) The
Cytokine Handbook (3d ed.) Academic Press, San Diego; Mire-Sluis
and Thorpe, (ed. 1998) Cytokines Academic Press, San Diego; Metcalf
and Nicola, (1995) The Hematopoietic Colony Stimulating Factors
Cambridge University Press; and Aggarwal and Gutterman, (1991)
Human Cytokines Blackwell Pub. Cytokine expression by cells of the
immune system plays an important role in the regulation of the
immune response. Most cytokines are pleiotropic and have multiple
biological activities, including antigen-presentation; activation;
proliferation and differentiation of CD4+ T cell subsets; antibody
response by B cells; and manifestations of hypersensitivity. In
addition cytokines may be used in the diagnosis and therapy of a
wide range of degenerative or abnormal conditions which directly or
indirectly involve the immune system and/or hematopoietic
cells.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and/or differentiation of pluripotential hematopoietic stem
cells into vast numbers of progenitors comprising diverse cellular
lineages making up a complex immune system. Proper and balanced
interactions between the cellular components are necessary for a
healthy immune response. The different cellular lineages often
respond in a different manner when lymphokines are administered in
conjunction with other agents.
[0006] Cell lineages especially important to the immune response
include two classes of lymphocytes: B-cells, which can produce and
secrete immunoglobulins (proteins with the capability of
recognizing and binding to foreign matter to effect its removal),
and T-cells of various subsets that secrete lymphokines and induce
or suppress the B-cells and various other cells (including other
T-cells) making up the immune network. These lymphocytes interact
with many other cell types.
[0007] From the foregoing, it is evident that the discovery and
development of new lymphokines, e.g., related to G-CSF and/or IL-6,
could contribute to new therapies for a wide range of degenerative
or abnormal conditions which directly or indirectly involve the
immune system and/or hematopoietic cells. In particular, the
discovery and development of lymphokines which enhance or
potentiate the beneficial activities of known lymphokines would be
highly advantageous. Originally the novel gene IL-B30 was
identified as a potential cytokine based on its predicted structure
and was classified as a long-chain cytokine like IL-6 and G-CSF
(International Patent Application PCT/US98/15423 (WO 99/05280).
IL-6 and related cytokines like Oncostatin M, leukemia inhibitory
factor (LIF), ciliary neurotrophic factor (CNTF) and
cardiothrophin-1 have biological activities on hematopoiesis,
thrombopoiesis, induction of an acute phase response, osteoclast
formation, neuron differentiation and survival, and cardiac
hypertrophy. Transgenic expression of IL-B30 in mice induced a
similar phenotype as that observed after overexpression of IL-6 in
mice, comprising runting, systemic inflammation, infertility and
death. IL-B30 appears to be a novel cytokine involved in
inflammation.
SUMMARY OF THE INVENTION
[0008] The present invention is based, in part, upon the discovery
of the physiological role of IL-B30, also referred to herein as the
IL-B30 protein, and its role in the immune response. In particular,
the role of IL-B30 has been elucidated in pathways involved in
inflammation, infectious disease, hematopoietic development, and
viral infection. The invention is specifically directed to
compositions comprising combinations of IL-12 p40 subunit with
interleukin-B30 (IL-B30) and their biological activities. It
includes nucleic acids coding for both polypeptides or fusion
proteins, and methods for their production and use. The nucleic
acids of the invention are characterized, in part, by their
homology to complementary DNA (cDNA) sequences disclosed herein,
and/or by functional assays. Also provided are polypeptides,
antibodies, and methods of using them, including using nucleic acid
expression methods. Methods for modulating or intervening in the
control of a growth factor dependent physiology or an immune
response are provided.
[0009] The present invention is based, in part, upon the discovery
that the p40 subunit of IL-12 also associates with the IL-B30
cytokine, described previously, e.g., in U.S. Ser. No. 08/900,905
and Ser. No. 09/122,443, in a natural form. Thus, the coexpression
of the two polypeptides together results in functional receptor
binding and signaling.
[0010] The present invention provides compositions comprising: a)
both a substantially pure polypeptide comprising a plurality of
distinct segments of at least 7 contiguous amino acid from IL-12
p40 and a substantially pure polypeptide comprising a plurality of
distinct segments of at least 7 contiguous amino acids from IL-B30;
b) both a substantially pure polypeptide comprising at least 11
contiguous amino acids from IL-12 p40 and a substantially pure
polypeptide comprising at least 11 contiguous amino acids from
IL-B30; c) a substantially pure polypeptide comprising both a
plurality of distinct segments of at least 7 contiguous amino acids
of IL-12 p40 and a plurality of distinct segments of at least 7
contiguous amino acids of IL-B30; or d) a substantially pure
polypeptide comprising both a segment of at least 11 contiguous
amino acids of IL-12 p40 and a segment of at least 11 contiguous
amino acids of IL-B30. Various embodiments include such
compositions: a) wherein the described plurality of distinct
segments of at least 7 contiguous amino acids comprise one segment
of at least 9 contiguous amino acids; b) wherein the described
plurality of distinct segments of at least 7 contiguous amino acids
are both at least 9 contiguous amino acids; c) wherein the
described segment of at least 11 contiguous amino acids of IL-12
p40 is at least 15 contiguous amino acids; d) wherein the described
segment of at least 11 contiguous amino acids of IL-B30 is at least
15 contiguous amino acids; e) further comprising a carrier selected
from an aqueous compound, including water, saline, and/or buffer; 0
formulated for oral, rectal, nasal, topical, or parenteral
administration; or g) which is sterile composition. Other
embodiments include those: a) wherein at least one of the described
polypeptides is: i) detectably labeled; ii) recombinantly produced;
iii) unglycosylated; iv) denatured; v) attached to a solid
substrate; or vi) conjugated to another chemical moiety; b)
comprising both a substantially pure IL-12 p40 polypeptide and a
substantially pure IL-B30 polypeptide; c) comprising a
substantially pure polypeptide comprising IL-12 p40 fused to
IL-B30; or d) combined with IL-18, IL-12, radiation or
chemotherapy, an immune adjuvant, or an anti-viral. Kit embodiments
include those comprising such a described composition and: a) a
compartment comprising the described polypeptide; or b)
instructions for use or disposal of reagents in the described
kit.
[0011] Nucleic acid compositions of the invention include, e.g., an
isolated or recombinant nucleic acid encoding: a) both a
substantially pure polypeptide comprising a plurality of distinct
segments of at least 7 contiguous amino acid from IL-12 p40 and a
substantially pure polypeptide comprising a plurality of distinct
segments of at least 7 contiguous amino acids from IL-B30; b) both
a substantially pure polypeptide comprising at least 11 contiguous
amino acids from IL-12 p40 and a substantially pure polypeptide
comprising at least 11 contiguous amino acids from IL-B30; c) a
substantially pure polypeptide comprising both a plurality of
distinct segments of at least 7 contiguous amino acids of IL-12 p40
and a plurality of distinct segments of at least 7 contiguous amino
acids of IL-B30; or d) a substantially pure polypeptide comprising
both a segment of at least 11 contiguous amino acids of IL-12 p40
and a segment of at least 11 contiguous amino acids of IL-B30.
Various embodiments include such a nucleic acid: a) wherein the
described plurality of distinct segments of at least 7 contiguous
amino acids comprise one segment of at least 9 contiguous amino
acids; b) wherein the described plurality of distinct segments of
at least 7 contiguous amino acids are both at least 9 contiguous
amino acids; c) wherein the described segment of at least 11
contiguous amino acids of IL-12 p40 is at least 15 contiguous amino
acids; d) wherein the described segment of at least 11 contiguous
amino acids of IL-B30 is at least 15 contiguous amino acids; e)
wherein the described IL-12 p40 is from a primate; f) wherein the
described IL-B30 is from a primate; g) which is an expression
vector; h) which further comprises an origin of replication; i)
which comprises a detectable label; j) which comprises synthetic
nucleotide sequence; k) which is less than 6 kb, preferably less
than 3 kb; or l) which is from primate. Also provided is a cell
comprising the described recombinant nucleic acid, including
wherein the described cell is: a prokaryotic, eukaryotic,
bacterial, yeast, insect, mammalian, mouse, primate, or human cell.
Kit embodiments include those comprising a described nucleic acid
and: a) a compartment comprising the described nucleic acid; b) a
compartment further comprising a primate IL-12 p40 polypeptide; c)
a compartment further comprising a primate IL-B30 polypeptide; or
d) instructions for use or disposal of reagents in the described
kit.
[0012] Alternatively, the invention provides a nucleic acid which
hybridizes: a) under wash conditions of 30 minutes at 50.degree. C.
and less than 1M salt to the natural mature coding portion of
primate IL-12 p40; and b) under wash conditions of 30 minutes at
50.degree. C. and less than 1M salt to the natural mature coding
portion of primate IL-B30. Various embodiments include such a
described nucleic acid wherein: a) the described wash conditions
for IL-12 p40 are at 60.degree. C. and less than 400 mM salt; b)
the described wash conditions for IL-B30 are at 60.degree. C. and
less than 400 mM salt; c) the described nucleic acid exhibits
identity over a stretch of at least 50 nucleotides to sequence
encoding primate IL-12 p40; and/or d) the described nucleic acid
exhibits identity over a stretch of at least 50 nucleotides to
sequence encoding primate IL-B30. Preferred embodiments include
such a nucleic acid wherein: a) the described wash conditions for
IL-12 p40 are at 65.degree. C. and less than 150 mM salt; b) the
described wash conditions for IL-B30 are at 65.degree. C. and less
than 150 mM salt; c) the described nucleic acid exhibits identity
over a stretch of at least 90 nucleotides to sequence encoding
primate IL-12 p40; and/or d) the described nucleic acid exhibits
identity over a stretch of at least 90 nucleotides to sequence
encoding primate IL-B30.
[0013] Antagonists of the IL-12 p40/IL-B30 compositions are
provided, combined with, e.g., a TNF.alpha. antagonist, an IL-12
antagonist, IL-10, or steroids.
[0014] The invention also provides a binding compound, e.g.,
comprising an antigen binding site from an antibody, which antibody
specifically binds to an IL-12 p40/IL-B30 composition, as
described, a) comprising a substantially pure polypeptide
comprising both a substantially pure IL-12 p40 polypeptide and a
substantially pure IL-B30 polypeptide; or b) comprising a
substantially pure polypeptide comprising IL-12 p40 fused to
IL-B30; but not to either IL-12 p40 or IL-B30 polypeptide. Other
binding compounds include those wherein: a) the described binding
compound is in a container; b) the described binding compound is an
Fv, Fab, or Fab2 fragment; c) the described binding compound is
conjugated to another chemical moiety; or d) the described
antibody: i) is raised against an IL-12 p40/IL-B30 composition; ii)
is immunoselected; iii) is a polyclonal antibody; iv) exhibits a Kd
to antigen of at least 30 mM; v) is attached to a solid substrate,
including a bead or plastic membrane; vi) is in a sterile
composition; or vii) is detectably labeled, including a radioactive
or fluorescent label. Certain preferred forms include compositions
comprising: a) a sterile binding compound, as described; or b) the
described binding compound and a carrier, wherein the described
carrier is: i) an aqueous compound, including water, saline, and/or
buffer; and/or ii) formulated for oral, rectal, nasal, topical, or
parenteral administration. Additionally, kit embodiments are
provided comprising the described binding compound and: a) a
compartment comprising the described binding compound; or b)
instructions for use or disposal of reagents in the described
kit.
[0015] Moreover, the invention provides methods for producing an
antigen:antibody complex, comprising contacting, under appropriate
conditions, a primate IL-12 p40/IL-B30 composition with a described
binding compound, thereby allowing the described complex to form.
Various methods include those wherein: a) the described complex is
purified from other cytokines; b) the described complex is purified
from other antibody; c) the described contacting is with a sample
comprising a cytokine; d) the described contacting allows
quantitative detection of the described antigen; e) the described
contacting is with a sample comprising the described antibody; or
f) the described contacting allows quantitative detection of the
described antibody.
[0016] The invention also provides methods of modulating physiology
or development of a cell or tissue comprising contacting the
described cell with an IL-12 p40/IL-B30 composition, or antagonist
thereof. One preferred method is modulating physiology or
development of a cell comprising contacting the described cell with
an IL-12 p40/IL-B30 composition, and the described contacting
results in an increase in production of IFN.gamma.. Typically, the
described cell is in a host organism, and the described organism
exhibits an enhanced Th1 response, e.g., one selected from an:
anti-tumor effect; adjuvant effect; anti-viral effect; or
antagonized allergic effect. Often, the contacting is in
combination with: IL-18; IL-12; radiation therapy or chemotherapy;
an immune adjuvant; or an anti-viral therapeutic.
[0017] In another embodiment, the described antagonist is an
antibody against IL-12 receptor subunit .beta.1. Thus, the
invention also embraces a method, as described, wherein the
described contacting is with an antagonist, and the described
contacting results in a relative decrease in production of
IFN.gamma.. Thus, the invention provides methods of modulating
physiology or development of a cell in a host organism, comprising
administering the described antagonist to the described organism,
wherein the described contacting results in amelioration of: an
autoimmune condition or a chronic inflammatory condition.
[0018] The identification of the association of the two subunits
provides methods of increasing the secretion of: a) a primate
IL-B30, such method comprising expressing the described polypeptide
with IL-12 p40; or b) a primate IL-12 p40, such method comprising
expressing the described IL-12 p40 with IL-B30. Preferably, either:
a) the described increasing is at least 3-fold; or b) the described
expressing is of a recombinant nucleic acid encoding IL-B30 and
IL-12 p40.
[0019] Methods for screening for a receptor which binds the
described IL-12 p40/IL-B30 composition are provided, e.g.,
comprising contacting the described complex to a cell expressing
the described receptor under conditions allowing the described
complex to bind to the described receptor, thereby forming a
detectable interaction. Preferably, the described interaction
results in a physiological response in the described cell.
[0020] The present invention also provides methods of modulating
the trafficking or activation of a leukocyte in an animal, the
methods comprising contacting monocyte/macrophage lineage cells in
the animal with a therapeutic amount of an agonist of a mammalian
IL-B30 protein; or an antagonist of a mammalian IL-B30 protein.
Preferred embodiments include where: the mammalian IL-B30 protein
is a primate protein; and/or the antagonist is an antibody which
binds to the mammalian IL-B30. Certain embodiments include where
the monocyte/macrophage lineage cells include a microglial cell or
a dendritic cell, or where the animal exhibits signs or symptoms of
an inflammatory, leukoproliferative, neurodegenerative, or
post-traumatic condition. Preferred embodiments include where the
sign or symptom is in lung tissue; liver tissue; neural tissue;
lymphoid tissue; myeloid tissue; pancreas; gastrointestinal tissue;
thyroid tissue; muscle tissue; or skin or collagenous tissue.
[0021] Other methods include where the modulating is inhibiting
function of the leukocyte cell; and/or where the administering is
the agonist. Preferably, the agonist is the mammalian IL-B30.
[0022] Certain embodiments include where the animal is experiencing
signs or symptoms of autoimmunity; an inflammatory condition;
tissue specific autoimmunity; degenerative autoimmunity; rheumatoid
arthritis; osteoarthritis; atherosclerosis; multiple sclerosis;
vasculitis; delayed hypersensitivities; skin grafting; a
transplant; spinal injury; stroke; neurodegeneration; an infectious
disease; ischemia; cancer; tumors; multiple myeloma; Castleman's
disease; postmenopausal osteoporosis or IL-6-associated diseases.
The administering may be in combination with: an anti-inflammatory
cytokine agonist or antagonist; an analgesic; an anti-inflammatory
agent; or a steroid.
[0023] Various other methods are provided where the modulating is
enhancing function of the leukocyte cell, and/or the administering
is the antagonist. Preferably, the antagonist is: an antibody which
binds to the mammalian IL-B30; or a mutein of the mammalian IL-B30
which competes with the mammalian IL-B30 in binding to an IL-B30
receptor, but does not substantially signal. In various
embodiments, the method is applied where the animal experiences
signs or symptoms of wound healing or clot formation. The
administering will often be in combination with: an angiogenic
factor; a growth factor, including FGF or PDGF; an antibiotic; or a
clotting factor.
[0024] Lastly, the present invention provides a method of inducing
the proliferation of memory T-cells by administering IL-B30 or an
agonist thereof.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference.
[0026] Outline
[0027] I. General
[0028] II. Purified IL-12 p40/IL-B30 complex [0029] A. physical
properties [0030] B. biological properties
[0031] III. Physical Variants [0032] A. sequence variants,
fragments [0033] B. post-translational variants [0034] 1.
glycosylation [0035] 2. others
[0036] IV. Functional Variants [0037] A. analogs, fragments [0038]
1. agonists [0039] 2. antagonists [0040] B. mimetics [0041] 1.
protein [0042] 2. chemicals [0043] C. species variants
[0044] V. Antibodies [0045] A. polyclonal [0046] B. monoclonal
[0047] C. fragments, binding compositions
[0048] VI. Nucleic Acids [0049] A. natural isolates; methods [0050]
B. synthetic genes [0051] C. methods to isolate
[0052] VII. Making p40/IL-B30 complex, mimetics [0053] A.
recombinant methods [0054] B. synthetic methods [0055] C. natural
purification
[0056] VIII. Uses [0057] A. diagnostic [0058] B. therapeutic
[0059] IX. Kits [0060] A. nucleic acid reagents [0061] B. protein
reagents [0062] C. antibody reagents
[0063] X. Isolating receptors for p40/IL-B30 complexes
I. General
[0064] The present invention provides description and teaching of
pairing of mammalian proteins to make a soluble cytokine, e.g., a
secreted molecule which can mediate a signal between immune or
other cells. See, e.g., Paul, (1998) Fundamental Immunology (4th
ed.) Raven Press, N.Y. Certain soluble factors are made up of
heterodimer polypeptides, e.g., IL-6 and IL-12. The dimer forms,
which are likely the physiological forms, and fragments, or
antagonists will be useful, e.g., in physiological modulation of
cells expressing a receptor. It is likely that the functional
cytokine comprising p40/IL-B30 complex has either stimulatory or
inhibitory effects on hematopoietic cells, including, e.g.,
lymphoid cells, such as T-cells, B-cells, natural killer (NK)
cells, macrophages, dendritic cells, hematopoietic progenitors,
etc. The proteins will also be useful as antigens, e.g.,
immunogens, for raising antibodies to various epitopes on the
protein, both linear and conformational epitopes.
[0065] The IL-12 p40 subunit has been described. See, e.g., Seiler
et al., U.S. Pat. No. 5,547,852; Scott and Trinchieri, U.S. Pat.
No. 5,571,515; Gately et al., U.S. Pat. No. 5,650,492; Liesehke and
Mulligan, U.S. Pat. No. 5,891,680; Warne et al., U.S. Pat. No.
5,744,132; and accession numbers gbM86671, gbAF133197, gbU16674,
gbU83184, embY07762, embY11129.1, gbM65272, gbAF007576, gbU19841,
gbU11815, gbU57752, gbAF004024, gbU49100, gbU19834, and embX97019.
A sequence encoding IL-B30 was identified from a human genomic
sequence. The molecule was designated huIL-B30. A rodent sequence,
e.g., from mouse, was also described. See, e.g., U.S. Ser. No.
08/900,905 and 09/122,443. The present invention embraces
compositions comprising combinations of these two polypeptides,
e.g., p40 and IL-B30, and nucleic acid constructs encoding both
sequences. Antibodies which recognize the combinations are also
provided, and methods of producing the two messages or
polypeptides, e.g., coordinately.
[0066] The human IL-B30 gene encodes a small soluble cytokine-like
protein, of about 198 amino acids. The psort predicted signal
sequence probably is about 17 residues, and would run from the Met
to about Ala. See Table 1 and SEQ. ID. NO: 1 and 2. IL-B30 exhibits
structural motifs characteristic of a member of the long chain
cytokines. Compare, e.g., IL-B30, G-CSF, and IL-6, sequences
available from GenBank. See also U.S. Ser. No. 08/900,905 and
09/122,443.
TABLE-US-00001 TABLE 1 Nucleic acid (SEQ ID NO: 1) encoding IL-B30
from a primate, e.g., human. Translated amino acid sequence is SEQ
ID NO: 2. ATG CTG GGG AGC AGA GCT GTA ATG CTG CTG TTG CTG CTG CCC
TGG ACA 48 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro
Trp Thr -21 -20 -15 -10 GCT CAG GGC AGA GCT GTG CCT GGG GGC AGC AGC
CCT GCC TGG ACT CAG 96 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser
Pro Ala Trp Thr Gln -5 1 5 10 TGC CAG CAG CTT TCA CAG AAG CTC TGC
ACA CTG GCC TGG AGT GCA CAT 144 Cys Gln Gln Leu Ser Gln Lys Leu Cys
Thr Leu Ala Trp Ser Ala His 15 20 25 CCA CTA GTG GGA CAC ATG GAT
CTA AGA GAA GAG GGA GAT GAA GAG ACT 192 Pro Leu Val Gly His Met Asp
Leu Arg Glu Glu Gly Asp Glu Glu Thr 30 35 40 ACA AAT GAT GTT CCC
CAT ATC CAG TGT GGA GAT GGC TGT GAC CCC CAA 240 Thr Asn Asp Val Pro
His Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln 45 50 55 GGA CTC AGG
GAC AAC AGT CAG TTC TGC TTG CAA AGG ATC CAC CAG GGT 288 Gly Leu Arg
Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly 60 65 70 75 CTG
ATT TTT TAT GAG AAG CTG CTA GGA TCG GAT ATT TTC ACA GGG GAG 336 Leu
Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85
90 CCT TCT CTG CTC CCT GAT AGC CCT GTG GCG CAG CTT CAT GCC TCC CTA
384 Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu
95 100 105 CTG GGC CTC AGC CAA CTC CTG CAG CCT GAG GGT CAC CAC TGG
GAG ACT 432 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp
Glu Thr 110 115 120 CAG CAG ATT CCA AGC CTC AGT CCC AGC CAG CCA TGG
CAG CGT CTC CTT 480 Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp
Gln Arg Leu Leu 125 130 135 CTC CGC TTC AAA ATC CTT CGC AGC CTC CAG
GCC TTT GTG GCT GTA GCC 528 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln
Ala Phe Val Ala Val Ala 140 145 150 155 GCC CGG GTC TTT GCC CAT GGA
GCA GCA ACC CTG AGT CCC TAA 570 Ala Arg Val Phe Ala His Gly Ala Ala
Thr Leu Ser Pro 160 165
MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEE
TTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVAQLHA
SLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP
coding sequence: ATGCTGGGGA GCAGAGCTGT AATGCTGCTG TTGCTGCTGC
CCTGGACAGC TCAGGGCAGA GCTGTGCCTG GGGGCAGCAG CCCTGCCTGG ACTCAGTGCC
AGCAGCTTTC ACAGAAGCTC TGCACACTGG CCTGGAGTGC ACATCCACTA GTGGGACACA
TGGATCTAAG AGAAGAGGGA GATGAAGAGA CTACAAATGA TGTTCCCCAT ATCCAGTGTG
GAGATGGCTG TGACCCCCAA GGACTCAGGG ACAACAGTCA GTTCTGCTTG CAAAGGATCC
ACCAGGGTCT GATTTTTTAT GAGAAGCTGC TAGGATCGGA TATTTTCACA GGGGAGCCTT
CTCTGCTCCC TGATAGCCCT GTGGCGCAGC TTCATGCCTC CCTACTGGGC CTCAGCCAAC
TCCTGCAGCC TGAGGGTCAC CACTGGGAGA CTCAGCAGAT TCCAAGCCTC AGTCCCAGCC
AGCCATGGCA GCGTCTCCTT CTCCGCTTCA AAATCCTTCG CAGCCTCCAG GCCTTTGTGG
CTGTAGCCGC CCGGGTCTTT GCCCATGGAG CAGCAACCCT GAGTCCCTAA Rodent,
e.g., mouse, IL-B30 (SEQ ID NO: 3 and 4): CGCTTAGAAG TCGGACTACA
GAGTTAGACT CAGAACCAAA GGAGGTGGAT AGGGGGTCCA 60 CAGGCCTGGT
GCAGATCACA GAGCCAGCCA GATCTGAGAA GCAGGGAACA AG ATG 115 Met -21 CTG
GAT TGC AGA GCA GTA ATA ATG CTA TGG CTG TTG CCC TGG GTC ACT 163 Leu
Asp Cys Arg Ala Val Ile Met Leu Trp Leu Leu Pro Trp Val Thr -20 -15
-10 -5 CAG GGC CTG GCT GTG CCT AGG AGT AGC AGT CCT GAC TGG GCT CAG
TGC 211 Gln Gly Leu Ala Val Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln
Cys 1 5 10 CAG CAG CTC TCT CGG AAT CTC TGC ATG CTA GCC TGG AAC GCA
CAT GCA 259 Gln Gln Leu Ser Arg Asn Leu Cys Met Leu Ala Trp Asn Ala
His Ala 15 20 25 CCA GCG GGA CAT ATG AAT CTA CTA AGA GAA GAA GAG
GAT GAA GAG ACT 307 Pro Ala Gly His Met Asn Leu Leu Arg Glu Glu Glu
Asp Glu Glu Thr 30 35 40 AAA AAT AAT GTG CCC CGT ATC CAG TGT GAA
GAT GGT TGT GAC CCA CAA 355 Lys Asn Asn Val Pro Arg Ile Gln Cys Glu
Asp Gly Cys Asp Pro Gln 45 50 55 60 GGA CTC AAG GAC AAC AGC CAG TTC
TGC TTG CAA AGG ATC CGC CAA GGT 403 Gly Leu Lys Asp Asn Ser Gln Phe
Cys Leu Gln Arg Ile Arg Gln Gly 65 70 75 CTG GCT TTT TAT AAG CAC
CTG CTT GAC TCT GAC ATC TTC AAA GGG GAG 451 Leu Ala Phe Tyr Lys His
Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu 80 85 90 CCT GCT CTA CTC
CCT GAT AGC CCC ATG GAG CAA CTT CAC ACC TCC CTA 499 Pro Ala Leu Leu
Pro Asp Ser Pro Met Glu Gln Leu His Thr Ser Leu 95 100 105 CTA GGA
CTC AGC CAA CTC CTC CAG CCA GAG GAT CAC CCC CGG GAG ACC 547 Leu Gly
Leu Ser Gln Leu Leu Gln Pro Glu Asp His Pro Arg Glu Thr 110 115 120
CAA CAG ATG CCC AGC CTG AGT TCT AGT CAG CAG TGG CAG CGC CCC CTT 595
Gln Gln Met Pro Ser Leu Ser Ser Ser Gln Gln Trp Gln Arg Pro Leu 125
130 135 140 CTC CGT TCC AAG ATC CTT CGA AGC CTC CAG GCC TTT TTG GCC
ATA GCT 643 Leu Arg Ser Lys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala
Ile Ala 145 150 155 GCC CGG GTC TTT GCC CAC GGA GCA GCA ACT CTG ACT
GAG CCC TTA GTG 691 Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Thr
Glu Pro Leu Val 160 165 170 CCA ACA GCT TAAGGATGCC CAGGTTCCCA
TGGCTACCAT GATAAGACTA 740 Pro Thr Ala 175 ATCTATCAGC CCAGACATCT
ACCAGTTAAT TAACCCATTA GGACTTGTGC TGTTCTTGTT 800 TCGTTTGTTT
TGCGTGAAGG GCAAGGACAC CATTATTAAA GAGAAAAGAA ACAAACCCCA 860
GAGCAGGCAG CTGGCTAGAG AAAGGAGCTG GAGAAGAAGA ATAAAGTCTC GAGCCCTTGG
920 CCTTGGAAGC GGGCAAGCAG CTGCGTGGCC TGAGGGGAAG GGGGCGGTGG
CATCGAGAAA 980 CTGTGAGAAA ACCCAGAGCA TCAGAAAAAG TGAGCCCAGG
CTTTGGCCAT TATCTGTAAG 1040 AAAAACAAGA AAAGGGGAAC ATTATACTTT
CCTGGGTGGC TCAGGGAAAT GTGCAGATGC 1100 ACAGTACTCC AGACAGCAGC
TCTGTACCTG CCTGCTCTGT CCCTCAGTTC TAACAGAATC 1160 TAGTCACTAA
GAACTAACAG GACTACCAAT ACGAACTGAC AAA 1203
MLDCRAVIMLWLLPWVTQGLAVPRSSSPDWAQCQQLSRNLCMLAWNAHAPAGHMNLLREEED
EETKNNVPRIQCEDGCDPQGLKDNSQFCLQRIRQGLAFYKHLLDSDIFKGEPALLPDSPMEQ
LHTSLLGLSQLLQPEDHPRETQQMPSLSSSQQWQRPLLRSKILRSLQAFLAIAARVFAHGAA
TLTEPLVPTA
[0067] The structural homology of IL-B30 to related cytokine
proteins suggests related function of this molecule. However,
recognition of the association of the IL-12 p40 polypeptide with
the IL-B30 polypeptide allows for biological assay of active
p40/IL-B30 dimers. IL-12 p40/IL-B30 compositions may be made up of
either distinct polypeptides representing each of the individual
polypeptides, or fusion constructs of IL-12 p40 with IL-B30.
Observations indicate that the dimer is capable of inducing
interferon-.gamma. (IFN.gamma.) production by various cell types,
e.g., PBMC, suggesting biological functions for which the dimer
will be used. Moreover, experiments indicate that the IL-12
receptor .beta.1 subunit is a component of the receptor for the
p40/IL-B30 dimer.
[0068] IFN.gamma. activates macrophages, stimulating tumoricidal
and microbicidal activities. It also modulates class I and II MHC
molecule expression, including up-regulation of class II molecules
on monocytes/macrophages and dendritic cells, and induces
expression on epithelial, endothelial, and other cells, rendering
them capable of antigen presentation. The cytokine is a Th1-like
cytokine which promotes the development of Th1-like CD4+ T cells,
but inhibits that of Th2-like T cells. It is a powerful and
relatively specific inhibitor of IL-4-induced IgE and IgG4
synthesis by B lymphocytes, although at higher concentrations it
non-specifically inhibits the production of all antibody isotypes.
IFN.gamma. augments cytotoxic immune responses against
intracellular organisms and tumors mediated by NK cells and CTLs.
Like IL-12, IFN.gamma. has the propensity to promote cell-mediated
cytotoxic response while inhibiting allergic inflammation and IgE
synthesis. See, e.g., Karupiah, (ed. 1997) Gamma Interferon in
Antiviral Defense Chapman & Hall; Jaffe, (ed. 1992)
Anti-Infective Applications of Interferon-Gamma Marcel Dekker
(ISBN: 0824786882); Sutterwala et al., (1999) J. Leukoc. Biol.
65:543-551; Billiau et al., (1998) Ann. NY Acad. Sci. 856:22-32;
and Gessani et al., (1998) Cytokine Growth Factor Rev.
9:117-123.
[0069] IL-B30 agonists, or antagonists, may also act as functional
or receptor antagonists, e.g., which block IL-6 or IL-12 binding to
their respective receptors, or mediating the opposite actions.
Thus, IL-B30, or its antagonists, may be useful in the treatment of
abnormal medical conditions, including immune disorders, e.g., T
cell immune deficiencies, chronic inflammation, or tissue
rejection, or in cardiovascular or neurophysiological conditions.
Agonists would be likely to be used in a therapeutic context of
enhancing cell mediated immunity, e.g., in anti-tumor, adjuvant,
and anti-viral situations, or to antagonize allergic responses.
Antagonists would likely be used in the context of blocking such
enhanced immunity, e.g., in cellular contributions to autoimmune
diseases or chronic inflammatory conditions.
[0070] The natural antigens are capable of mediating various
biochemical responses which lead to biological or physiological
responses in target cells. The preferred embodiments would be from
human, but other primate, or other species counterparts exist in
nature. Additional sequences for proteins in other mammalian
species, e.g., primates, canines, felines, and rodents, should also
be available.
[0071] In particular, the association of the IL-12 p40 subunit with
IL-B30 has been confirmed. The IL-12 p40 and IL-B30 molecules
should have evolved together. If the two functionally associate,
they might act together in the fashion of IL-12. See, e.g.,
Trinchieri (1998) Adv. Immunol. 70:83-243; Gately et al., (1998)
Ann. Rev. Immunol. 16:495-521; and Trinchieri (1998) Int. Rev.
Immunol. 16:365-396.
[0072] As a complex, however, the complex would be expected to
interact with two tall signaling receptors in the cytokine receptor
family. This has been confirmed in the case of IL-12 receptor
subunit .beta.1. Other related receptors can be tested for binding
to the soluble complex. A series of cells, e.g., BAF/3, that stably
express various of these tall receptors capable of signal
transduction have been constructed.
[0073] The supernatants of transfectants of both IL-12 p40 and
IL-B30 (or a single combination construct) in the same cell, were
used to test these various cells to see if there is a proliferative
or other signaling response. As such, most of the physiological
effects of the cytokine may be due to the complex of the proteins.
As such, many of the descriptions below of biology resulting from
the cytokine may actually be physiologically effected by the
complex comprising the combination of the subunits.
[0074] The descriptions below may also be applied to the IL-12
p40/IL-B30 complex. A fusion of the IL-12 p40 subunit with the
IL-B30 was constructed, as, e.g., the hyper IL-6. See, e.g.,
Fischer et al., (1997) Nature Biotechnol. 15:142-145; Rakemann et
al., (1999) J. Biol. Chem. 274:1257-1266; and Peters et al., (1998)
J. Immunol. 161:3575-3581; which are incorporated herein by
reference. Moreover, matching of the cytokine complex with a
receptor comprising the IL-12 receptor subunit .beta.1 allows for
identification of antibodies to that subunit as a receptor
antagonist of the cytokine complex.
II. Purified p40/IL-B30 Complex
[0075] Human IL-B30 amino acid sequence, is shown as one embodiment
within SEQ ID NO: 2. Other naturally occurring nucleic acids which
encode the protein can be isolated by standard procedures using the
provided sequence, e.g., PCR techniques, or by hybridization. These
amino acid sequences, provided amino to carboxy, are important in
providing sequence information for the cytokine subunit allowing
for distinguishing the protein antigen from other proteins and
exemplifying numerous variants. Moreover, the peptide sequences
allow preparation of peptides to generate antibodies to recognize
segments, and nucleotide sequences allow preparation of
oligonucleotide probes, both of which are strategies for detection
or isolation, e.g., cloning, of genes encoding such sequences.
[0076] As used herein, the term "human soluble IL-B30" shall
encompass, when used in a protein context, a protein having amino
acid sequence corresponding to a soluble polypeptide from SEQ ID
NO: 2. Significant fragments thereof will often retain similar
functions, e.g., antigenicity. Preferred embodiments comprise a
plurality of distinct, e.g., nonoverlapping, segments of the
specified length. Typically, the plurality will be at least two,
more usually at least three, and preferably 5, 7, or even more.
While the length minima may be recited, longer lengths, of various
sizes, may be appropriate, e.g., one of length 7, and two of length
12. Similar features apply to the IL-12 p40 polypeptide, and to
polynucleotides of either or both.
[0077] Binding components, e.g., antibodies, typically bind to an
IL-12 p40/IL-B30 complex with high affinity, e.g., at least about
100 nM, usually better than about 30 nM, preferably better than
about 10 nM, and more preferably at better than about 3 nM.
Counterpart protein complexes will be found in mammalian species
other than human, e.g., other primates, ungulates, or rodents.
Non-mammalian species should also possess structurally or
functionally related genes and proteins, e.g., birds or
amphibians.
[0078] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least about
12 amino acids, typically at least about 16 amino acids, preferably
at least about 20 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 35, 40,
45, 50, etc. Such fragments may have ends which begin and/or end at
virtually all positions, e.g., beginning at residues 1, 2, 3, etc.,
and ending at, e.g., 175, 174, 173, etc., in all practical
combinations for either the IL-B30 or the IL-12 p40 subunit.
Particularly interesting peptides have ends corresponding to
structural domain boundaries, e.g., helices A, B, C, and/or D of
the IL-B30 or the Ig domains of the IL-12 p40. See below.
[0079] The term "binding composition" refers to molecules that bind
with specificity to the IL-12 p40/IL-B30 complex, e.g., in an
antibody-antigen interaction, but not to the individual components
alone. The specificity may be more or less inclusive, e.g.,
specific to a particular embodiment, or to groups of related
embodiments, e.g., primate, rodent, etc. Depletion or absorptions
can provide desired selectivities, e.g., to deplete antibodies
which bind to either polypeptide component alone. Also provided are
compounds, e.g., proteins, which specifically associate with the
IL-12 p40/IL-B30 complex, including in a natural physiologically
relevant protein-protein interaction, either covalent or
non-covalent. The molecule may be a polymer, or chemical reagent. A
functional analog may be a protein with structural modifications,
or it may be a molecule which has a molecular shape which interacts
with the appropriate binding determinants. The compounds may serve
as agonists or antagonists of a receptor binding interaction, see,
e.g., Goodman et al., (eds.), Goodman & Gilman's: The
Pharmacological Bases of Therapeutics (current ed.) Pergamon
Press.
[0080] Substantially pure, e.g., in a protein context, typically
means that the protein is free from other contaminating proteins,
nucleic acids, or other biologicals derived from the original
source organism. Purity may be assayed by standard methods,
typically by weight, and will ordinarily be at least about 40%
pure, generally at least about 50% pure, often at least about 60%
pure, typically at least about 80% pure, preferably at least about
90% pure, and in most preferred embodiments, at least about 95%
pure. Carriers or excipients will often be added. A composition
comprising a substantially pure IL-12 p40 and IL-B30 will not have
large amounts of extraneous polypeptides which are not naturally
associated with the complex of the two polypeptides.
[0081] Solubility of a polypeptide or fragment depends upon the
environment and the polypeptide. Many parameters affect polypeptide
solubility, including temperature, electrolyte environment, size
and molecular characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the polypeptide is
used ranges from about 4.degree. C. to about 65.degree. C. Usually
the temperature at use is greater than about 18.degree. C. For
diagnostic purposes, the temperature will usually be about room
temperature or warmer, but less than the denaturation temperature
of components in the assay. For therapeutic purposes, the
temperature will usually be body temperature, typically about
37.degree. C. for humans and mice, though under certain situations
the temperature may be raised or lowered in situ or in vitro.
[0082] The size and structure of the polypeptides should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents. In particular, a complex made up of the
association of the two polypeptides is preferred, as is a fusion
composition.
[0083] The solvent and electrolytes will usually be a biologically
compatible buffer, of a type used for preservation of biological
activities, and will usually approximate a physiological aqueous
solvent. Usually the solvent will have a neutral pH, typically
between about 5 and 10, and preferably about 7.5. On some
occasions, one or more detergents will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein. In other
instances, a harsh detergent may be used to effect significant
denaturation.
[0084] An IL-B30 polypeptide that specifically binds to or that is
specifically immunoreactive with an antibody, e.g., such as a
polyclonal antibody, generated against a defined immunogen, e.g.,
such as an immunogen consisting of an amino acid sequence of SEQ ID
NO: 2 or fragments thereof or a polypeptide generated from the
nucleic acid of SEQ ID NO: 1 is typically determined in an
immunoassay. Included within the metes and bounds of the present
invention are those nucleic acid sequences described herein,
including functional variants, that encode polypeptides that
selectively bind to polyclonal antibodies generated against the
prototypical IL-B30 polypeptide as structurally and functionally
defined herein. The immunoassay typically uses a polyclonal
antiserum which was raised, e.g., to a complex comprising a protein
of SEQ ID NO: 2. This antiserum is selected, or depleted, to have
low crossreactivity against appropriate other closely related
family members, preferably from the same species, and any such
crossreactivity is removed by immunoabsorption or depletion prior
to use in the immunoassay. In particular, antibodies which bind to
the IL-12 p40 or the IL-B30 polypeptides alone are targets for
immunodepletion. Appropriate selective serum preparations can be
isolated, and characterized.
[0085] In order to produce antisera for use in an immunoassay, the
compls comprising the protein, e.g., of SEQ ID NO: 2, is isolated
as described herein. For example, recombinant protein may be
produced in a mammalian cell line. An appropriate host, e.g., an
inbred strain of mice such as Balb/c, is immunized with the complex
comprising a protein of SEQ ID NO: 2 using a standard adjuvant,
such as Freund's adjuvant, and a standard mouse immunization
protocol (see Harlow and Lane). Alternatively, a substantially
full-length synthetic peptide construct derived from the sequences
disclosed herein can be used as an immunogen. Polyclonal sera are
collected and titered against the immunogen protein in an
immunoassay, e.g., a solid phase immunoassay with the immunogen
immobilized on a solid support, along with appropriate depletions
or selections. Polyclonal antisera with a titer of 10.sup.4 or
greater are selected and tested for their cross reactivity against
other closely related family members, e.g., LIF, CT-1, CNTF, or
other members of the IL-6 family, using a competitive binding
immunoassay such as the one described in Harlow and Lane, supra, at
pages 570-573. Preferably at least two individual IL-6/IL-12 family
members are used in this determination in conjunction with the
target. These long chain cytokine family members can be produced as
recombinant proteins and isolated using standard molecular biology
and protein chemistry techniques as described herein. Thus,
antibody preparations can be identified or produced having desired
selectivity or specificity for subsets of IL-12 p40/IL-B30 family
members. Alternatively, antibodies may be prepared which bind to
fusion polypeptide forms of the complex comprising the IL-12 p40
and IL-B30.
[0086] Immunoassays in the competitive binding format can be used
for the crossreactivity determinations. For example, the fusion
protein can be immobilized to a solid support. Proteins added to
the assay compete with the binding of the selective antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the selective antisera to the immobilized
protein is compared to the fusion protein. The percent
crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
crossreactivity with each of the proteins listed above are selected
and pooled. The cross-reacting selective antibodies are then
removed from the pooled antisera by immunoabsorption with the
above-listed proteins.
[0087] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein to the immunogen fusion protein. In order to make
this comparison, the two proteins are each assayed at a wide range
of concentrations and the amount of each protein required to
inhibit 50% of the binding of the selective antisera to the
immobilized fusion protein is determined. If the amount of the
second protein required is less than twice the amount of the fusion
protein that is required, then the second protein is said to
specifically bind to a selective antibody generated to the
immunogen.
III. Physical Variants
[0088] This invention also encompasses complexes comprising
proteins or peptides having substantial amino acid sequence
identity with the amino acid sequences of the IL-12 p40/IL-B30
antigen. The variants include species, polymorphic, or allelic
variants.
[0089] Amino acid sequence homology, or sequence identity, is
determined by optimizing residue matches, if necessary, by
introducing gaps as required. See also Needleham et al., (1970) J.
Mol. Biol. 48:443-453; Sankoff et al., (1983) Chapter One in Time
Warps, String Edits, and Macromolecules: The Theory and Practice of
Sequence Comparison, Addison-Wesley, Reading, Mass.; and software
packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
Sequence identity changes when considering conservative
substitutions as matches. Conservative substitutions typically
include substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
phenylalanine, tyrosine. The conservation may apply to biological
features, functional features, or structural features. Homologous
amino acid sequences are typically intended to include natural
polymorphic or allelic and interspecies variations of a protein
sequence. Typical homologous proteins or peptides will have from
25-100% identity (if gaps can be introduced), to 50-100% identity
(if conservative substitutions are included) with the amino acid
sequence of the IL-B30. Identity measures will be at least about
35%, generally at least about 40%, often at least about 50%,
typically at least about 60%, usually at least about 70%,
preferably at least about 80%, and more preferably at least about
90%.
[0090] The isolated IL-12 p40 or IL-B30 DNA can be readily modified
by nucleotide substitutions, nucleotide deletions, nucleotide
insertions, and inversions of short nucleotide stretches. These
modifications result in novel DNA sequences which encode these
antigens, their derivatives, or proteins having similar
physiological, immunogenic, antigenic, or other functional
activity. These modified sequences can be used to produce mutant
antigens or to enhance expression. Enhanced expression may involve
gene amplification, increased transcription, increased translation,
and other mechanisms. "Mutant IL-B30" encompasses a polypeptide
otherwise falling within the sequence identity definition of the
IL-B30 as set forth above, but having an amino acid sequence which
differs from that of IL-B30 as normally found in nature, whether by
way of deletion, substitution, or insertion. This generally
includes proteins having significant identity with a protein having
sequence of SEQ ID NO: 2, and as sharing various biological
activities, e.g., antigenic or immunogenic, with those sequences,
and in preferred embodiments contain most of the natural
full-length disclosed sequences. Full-length sequences will
typically be preferred, though truncated versions will also be
useful, likewise, genes or proteins found from natural sources are
typically most desired. Similar concepts apply to different IL-B30
proteins, particularly those found in various warm-blooded animals,
e.g., mammals and birds. These descriptions are generally meant to
encompass various IL-B30 proteins, not limited to the particular
primate embodiments specifically discussed.
[0091] IL-12 p40 or IL-B30 mutagenesis can also be conducted by
making amino acid insertions or deletions. Substitutions,
deletions, insertions, or any combinations may be generated to
arrive at a final construct. Insertions include amino- or
carboxy-terminal fusions. Random mutagenesis can be conducted at a
target codon and the expressed mutants can then be screened for the
desired activity. Methods for making substitution mutations at
predetermined sites in DNA having a known sequence are well known
in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. See, e.g., Sambrook et al., (1989);
Ausubel et al., (1987 and Supplements); and Kunkel et al., (1987)
Methods in Enzymol. 154:367-382. Preferred embodiments include,
e.g., 1-fold, 2-fold, 3-fold, 5-fold, 7-fold, etc., preferably
conservative substitutions at the nucleotide or amino acid levels.
Preferably the substitutions will be away from the conserved
cysteines, and often will be in the regions away from the helical
structural domains. Such variants may be useful to produce specific
antibodies, and often will share many or all biological properties.
Recognition of the cytokine structure provides important insight
into the structure and positions of residues which may be modified
to effect desired changes in receptor interaction. Also, the
interaction of the IL-12 p40 with the IL-B30 protein requires
complementary structural features in the interacting surface.
Structural analysis will further allow prediction of the surface
residues critical in both complex formation and complex to receptor
interaction.
[0092] The present invention also provides recombinant proteins,
e.g., heterologous fusion proteins using segments from these
proteins. A heterologous fusion protein is a fusion of proteins or
segments which are naturally not normally fused in the same manner.
A similar concept applies to heterologous nucleic acid
sequences.
[0093] In addition, new constructs may be made from combining
similar functional domains from other proteins. For example,
target-binding or other segments may be "swapped" between different
new fusion polypeptides or fragments. See, e.g., Cunningham et al.,
(1989) Science 243:1330-1336; and O'Dowd et al., (1988) J. Biol.
Chem. 263:15985-15992.
[0094] The phosphoramidite method described by Beaucage and
Carruthers, (1981) Tetra. Letts. 22:1859-1862, will produce
suitable synthetic DNA fragments. A double stranded fragment will
often be obtained either by synthesizing the complementary strand
and annealing the strand together under appropriate conditions or
by adding the complementary strand using DNA polymerase with an
appropriate primer sequence, e.g., PCR techniques.
[0095] Structural analysis can be applied to this gene, in
comparison to the IL-6 family of cytokines. The family includes,
e.g., IL-6, IL-11, IL-12, G-CSF, LIF, OSM, CNTF, and Ob. Alignment
of the human and mouse IL-B30 sequences with other members of the
IL-6 family should allow definition of structural features. In
particular, .beta.-sheet and .alpha.-helix residues can be
determined using, e.g., RASMOL program, see Bazan et al., (1996)
Nature 379:591; Lodi et al., (1994) Science 263:1762-1766; Sayle
and Milner-White, (1995) TIBS 20:374-376; and Gronenberg et al.,
(1991) Protein Engineering 4:263-269. See, also, Wilkins et al.,
(eds. 1997) Proteome Research: New Frontiers in Functional Genomics
Springer-Verlag, NY. Preferred residues for substitutions include
the surface exposed residues which would be predicted to interact
with receptor. Other residues which should conserve function will
be conservative substitutions, particularly at a position far from
the surface exposed residues.
IV. Functional Variants
[0096] The blocking of physiological response to the IL-12
p40/IL-B30 complexes may result from the competitive inhibition of
binding of the ligand to its receptor. Identification of one
subunit of the receptor allows for further characterization, as
described, and use of antibodies to that subunit to block binding
and/or signaling with the complex.
[0097] In vitro assays of the present invention will often use
isolated complex, protein, soluble fragments comprising receptor
binding segments of these proteins, or fragments attached to solid
phase substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations
and modifications, or cytokine mutations and modifications, e.g.,
IL-12 p40/IL-B30 complex analogs.
[0098] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
cytokine complex, or receptor binding fragments compete with a test
compound.
[0099] "Derivatives" of IL-12 p40/IL-B30 antigens include amino
acid sequence mutants from naturally occurring forms, glycosylation
variants, and covalent or aggregate conjugates with other chemical
moieties. Covalent derivatives can be prepared by linkage of
functionalities to groups which are found in IL-12 p40/IL-B30
complex amino acid side chains or at the N- or C-termini, e.g., by
standard means. See, e.g., Lundblad and Noyes, (1988) Chemical
Reagents for Protein Modification, vols. 1-2, CRC Press, Inc., Boca
Raton, Fla.; Hugli, (ed. 1989) Techniques in Protein Chemistry,
Academic Press, San Diego, Calif.; and Wong, (1991) Chemistry of
Protein Conjugation and Cross Linking, CRC Press, Boca Raton,
Fla.
[0100] In particular, glycosylation alterations are included, e.g.,
made by modifying the glycosylation patterns of a polypeptide
during its synthesis and processing, or in further processing
steps. See, e.g., Elbein, (1987) Ann. Rev. Biochem. 56:497-534.
Also embraced are versions of the peptides with the same primary
amino acid sequence which have other minor modifications, including
phosphorylated amino acid residues, e.g., phosphotyrosine,
phosphoserine, or phosphothreonine.
[0101] Fusion polypeptides between the IL-12 p40 and IL-B30 are
also provided. Many cytokine receptors or other surface proteins
are multimeric, e.g., homodimeric entities, and a repeat construct
may have various advantages, including lessened susceptibility to
proteolytic cleavage. Typical examples are fusions of a reporter
polypeptide, e.g., luciferase, with a segment or domain of a
protein, e.g., a receptor-binding segment, so that the presence or
location of the fused ligand may be easily determined. See, e.g.,
Dull et al., U.S. Pat. No. 4,859,609. Other gene fusion partners
include bacterial .beta.-galactosidase, trpE, Protein A,
.beta.-lactamase, alpha amylase, alcohol dehydrogenase, yeast alpha
mating factor, and detection or purification tags such as a FLAG
sequence of His6 sequence. See, e.g., Godowski et al., (1988)
Science 241:812-816. Fusion constructs with other therapeutic
entities, e.g., which are to be coadministered, but proteolytically
cleaved, are also provided.
[0102] Fusion peptides will typically be made by either recombinant
nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are
described generally, e.g., in Sambrook et al., (1989) Molecular
Cloning: A Laboratory Manual (2d ed.), vols. 1-3, Cold Spring
Harbor Laboratory; and Ausubel et al., (eds. 1993) Current
Protocols in Molecular Biology, Greene and Wiley, NY. Techniques
for synthesis of polypeptides are described, e.g., in Merrifield,
(1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield, (1986) Science
232: 341-347; Atherton et al., (1989) Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford; and Grant,
(1992) Synthetic Peptides: A User's Guide, W.H. Freeman, NY.
Refolding methods may be applicable to synthetic proteins.
[0103] This invention also contemplates the use of derivatives of
IL-12 p40 or IL-B30 proteins other than variations in amino acid
sequence or glycosylation. Such derivatives may involve covalent or
aggregative association with chemical moieties or protein carriers.
Covalent or aggregative derivatives will be useful as immunogens,
as reagents in immunoassays, or in purification methods such as for
affinity purification of binding partners, e.g., other antigens. An
IL-12 p40 or IL-B30 can be immobilized by covalent bonding to a
solid support such as cyanogen bromide-activated SEPHAROSE, by
methods which are well known in the art, or adsorbed onto
polyolefin surfaces, with or without glutaraldehyde cross-linking,
for use in the assay or purification of anti-IL-12 p40 or IL-B30
antibodies or an alternative binding composition. The IL-12 p40,
IL-B30, or fusion proteins can also be labeled with a detectable
group, e.g., for use in diagnostic assays. Purification of IL-12
p40/IL-B30 complex may be effected by an immobilized antibody to
either polypeptide or sequence component or complementary binding
partner, e.g., binding portion of a receptor.
[0104] A solubilized IL-12 p40/IL-B30 polypeptide or fragment of
this invention can be used as an immunogen for the production of
antisera or antibodies specific for binding. Purified antigen can
be used to screen monoclonal antibodies or antigen-binding
fragments, encompassing antigen binding fragments of natural
antibodies, e.g., Fab, Fab', F(ab).sub.2, etc. Purified IL-12
p40/IL-B30 antigens can also be used as a reagent to detect
antibodies generated in response to the presence of elevated levels
of the cytokine complex, which may be diagnostic of an abnormal or
specific physiological or disease condition. This invention
contemplates antibodies raised against amino acid sequences encoded
by nucleotide sequence shown in SEQ ID NO: 1, or fragments of
proteins containing it. In particular, this invention contemplates
antibodies having binding affinity to or being raised against
specific domains, e.g., helices A, B, C, or D of the IL-B30, or the
Ig domains of the IL-12 p40.
[0105] The present invention contemplates the isolation of
additional closely related species variants. Southern and Northern
blot analysis will establish that similar genetic entities exist in
other mammals. It is likely that IL-B30s are widespread in species
variants, e.g., rodents, lagomorphs, carnivores, artiodactyla,
perissodactyla, and primates.
[0106] The invention also provides means to isolate a group of
related antigens displaying both distinctness and similarities in
structure, expression, and function. Elucidation of many of the
physiological effects of the molecules will be greatly accelerated
by the isolation and characterization of additional distinct
species or polymorphic variants of them. In particular, the present
invention provides useful probes for identifying additional
homologous genetic entities in different species.
[0107] The isolated genes will allow transformation of cells
lacking expression of an IL-B30, e.g., either species types or
cells which lack corresponding proteins and exhibit negative
background activity. This should allow analysis of the function of
IL-B30 in comparison to untransformed control cells.
[0108] Dissection of critical structural elements which effect the
various physiological functions mediated through these antigens is
possible using standard techniques of modern molecular biology,
particularly in comparing members of the related class. See, e.g.,
the homolog-scanning mutagenesis technique described in Cunningham
et al., (1989) Science 243:1339-1336; and approaches used in O'Dowd
et al., (1988) J. Biol. Chem. 263:15985-15992; and Lechleiter et
al., (1990) EMBO J. 9:4381-4390.
[0109] Intracellular functions would probably involve receptor
signaling. However, protein internalization may occur under certain
circumstances, and interaction between intracellular components and
cytokine may occur. Specific segments of interaction of IL-B30 with
interacting components may be identified by mutagenesis or direct
biochemical means, e.g., cross-linking or affinity methods.
Structural analysis by crystallographic or other physical methods
will also be applicable. Further investigation of the mechanism of
signal transduction will include study of associated components
which may be isolatable by affinity methods or by genetic means,
e.g., complementation analysis of mutants.
[0110] Further study of the expression and control of IL-B30 will
be pursued. The controlling elements associated with the antigens
should exhibit differential physiological, developmental, tissue
specific, or other expression patterns. Upstream or downstream
genetic regions, e.g., control elements, are of interest.
[0111] Structural studies of the IL-B30 antigens will lead to
design of new antigens, particularly analogs exhibiting agonist or
antagonist properties on the molecule. This can be combined with
previously described screening methods to isolate antigens
exhibiting desired spectra of activities.
V. Antibodies
[0112] Antibodies can be raised to various epitopes of the
p40/IL-B30 proteins, including species, polymorphic, or allelic
variants, and fragments thereof, both in their naturally occurring
forms and in their recombinant forms. Additionally, antibodies can
be raised to IL-B30s in either their active forms or in their
inactive forms, including native or denatured versions.
Anti-idiotypic antibodies are also contemplated.
[0113] Antibodies, including binding fragments and single chain
versions, against predetermined fragments of the antigens can be
raised by immunization of animals with conjugates of the fragments
with immunogenic proteins. Monoclonal antibodies are prepared from
cells secreting the desired antibody. These antibodies can be
screened for binding to normal or defective IL-B30s, or screened
for agonistic or antagonistic activity, e.g., mediated through a
receptor. Antibodies may be agonistic or antagonistic, e.g., by
sterically blocking binding to a receptor. These monoclonal
antibodies will usually bind with at least a K.sub.D of about 1 mM,
more usually at least about 300 .mu.M, typically at least about 100
.mu.M, more typically at least about 30 .mu.M, preferably at least
about 10 .mu.M, and more preferably at least about 3 .mu.M or
better.
[0114] The antibodies of this invention can also be useful in
diagnostic applications. As capture or non-neutralizing antibodies,
they can be screened for ability to bind to the antigens without
inhibiting binding to a receptor. As neutralizing antibodies, they
can be useful in competitive binding assays. They will also be
useful in detecting or quantifying IL-B30 protein or its receptors.
See, e.g., Chan, (ed. 1987) Immunology: A Practical Guide, Academic
Press, Orlando, Fla.; Price and Newman, (eds. 1991) Principles and
Practice of Immunoassay, Stockton Press, N.Y.; and Ngo, (ed. 1988)
Nonisotopic Immunoassay, Plenum Press, N.Y. Cross absorptions or
other tests will identify antibodies which exhibit various spectra
of specificities, e.g., unique or shared species specificities.
[0115] Further, the antibodies, including antigen binding
fragments, of this invention can be potent antagonists that bind to
the antigen and inhibit functional binding, e.g., to a receptor
which may elicit a biological response. They also can be useful as
non-neutralizing antibodies and can be coupled to toxins or
radionuclides so that when the antibody binds to antigen, a cell
expressing it, e.g., on its surface, is killed. Further, these
antibodies can be conjugated to drugs or other therapeutic agents,
either directly or indirectly by means of a linker, and may effect
drug targeting.
[0116] Antigen fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as immunogens. An antigen and its fragments
may be fused or covalently linked to a variety of immunogens, such
as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid,
etc. See Microbiology, Hoeber Medical Division, Harper and Row,
1969; Landsteiner, (1962) Specificity of Serological Reactions,
Dover Publications, New York; Williams et al., (1967) Methods in
Immunology and Immunochemistry, vol. 1, Academic Press, New York;
and Harlow and Lane, (1988) Antibodies: A Laboratory Manual, CSH
Press, NY, for descriptions of methods of preparing polyclonal
antisera.
[0117] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies may be found in, e.g., Stites et al., (eds.)
Basic and Clinical Immunology (4th ed.), Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane, (1988) Antibodies: A Laboratory Manual, CSH Press;
Goding, (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.), Academic Press, New York; and particularly in Kohler and
Milstein, (1975) in Nature 256:495-497, which discusses one method
of generating monoclonal antibodies.
[0118] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse et al., (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward et al., (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; Moore et al., U.S. Pat. No. 4,642,334; and Queen et al.,
(1989) Proc. Natl. Acad. Sci. USA 86:10029-10033.
[0119] The antibodies of this invention can also be used for
affinity chromatography in isolating the protein. Columns can be
prepared where the antibodies are linked to a solid support. See,
e.g., Wilchek et al., (1984) Meth. Enzymol. 104:3-55. Conversely,
protein can be used for depletion or cross absorptions to prepare
selectively specific binding compositions.
[0120] Antibodies raised against each IL-B30 will also be useful to
raise anti-idiotypic antibodies. These will be useful in detecting
or diagnosing various immunological conditions related to
expression of the respective antigens.
VI. Nucleic Acids
[0121] The described peptide sequences and the related reagents are
useful in detecting, isolating, or identifying a DNA clone encoding
both IL-12 p40 and IL-B30, e.g., from a natural source. Typically,
it will be useful in isolating genes from a mammal, and similar
procedures will be applied to isolate genes from other species,
e.g., warm-blooded animals, such as birds and mammals. Cross
hybridization will allow isolation of IL-12 p40 or IL-B30 from the
same, e.g., polymorphic variants, or other species. A number of
different approaches will be available to successfully isolate a
suitable nucleic acid clone. Such genes allow construction of
coexpression constructs or fusion constructs.
[0122] The purified protein or polypeptides are useful for
generating antibodies by standard methods, as described above.
Synthetic peptides or purified protein can be presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan, (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane, (1989) Antibodies: A Laboratory Manual, Cold
Spring Harbor Press.
[0123] For example, a specific binding composition could be used
for screening of an expression library made from a cell line which
expresses both IL-12 p40 and IL-B30. Screening of intracellular
expression can be performed by various staining or
immunofluorescence procedures. Binding compositions could be used
to affinity purify or sort out cells expressing a surface fusion
protein.
[0124] The peptide segments can also be used to select or identify
appropriate oligonucleotides to screen a library. The genetic code
can be used to select appropriate oligonucleotides useful as probes
for screening. See, e.g., GenBank and SEQ ID NO: 1. In combination
with polymerase chain reaction (PCR) techniques, synthetic
oligonucleotides will be useful in selecting correct clones from a
library. Complementary sequences will also be used as probes,
primers, or antisense strands. Various fragments should be
particularly useful, e.g., coupled with anchored vector or poly-A
complementary PCR techniques or with complementary DNA of other
peptides.
[0125] This invention contemplates use of isolated DNA or fragments
to encode a biologically active complex of the corresponding IL-12
p40 and IL-B30 polypeptide, particularly lacking the portion coding
the untranslated portions of the described sequences. In addition,
this invention covers isolated or recombinant DNA which encodes a
biologically active fusion protein or polypeptide and which is
capable of hybridizing under appropriate conditions with the DNA
sequences described herein. Said biologically active protein or
polypeptide can be an intact antigen, or fragment, and have an
amino acid sequence disclosed in, e.g., SEQ ID NO: 2, particularly
a mature, secreted polypeptide. Further, this invention covers the
use of isolated or recombinant DNA, or fragments thereof, which
encode proteins which exhibit high identity to a secreted IL-12
p49/IL-B30 complex. The isolated DNA can have the respective
regulatory sequences in the 5' and 3' flanks, e.g., promoters,
enhancers, poly-A addition signals, and others. Alternatively,
expression may be effected by operably linking a coding segment to
a heterologous promoter, e.g., by inserting a promoter upstream
from an endogenous gene. See, e.g., Treco et al., WO96/29411 or
U.S. Ser. No. 08/406,030.
[0126] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other extraneous components which naturally accompany a native
sequence, e.g., ribosomes, polymerases, and/or flanking genomic
sequences from the originating species. The term embraces a nucleic
acid sequence which has been removed from its naturally occurring
environment, and includes recombinant or cloned DNA isolates and
chemically synthesized analogs or analogs biologically synthesized
by heterologous systems. A substantially pure molecule includes
isolated forms of the molecule, e.g., distinct from an isolated
chromosome. Generally, the nucleic acid will be in a vector or
fragment less than about 50 kb, usually less than about 30 kb,
typically less than about 10 kb, and preferably less than about 6
kb.
[0127] An isolated nucleic acid will generally be a homogeneous
composition of molecules, but will, in some embodiments, contain
minor heterogeneity. This heterogeneity is typically found at the
polymer ends or portions not critical to a desired biological
function or activity.
[0128] A "recombinant" nucleic acid is defined either by its method
of production or its structure. In reference to its method of
production, e.g., a product made by a process, the process is use
of recombinant nucleic acid techniques, e.g., involving human
intervention in the nucleotide sequence, typically selection or
production. Alternatively, it can be a nucleic acid made by
generating a sequence comprising fusion of two fragments which are
not naturally contiguous to each other, but is meant to exclude
products of nature, e.g., naturally occurring mutants. Thus, e.g.,
products made by transforming cells with any unnaturally occurring
vector is encompassed, as are nucleic acids comprising sequence
derived using any synthetic oligonucleotide process. Such is often
done to replace a codon with a redundant codon encoding the same or
a conservative amino acid, while typically introducing or removing
a sequence recognition site.
[0129] Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a single genetic entity
comprising a desired combination of functions not found in the
commonly available natural forms. Restriction enzyme recognition
sites are often the target of such artificial manipulations, but
other site specific targets, e.g., promoters, DNA replication
sites, regulation sequences, control sequences, or other useful
features may be incorporated by design. A similar concept is
intended for a recombinant, e.g., fusion, polypeptide. Specifically
included are synthetic nucleic acids which, by genetic code
redundancy, encode polypeptides similar to fragments of these
antigens, and fusions of sequences from various different species
or polymorphic variants.
[0130] A significant "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides, generally at
least about 22 nucleotides, ordinarily at least about 29
nucleotides, more often at least about 35 nucleotides, typically at
least about 41 nucleotides, usually at least about 47 nucleotides,
preferably at least about 55 nucleotides, and in particularly
preferred embodiments will be at least about 60 or more
nucleotides, e.g., 67, 73, 81, 89, 95, etc., including hundreds
and/or thousands.
[0131] A DNA which codes for an IL-B30 protein will be particularly
useful to identify genes, mRNA, and cDNA species which code for
related or similar proteins, as well as DNAs which code for
homologous proteins from different species. There will be homologs
in other species, including primates, rodents, canines, felines,
and birds. Various IL-B30 proteins should be homologous and are
encompassed herein. However, even proteins that have a more distant
evolutionary relationship to the antigen can readily be isolated
under appropriate conditions using these sequences if they are
sufficiently homologous. Primate IL-B30 proteins are of particular
interest. Likewise with the IL-12 p40, which proteins are prime
targets for the fusion constructs or combination compositions.
[0132] Recombinant clones derived from the genomic sequences, e.g.,
containing introns, will be useful for transgenic studies,
including, e.g., transgenic cells and organisms, and for gene
therapy. See, e.g., Goodnow, (1992) "Transgenic Animals" in Roitt
(ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp.
1502-1504; Travis, (1992) Science 256:1392-1394; Kuhn et al.,
(1991) Science 254:707-710; Capecchi (1989) Science 244:1288;
Robertson, (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, IRL Press, Oxford; and Rosenberg, (1992) J.
Clinical Oncology 10:180-199.
[0133] Substantial homology, e.g., identity, in the nucleic acid
sequence comparison context means either that the segments, or
their complementary strands, when compared, are identical when
optimally aligned, with appropriate nucleotide insertions or
deletions, in at least about 50% of the nucleotides, generally at
least about 58%, ordinarily at least about 65%, often at least
about 71%, typically at least about 77%, usually at least about
85%, preferably at least about 95 to 98% or more, and in particular
embodiments, as high as about 99% or more of the nucleotides.
Alternatively, substantial homology exists when the segments will
hybridize under selective hybridization conditions, to a strand, or
its complement, typically using a sequence of IL-12 p40 and/or
IL-B30, e.g., in SEQ ID NO: 1. Typically, selective hybridization
will occur when there is at least about 55% identity over a stretch
of at least about 30 nucleotides, preferably at least about 75%
over a stretch of about 25 nucleotides, and most preferably at
least about 90% over about 20 nucleotides. See, Kanehisa, (1984)
Nuc. Acids Res. 12:203-213. The length of identity comparison, as
described, may be over longer stretches, and in certain embodiments
will be over a stretch of at least about 17 nucleotides, usually at
least about 28 nucleotides, typically at least about 40
nucleotides, and preferably at least about 75 to 100 or more
nucleotides.
[0134] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., usually in excess of about 37.degree. C.,
typically in excess of about 55.degree. C., more typically in
excess of about 60 or 65.degree. C., and preferably in excess of
about 70.degree. C. Stringent salt conditions will ordinarily be
less than about 1000 mM, usually less than about 400 mM, typically
less than about 250 mM, preferably less than about 150 mM,
including about 100, 50, or even 20 mM. However, the combination of
parameters is much more important than the measure of any single
parameter. See, e.g., Wetmur and Davidson, (1968) J. Mol. Biol.
31:349-370. Hybridization under stringent conditions should give a
background of at least 2-fold over background, preferably at least
3-5 or more.
[0135] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0136] Optical alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman, (1981) Adv. Appl. Math. 2:482, by the homology alignment
algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman, (1988)
Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by visual inspection (see generally
Ausubel et al., supra).
[0137] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendrogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle, (1987) J. Mol. Evol. 35:351-360. The method
used is similar to the method described by Higgins and Sharp,
(1989) CABIOS 5:151-153. The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0138] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described Altschul et al., (1990) J.
Mol. Biol. 215:403-410. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http:www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pairs (HSPs) by identifying
short words of length W in the query sequence, which either match
or satisfy some positive-valued threshold score T when aligned with
a word of the same length in a database sequence. T is referred to
as the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
then extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of the
word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLAST program uses as defaults a
wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and
Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments
(B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of
both strands.
[0139] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin and Altschul,
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0140] A further indication that two nucleic acid sequences of
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, e.g., where the two peptides
differ only by conservative substitutions. Another indication that
two nucleic acid sequences are substantially identical is that they
hybridize to each other under stringent conditions, as described
below.
[0141] IL-B30 from other mammalian species can be cloned and
isolated by cross-species hybridization of closely related species.
Homology may be relatively low between distantly related species,
and thus hybridization of relatively closely related species is
advisable. Alternatively, preparation of an antibody preparation
which exhibits less species specificity may be useful in expression
cloning approaches.
VII. Making p40/IL-B30 Combinations; Mimetics
[0142] DNA which encodes the IL-12 p40 or IL-B30 or fragments
thereof can be obtained by chemical synthesis, screening cDNA
libraries, or screening genomic libraries prepared from a wide
variety of cell lines or tissue samples. See, e.g., Okayama and
Berg, (1982) Mol. Cell. Biol. 2:161-170; Gubler and Hoffman, (1983)
Gene 25:263-269; and Glover, (ed. 1984) DNA Cloning: A Practical
Approach, IRL Press, Oxford. Alternatively, the sequences provided
herein provide useful PCR primers or allow synthetic or other
preparation of suitable genes encoding an IL-12 p40 or IL-B30;
including naturally occurring embodiments.
[0143] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length IL-12 p40 and IL-B30 or
fragments which can, in turn, e.g., be used to generate polyclonal
or monoclonal antibodies; for binding studies; for construction and
expression of modified molecules; and for structure/function
studies.
[0144] Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other vehicles which
enable the integration of DNA fragments into the genome of the
host. See, e.g., Pouwels et al., (1985 and Supplements) Cloning
Vectors: A Laboratory Manual, Elsevier, N.Y.; and Rodriguez et al.,
(eds. 1988) Vectors: A Survey of Molecular Cloning Vectors and
Their Uses, Buttersworth, Boston, Mass.
[0145] For purposes of this invention, DNA sequences are operably
linked when they are functionally related to each other. For
example, DNA for a presequence or secretory leader is operably
linked to a polypeptide if it is expressed as a preprotein or
participates in directing the polypeptide to the cell membrane or
in secretion of the polypeptide. A promoter is operably linked to a
coding sequence if it controls the transcription of the
polypeptide; a ribosome binding site is operably linked to a coding
sequence if it is positioned to permit translation. Usually,
operably linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences that in
turn control expression. See, e.g., Rodriguez et al., Chapter 10,
pp. 205-236; Balbas and Bolivar, (1990) Methods in Enzymology
185:14-37; and Ausubel et al., (1993) Current Protocols in
Molecular Biology, Greene and Wiley, NY. Coexpression of the two
coding sequences is particularly of interest herein.
[0146] Representative examples of suitable expression vectors
include pcDNA1; pCD, see Okayama et al., (1985) Mol. Cell Biol.
5:1136-1142; pMC1neo Poly-A, see Thomas et al., (1987) Cell
51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
See, e.g., Miller, (1988) Ann. Rev. Microbiol. 42:177-199.
[0147] It will often be desired to express an IL-12 p40 and/or
IL-B30 polypeptide in a system which provides a specific or defined
glycosylation pattern. See, e.g., Luckow and Summers, (1988)
Bio/Technology 6:47-55; and Kaufman, (1990) Meth. Enzymol.
185:487-511.
[0148] The IL-12 p40 and/or IL-B30, or a fragment thereof, may be
engineered to be phosphatidyl inositol (PI) linked to a cell
membrane, but can be removed from membranes by treatment with a
phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a biologically active
form, and allows purification by standard procedures of protein
chemistry. See, e.g., Low, (1989) Biochim. Biophys. Acta
988:427-454; Tse et al., (1985) Science 230:1003-1008; and Brunner
et al., (1991) J. Cell Biol. 114:1275-1283.
[0149] Now that the IL-12 p40 and IL-B30 have been characterized,
fragments or derivatives thereof can be prepared by conventional
processes for synthesizing peptides. These include processes such
as are described in Stewart and Young, (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky, (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; Bodanszky, (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; and Villafranca, (ed.
1991) Techniques in Protein Chemistry II, Academic Press, San
Diego, Calif.
VIII. Uses
[0150] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
IL-12 p40/IL-B30 complex mediated conditions, or below in the
description of kits for diagnosis. The gene may be useful in
forensic sciences, e.g., to distinguish rodent from human, or as a
marker to distinguish between different cells exhibiting
differential expression or modification patterns. The provided
compositions are useful reagents for, e.g., in vitro assays,
scientific research, and the synthesis or manufacture of nucleic
acids, polypeptides, or antibodies.
[0151] This invention also provides reagents with significant
commercial and/or therapeutic potential. The IL-12 p40/IL-B30
complex (naturally occurring or recombinant), fragments thereof,
and antibodies thereto, along with compounds identified as having
binding affinity to the complex or individual components thereof,
should be useful as reagents for teaching techniques of molecular
biology, immunology, or physiology. Appropriate kits may be
prepared with the reagents, e.g., in practical laboratory exercises
in production or use of proteins, antibodies, cloning methods,
histology, etc.
[0152] The reagents will also be useful in the treatment of
conditions associated with abnormal physiology or development,
including inflammatory conditions. They may be useful in vitro
tests for presence or absence of interacting components, which may
correlate with success of particular treatment strategies. In
particular, modulation of physiology of various, e.g.,
hematopoietic or lymphoid, cells will be achieved by appropriate
methods for treatment using the compositions provided herein. See,
e.g., Thomson, (1994; ed.) The Cytokine Handbook (2d ed.) Academic
Press, San Diego; Metcalf and Nicola, (1995) The Hematopoietic
Colony Stimulating Factors Cambridge University Press; and Aggarwal
and Gutterman, (1991) Human Cytokines Blackwell Pub.
[0153] Observations that the cytokine complex can induce IFN.gamma.
levels provides useful insight into therapeutic potential. In
particular, IFN.gamma. production results in enhanced cell mediated
immunity. See, e.g., Paul, (1998) Fundamental Immunology (4th ed.)
Raven Press, NY; and Delves and Roitt (eds. 1998) The Encyclopedia
of Immunology Academic Press (ISBN: 0122267656). Thus, enhancement
of cellular responses will be useful in contexts to enhance
anti-tumor activity, enhance vaccine responses (both humoral and
cellular immunity), enhance anti-viral effects, and to antagonize
allergic responses in certain windows of development. See, e.g.,
Rose and Mackay (eds. 1998) The Autoimmune Diseases (3d ed.)
Academic Press, San Diego; and Kay, (ed. 1997) Allergy and Allergic
Diseases Blackwell Science, Malden Mass. Conversely, antagonists
would be used to block or prevent such IFN.gamma. enhancement,
thereby reducing the strength or intensity of the cellular
enhancement. Such may be useful in, e.g., autoimmune situations
(such as multiple sclerosis or psoriasis) or chronic inflammatory
conditions (such as rheumatoid arthritis or inflammatory bowel
disease). See, e.g., Samter et al., (eds.) Immunological Diseases
vols. 1 and 2, Little, Brown and Co. The initial results suggest
that the role of the p40/IL-B30 is more critical in the maintenance
of the chronic inflammatory condition. Thus, blockage may be
effective after initial development of the condition.
[0154] With such therapeutic targets, the agonists or antagonists
will be combined with existing therapeutics, e.g., with other
modulators of inflammation. Thus, the agonists will often be
combined, e.g., with IL-18, IL-12, radiation or chemotherapy
treatments, vaccine adjuvants, and/or anti-viral therapeutics.
Alternatively, the antagonists may be combined with TNF.alpha.
antagonists, IL-12 antagonists, with IL-10, and/or steroids. Viral
homologs of the cytokines might also be used.
[0155] For example, a disease or disorder associated with abnormal
expression or abnormal signaling by an IL-12 p40/IL-B30 should be a
likely target for an agonist or antagonist. The new cytokine should
play a role in regulation or development of hematopoietic cells,
e.g., lymphoid cells, which affect immunological responses, e.g.,
inflammation and/or autoimmune disorders. Alternatively, it may
affect vascular physiology or development, or neuronal effects.
Timing of administration of the therapeutic relative to initiation
or maintenance of the condition may also be important. In
particular, the cytokine complex should mediate, in various
contexts, cytokine synthesis by the cells, proliferation, etc.
Antagonists of IL-12 p40/IL-B30, such as mutein variants of a
naturally occurring form or blocking antibodies, may provide a
selective and powerful way to block immune responses, e.g., in
situations as inflammatory or autoimmune responses. See also Samter
et al., (eds.) Immunological Diseases vols. 1 and 2, Little, Brown
and Co.
[0156] Particular targets for therapeutic application include,
e.g., lung conditions, both asthma and fibrosis, in EAE models
(which may be useful models for multiple sclerosis), diabetes, and
gut inflammations. See, e.g., Barnes et al., (1998) Mol. Med. Today
4:452-458; Pauwels et al., (1998) Clin. Exp. Allergy August 28
Suppl 3:1-5; Durham, (1998) Clin. Exp. Allergy June 28 Suppl
2:11-16; Leung, (1997) Pediatr. Res. 42:559-568; Pretolani et al.,
(1997) Res. Immunol. 148:33-38; Lamkhioued et al., (1996) Ann. NY
Acad. Sci. 796:203-208; Erb et al., (1996) Immunol. Cell. Biol.
74:206-208; and Anderson et al., (1994) Trends Pharmacol. Sci.
15:324-332 for asthma; Coker et al., (1998) Eur. Respir. J.
11:1218-1221; and Bienkowski et al., (1995) Proc. Soc. Exp. Biol.
Med. 209:118-140 for lung fibrosis; Pearson and McDevitt, (1999)
Curr. Top. Microbiol. Immunol. 238:79-122; Miller and Shevach,
(1998) Res. Immunol. 149:753-759; Hoffman and Karpus, (1998) Res.
Immunol. 149:790-794 (with discussion 846-847 and 855-860); Segal,
(1998) Res. Immunol. 149:811-820 (with discussion 850-851 and
855-860); Liblau et al., (1997) Immunol. Today 18:599-604; Gold et
al., (1997) Crit. Rev. Immunol. 17:507-510; Spack, (1997) Crit.
Rev. Immunol. 17:529-536; and Leonard et al., (1997) Crit. Rev.
Immunol. 17:545-553 for EAE models (for multiple sclerosis); Almawi
et al., (1999) J. Clin. Endocrinol. Metab. 84:1497-1502;
Rabinovitch et al., (1998) Biochem. Pharmacol. 55:1139-1149; and
Rabinovitch, (1998) Diabetes Metab. Rev. 14:129-151 for diabetes;
and Leach et al., (1999) Toxicol. Pathol. 27:123-133; Braun et al.,
(1999) Curr. Opin. Rheumatol. 11:68-74; Rugtveit et al., (1997)
Gastroenterology 112:1493-1505; Strober et al., (1997) Immunol.
Today 18:61-64; and Ford et al., (1996) Semin. Pediatr. Surg.
5:155-159 for gut/intestinal inflammatory conditions.
[0157] The p40/IL-B30 stimulation of memory activated cells results
in phenotypic changes which include adhesion molecules. CD69L is
highly expressed following stimulation with p40/IL-B30, and CD54 is
dramatically decreased. These changes in expression of adhesion
molecules may allow modulating memory cells to enter the T/DC cell
rich region of primary and secondary lymph nodes, e.g., via high
endothelial venules (HEV). The memory cells are also primed to
become sensitive to IL-12 stimulation. Thus, rapid and high IFN
production would quickly follow IL-12 induction by antigen. Thus
p40/IL-B30 may accelerate an immune response by memory cells,
either by increasing response rate, increasing memory cell numbers,
or both. The p40/IL-B30 may have differential effects specific for
memory cells, with lesser or no effect on naive cells. Conversely,
in many chronic inflammatory conditions, e.g., rheumatoid
arthritis, inflammatory bowel disease, psoriasis, etc., the active
lesions are dependent upon memory CD45Rb.sup.low cells. As such,
antagonists may effectively block the chronic phase of such an
inflammatory condition.
[0158] Various abnormal conditions are known in each of the cell
types shown to produce both IL-12 p40 and/or IL-B30 mRNA by
Northern blot analysis. See Berkow (ed.) The Merck Manual of
Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn et al.,
Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.; and
Weatherall et al., (eds.) Oxford Textbook of Medicine, Oxford
University Press, Oxford. Many other medical conditions and
diseases involve activation by macrophages or monocytes, and many
of these will be responsive to treatment by an agonist or
antagonist provided herein. See, e.g., Stites and Terr (eds. 1991)
Basic and Clinical Immunology Appleton and Lange, Norwalk, Conn.;
and Samter et al. (eds.), Immunological Diseases Little, Brown and
Co. These problems should be susceptible to prevention or treatment
using compositions provided herein.
[0159] The IL-12 p40/IL-B30 cytokine complex, antagonists,
antibodies, etc., can be purified and then administered to a
patient, veterinary or human. These reagents can be combined for
therapeutic use with additional active or inert ingredients, e.g.,
in conventional pharmaceutically acceptable carriers or diluents,
e.g., immunogenic adjuvants, along with physiologically innocuous
stabilizers, excipients, or preservatives. These combinations can
be sterile filtered and placed into dosage forms as by
lyophilization in dosage vials or storage in stabilized aqueous
preparations. This invention also contemplates use of antibodies or
binding fragments thereof, including forms which are not complement
binding.
[0160] Drug screening using IL-12 p40/IL-B30, fusion protein, or
fragments thereof, can be performed to identify compounds having
binding affinity to or other relevant biological effects on IL-12
p40/IL-B30 functions, including isolation of associated components.
Subsequent biological assays can then be utilized to determine if a
candidate compound has intrinsic stimulating activity and is
therefore a blocker or antagonist in that it blocks the activity of
the cytokine complex. Likewise, a compound having intrinsic
stimulating activity can activate the signal pathway and is thus an
agonist in that it simulates the activity of the cytokine complex.
This invention further contemplates the therapeutic use of blocking
antibodies to IL-12 p40, IL-B30, or the complex, as antagonists and
of stimulatory antibodies as agonists. This approach should be
particularly useful with other IL-12 p40 or IL-B30 species
variants.
[0161] The quantities of reagents necessary for effective therapy
will depend upon many different factors, including means of
administration, target site, physiological state of the patient,
and other medicants administered. Thus, treatment dosages should be
titrated to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts useful for in
situ administration of these reagents. Animal testing of effective
doses for treatment of particular disorders will provide further
predictive indication of human dosage. Various considerations are
described, e.g., in Gilman et al., (eds. 1990) Goodman and
Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,
Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.
(1990), Mack Publishing Co., Easton, Pa. Methods for administration
are discussed therein and below, e.g., for oral, intravenous,
intraperitoneal, or intramuscular administration, transdermal
diffusion, and others. Pharmaceutically acceptable carriers will
include water, saline, buffers, and other compounds described,
e.g., in the Merck Index, Merck & Co., Rahway, N.J. Dosage
ranges would ordinarily be expected to be in amounts lower than 1
mM concentrations, typically less than about 10 .mu.M
concentrations, usually less than about 100 nM, preferably less
than about 10 pM (picomolar), and most preferably less than about 1
fM (femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be utilized
for continuous or long term administration. See, e.g., Langer,
(1990) Science 249:1527-1533.
[0162] IL-12 p40, IL-B30, cytokine complex, fusion proteins,
fragments thereof, and antibodies to it or its fragments,
antagonists, and agonists, may be administered directly to the host
to be treated or, depending on the size of the compounds, it may be
desirable to conjugate them to carrier proteins such as ovalbumin
or serum albumin prior to their administration. Therapeutic
formulations may be administered in many conventional dosage
formulations. While it is possible for the active ingredient to be
administered alone, it is preferable to present it as a
pharmaceutical formulation. Formulations typically comprise at
least one active ingredient, as defined above, together with one or
more acceptable carriers thereof. Each carrier should be both
pharmaceutically and physiologically acceptable in the sense of
being compatible with the other ingredients and not injurious to
the patient. Formulations include those suitable for oral, rectal,
nasal, topical, or parenteral (including subcutaneous,
intramuscular, intravenous and intradermal) administration. The
formulations may conveniently be presented in unit dosage form and
may be prepared by many methods well known in the art of pharmacy.
See, e.g., Gilman et al., (eds. 1990) Goodman and Gilman's: The
Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and
Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack
Publishing Co., Easton, Pa.; Avis et al., (eds. 1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New
York; Lieberman et al., (eds. 1990) Pharmaceutical Dosage Forms:
Tablets, Dekker, New York; and Lieberman et al., (eds. 1990)
Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.
The therapy of this invention may be combined with or used in
association with other agents, e.g., other cytokines, including
IL-6 or G-CSF, or their respective antagonists.
[0163] Both naturally occurring and recombinant forms of the
IL-B30s of this invention are particularly useful in kits and assay
methods which are capable of screening compounds for binding
activity to the proteins. Several methods of automating assays have
been developed in recent years so as to permit screening of tens of
thousands of compounds in a short period. See, e.g., Fodor et al.,
(1991) Science 251:767-773, which describes means for testing of
binding affinity by a plurality of defined polymers synthesized on
a solid substrate. The development of suitable assays can be
greatly facilitated by the availability of large amounts of
purified, soluble IL-12 p40/IL-B30 cytokine complex as provided by
this invention.
[0164] Other methods can be used to determine the critical residues
in IL-12 p40/IL-B30 complex-receptor interactions. Mutational
analysis can be performed, e.g., see Somoza et al., (1993) J.
Exptl. Med. 178:549-558, to determine specific residues critical in
the interaction and/or signaling. PHD (Rost and Sander, (1994)
Proteins 19:55-72) and DSC (King and Sternberg, (1996) Protein Sci.
5:2298-2310) can provide secondary structure predictions of
.alpha.-helix (H), .beta.-strand (E), or coil (L). Helices A and D
are typically most important in receptor interaction, with the D
helix the more important region.
[0165] For example, antagonists can normally be found once the
antigen and/or receptor has been structurally defined, e.g., by
tertiary structure data. Testing of potential interacting analogs
is now possible upon the development of highly automated assay
methods using a purified IL-12 p40/IL-B30 complex. In particular,
new agonists and antagonists will be discovered by using screening
techniques described herein. Of particular importance are compounds
found to have a combined binding affinity for a spectrum of IL-12
p40/IL-B30 molecules, e.g., compounds which can serve as
antagonists for species variants of the cytokine complex.
[0166] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing an IL-20 p40/IL-B30. Cells may
be isolated which express an IL-12 p40/IL-B30 in isolation from
other molecules. Such cells, either in viable or fixed form, can be
used for standard binding partner binding assays. See also, Parce
et al., (1989) Science 246:243-247; and Owicki et al., (1990) Proc.
Natl. Acad. Sci. USA 87:4007-4011, which describe sensitive methods
to detect cellular responses.
[0167] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to an IL-12 p40/IL-B30 and is described
in detail in Geysen, European Patent Application 84/03564,
published on Sep. 13, 1984. First, large numbers of different small
peptide test compounds are synthesized on a solid substrate, e.g.,
plastic pins or some other appropriate surface, see Fodor et al.,
(1991). Then all the pins are reacted with solubilized, unpurified
or solubilized, purified p40/IL-B30, and washed. The next step
involves detecting bound p40/IL-B30.
[0168] Rational drug design may also be based upon structural
studies of the molecular shapes of the p40/IL-B30 and other
effectors or analogs. Effectors may be other proteins which mediate
other functions in response to binding, or other proteins which
normally interact with p40/IL-B30, e.g., a receptor. One means for
determining which sites interact with specific other proteins is a
physical structure determination, e.g., x-ray crystallography or 2
dimensional NMR techniques. These will provide guidance as to which
amino acid residues form molecular contact regions, as modeled,
e.g., against other cytokine-receptor models. For a detailed
description of protein structural determination, see, e.g.,
Blundell and Johnson, (1976) Protein Crystallography, Academic
Press, New York.
IX. Kits
[0169] This invention also contemplates use of p40/IL-B30 proteins,
fragments thereof, peptides, and their fusion products in a variety
of diagnostic kits and methods for detecting the presence of
another p40/IL-B30 or binding partner. Typically the kit will have
a compartment containing either a defined p40, p40/IL-B30, or
IL-B30 peptide or gene segment or a reagent which recognizes one or
the other, e.g., p40/IL-B30 fusion fragments or antibodies.
[0170] A kit for determining the binding affinity of a test
compound to an IL-12 p40/IL-B30 would typically comprise a test
compound; a labeled compound, for example a binding partner or
antibody having known binding affinity for p40/IL-B30; a source of
p40/IL-B30 (naturally occurring or recombinant); and a means for
separating bound from free labeled compound, such as a solid phase
for immobilizing the molecule. Once compounds are screened, those
having suitable binding affinity to the antigen can be evaluated in
suitable biological assays, as are well known in the art, to
determine whether they act as agonists or antagonists to the
p40/IL-B30 signaling pathway. The availability of recombinant IL-12
p40/IL-B30 fusion polypeptides also provide well defined standards
for calibrating such assays.
[0171] A preferred kit for determining the concentration of, e.g.,
a p40/IL-B30 in a sample would typically comprise a labeled
compound, e.g., binding partner or antibody, having known binding
affinity for the antigen, a source of cytokine (naturally occurring
or recombinant) and a means for separating the bound from free
labeled compound, e.g., a solid phase for immobilizing the
p40/IL-B30. Compartments containing reagents, and instructions,
will normally be provided.
[0172] Antibodies, including antigen binding fragments, specific
for the p40/IL-B30 or fragments are useful in diagnostic
applications to detect the presence of elevated levels of p40,
IL-B30, p40/IL-B30, and/or its fragments. Such diagnostic assays
can employ lysates, live cells, fixed cells, immunofluorescence,
cell cultures, body fluids, and further can involve the detection
of antigens related to the antigen in serum, or the like.
Diagnostic assays may be homogeneous (without a separation step
between free reagent and antigen-binding partner complex) or
heterogeneous (with a separation step). Various commercial assays
exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied
immunoassay technique (EMIT), substrate-labeled fluorescent
immunoassay (SLFIA), and the like. See, e.g., Van Vunakis et al.,
(1980) Meth Enzymol. 70:1-525; Harlow and Lane, (1980) Antibodies:
A Laboratory Manual, CSH Press, NY; and Coligan et al., (eds. 1993)
Current Protocols in Immunology, Greene and Wiley, NY.
[0173] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against a p40/IL-B30, as such may be
diagnostic of various abnormal states. For example, overproduction
of p40/IL-B30 may result in production of various immunological
reactions which may be diagnostic of abnormal physiological states,
particularly in proliferative cell conditions such as cancer or
abnormal activation or differentiation.
[0174] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody or
binding partner, or labeled p40/IL-B30 is provided. This is usually
in conjunction with other additives, such as buffers, stabilizers,
materials necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also contain
instructions for proper use and disposal of the contents after use.
Typically the kit has compartments for each useful reagent.
Desirably, the reagents are provided as a dry lyophilized powder,
where the reagents may be reconstituted in an aqueous medium
providing appropriate concentrations of reagents for performing the
assay.
[0175] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In many of these assays, the binding partner, test compound,
p40/IL-B30, or antibodies thereto can be labeled either directly or
indirectly. Possibilities for direct labeling include label groups:
radiolabels such as .sup.125I, enzymes such as peroxidase and
alkaline phosphatase, and fluorescent labels (U.S. Pat. No.
3,940,475) capable of monitoring the change in fluorescence
intensity, wavelength shift, or fluorescence polarization.
Possibilities for indirect labeling include biotinylation of one
constituent followed by binding to avidin coupled to one of the
above label groups.
[0176] There are also numerous methods of separating the bound from
the free p40/IL-B30, or alternatively the bound from the free test
compound. The p40/IL-B30 can be immobilized on various matrixes
followed by washing. Suitable matrixes include plastic such as an
ELISA plate, filters, and beads. See, e.g., Coligan et al., (eds.
1993) Current Protocols in Immunology, Vol. 1, Chapter 2, Greene
and Wiley, NY. Other suitable separation techniques include,
without limitation, the fluorescein antibody magnetizable particle
method described in Rattle et al., (1984) Clin. Chem. 30:1457-1461,
and the double antibody magnetic particle separation as described
in U.S. Pat. No. 4,659,678.
[0177] Methods for linking proteins or their fragments to the
various labels have been extensively reported in the literature and
do not require detailed discussion here. Many of the techniques
involve the use of activated carboxyl groups either through the use
of carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction of a mercapto group with an
activated halogen such as chloroacetyl, or an activated olefin such
as maleimide, for linkage, or the like. Fusion proteins will also
find use in these applications.
[0178] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of a p40/IL-B30. These sequences can be used as probes for
detecting levels of the p40 or IL-B30 messages in samples from
patients suspected of having an abnormal condition, e.g.,
inflammatory or autoimmune. Since the cytokine may be a marker or
mediator for activation, it may be useful to determine the numbers
of activated cells to determine, e.g., when additional therapy may
be called for, e.g., in a preventative fashion before the effects
become and progress to significance. The preparation of both RNA
and DNA nucleotide sequences, the labeling of the sequences, and
the preferred size of the sequences has received ample description
and discussion in the literature. See, e.g., Langer-Safer et al.,
(1982) Proc. Natl. Acad. Sci. 79:4381-4385; Caskey, (1987) Science
236:962-967; and Wilchek et al., (1988) Anal. Biochem.
171:1-32.
[0179] Diagnostic kits which also test for the qualitative or
quantitative expression of other molecules are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple
indications used as markers. Thus, kits may test for combinations
of markers. See, e.g., Viallet et al., (1989) Progress in Growth
Factor Res. 1:89-97. Other kits may be used to evaluate other cell
subsets.
X. Isolating a p40/IL-B30 Receptor
[0180] Having isolated a ligand of a specific ligand-receptor
interaction, methods exist for isolating the receptor. See, Gearing
et al., (1989) EMBO J. 8:3667-3676. For example, means to label the
IL-B30 cytokine without interfering with the binding to its
receptor can be determined. For example, an affinity label can be
fused to either the amino- or carboxyl-terminus of the ligand. Such
label may be a FLAG epitope tag, or, e.g., an Ig or Fc domain. An
expression library can be screened for specific binding of the
cytokine, e.g., by cell sorting, or other screening to detect
subpopulations which express such a binding component. See, e.g.,
Ho et al., (1993) Proc. Natl. Acad. Sci. USA 90:11267-11271; and
Liu et al., (1994) J. Immunol. 152:1821-29. Alternatively, a
panning method may be used. See, e.g., Seed and Aruffo, (1987)
Proc. Natl. Acad. Sci. USA 84:3365-3369.
[0181] Protein cross-linking techniques with label can be applied
to isolate binding partners of the p40/IL-B30 cytokine complex.
This would allow identification of proteins which specifically
interact with the cytokine, e.g., in a ligand-receptor like
manner.
[0182] Early experiments will be performed to determine whether the
known IL-6 or G-CSF receptor components are involved in response(s)
to p40/IL-B30. It is also quite possible that these functional
receptor complexes may share many or all components with a
p40/IL-B30 receptor complex, either a specific receptor subunit or
an accessory receptor subunit.
[0183] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
EXAMPLES
I. General Methods
[0184] Many of the standard methods below are described or
referenced, e.g., in Maniatis et al., (1982) Molecular Cloning, A
Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor
Press, NY; Sambrook et al., (1989) Molecular Cloning: A Laboratory
Manual (2d ed.) Vols. 1-3, CSH Press, NY; Ausubel et al., Biology
Greene Publishing Associates, Brooklyn, N.Y.; Ausubel et al., (1987
and Supplements) Current Protocols in Molecular Biology
Wiley/Greene, NY; Innis et al., (eds. 1990) PCR Protocols: A Guide
to Methods and Applications Academic Press, NY; Bonifacino et al.,
Current Protocols in Cell Biology Wiley, NY; and Doyle et al., Cell
and Tissue Culture: Laboratory Protocols Wiley, NY. Methods for
protein purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel et
al., (1987 and periodic supplements); Deutscher, (1990) "Guide to
Protein Purification," Methods in Enzymology vol. 182, and other
volumes in this series; Coligan et al., (1995 and supplements)
Current Protocols in Protein Science John Wiley and Sons, New York,
N.Y.; Matsudaira, (ed. 1993) A Practical Guide to Protein and
Peptide Purification for Microsequencing, Academic Press, San
Diego, Calif.; and manufacturer's literature on use of protein
purification products, e.g., Pharmacia, Piscataway, N.J., or
Bio-Rad, Richmond, Calif. Combination with recombinant techniques
allow fusion to appropriate segments (epitope tags), e.g., to a
FLAG sequence or an equivalent which can be fused, e.g., via a
protease-removable sequence. See, e.g., Hochuli (1990)
"Purification of Recombinant Proteins with Metal Chelate Absorbent"
in Setlow (ed.) Genetic Engineering, Principle and Methods
12:87-98, Plenum Press, NY; and Crowe et al., (1992) QIAexpress:
The High Level Expression & Protein Purification System
QUIAGEN, Inc., Chatsworth, Calif.
[0185] Computer sequence analysis is performed, e.g., using
available software programs, including those from the University of
Wisconsin Genetics Computer Group (GCG), Madison, Wis., the NCBI at
NIH, and GenBank, NCBI, EMBO, and other sources of public sequence.
Other analysis sources include, e.g., RASMOL program, see Bazan et
al., (1996) Nature 379:591; Lodi et al., (1994) Science
263:1762-1766; Sayle and Milner-White, (1995) TIBS 20:374-376; and
Gronenberg et al., (1991) Protein Engineering 4:263-269; and DSC,
see King and Sternberg, (1996) Protein Sci. 5:2298-2310. See, also,
Wilkins et al., (eds. 1997) Proteome Research: New Frontiers in
Functional Genomics Springer-Verlag, NY; Salzberg et al., (eds.
1998) Computational Methods in Molecular Biology Elsevier, NY; and
Birren et al., (eds. 1997) Genome Analysis: A Laboratory Manual
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
[0186] Standard immunological techniques are described, e.g., in
Hertzenberg et al., (eds. 1996) Weir's Handbook of Experimental
Immunology vols. 1-4, Blackwell Science; Coligan, (1991 and
updates) Current Protocols in Immunology Wiley/Greene, NY; and
Methods in Enzymology vols. 70, 73, 74, 84, 92, 93, 108, 116, 121,
132, 150, 162, and 163. Cytokine assays are described, e.g., in
Thomson, (ed. 1994) The Cytokine Handbook (2d ed.) Academic Press,
San Diego; Metcalf and Nicola, (1995) The Hematopoietic Colony
Stimulating Factors Cambridge University Press; and Aggarwal and
Gutterman, (1991) Human Cytokines Blackwell Pub.
[0187] Assays for vascular biological activities are well known in
the art. They will cover angiogenic and angiostatic activities in
tumor, or other tissues, e.g., arterial smooth muscle proliferation
(see, e.g., Koyoma et al., (1996) Cell 87:1069-1078), monocyte
adhesion to vascular epithelium (see McEvoy et al., (1997) J. Exp.
Med. 185:2069-2077), etc. See also Ross, (1993) Nature 362:801-809;
Rekhter and Gordon, (1995) Am. J. Pathol. 147:668-677; Thyberg et
al., (1990) Atherosclerosis 10:966-990; and Gumbiner, (1996) Cell
84:345-357.
[0188] Assays for neural cell biological activities are described,
e.g., in Wouterlood, (ed. 1995) Neuroscience Protocols modules 10,
Elsevier; Methods in Neurosciences Academic Press; and Neuromethods
Humana Press, Totowa, N.J. Methodology of developmental systems is
described, e.g., in Meisami (ed.) Handbook of Human Growth and
Developmental Biology CRC Press; and Chrispeels (ed.) Molecular
Techniques and Approaches in Developmental Biology
Interscience.
[0189] FACS analyses are described in Melamed et al., (1990) Flow
Cytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro,
(1988) Practical Flow Cytometry Liss, New York, N.Y.; and Robinson
et al., (1993) Handbook of Flow Cytometry Methods Wiley-Liss, New
York, N.Y.
II. Cloning of Human p40 and/or IL-B30
[0190] The IL-12 p40 sequences are available from various sequence
databases, as described above. The sequence of the IL-B30 gene is
provided in Table 1. The sequence is derived from a genomic human
sequence.
[0191] These sequences allow preparation of PCR primers, or probes,
to determine cellular distribution of the genes. The sequences
allow isolation of genomic DNA which encode the messages.
[0192] Using the probe or PCR primers, various tissues or cell
types are probed to determine cellular distribution. PCR products
are cloned using, e.g., a TA cloning kit (Invitrogen). The
resulting cDNA plasmids are sequenced from both termini on an
automated sequencer (Applied Biosystems).
III. Cellular Expression of p40 and IL-B30
[0193] An appropriate probe or primers specific for cDNA encoding
the respective genes are prepared. Typically, the probe is labeled,
e.g., by random priming. Coordinate expression of both subunits is
most important where the p40/IL-B30 complex is of interest.
IV. Purification of p40/IL-B30 Protein
[0194] Multiple transfected cell lines are screened for one which
expresses the cytokine or complex at a high level compared with
other cells. Alternatively, a combination recombinant construct can
be made. Various cell lines are screened and selected for their
favorable properties in handling. Individual isolation of the
respective subunits and combination thereafter may result in some
dimer formation. Natural IL-B30 can be isolated from natural
sources, or by expression from a transformed cell using an
appropriate expression vector. Adenovirus constructs can also be
used for production/expression.
[0195] Purification of the expressed subunits or complex is
achieved by standard procedures, or may be combined with engineered
means for effective purification at high efficiency from cell
lysates or supernatants. In particular, fusion of p40 to IL-B30,
with or without appropriate linker, can result in high efficiency
methods for processing or purification. FLAG or His.sub.6 segments
can be used for such purification features. Alternatively, affinity
chromatography may be used with specific antibodies, see below.
[0196] Protein is produced in coli, insect cell; or mammalian
expression systems, as desired.
V. Preparation of Antibodies Specific for p40/IL-B30
[0197] Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan, (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane, (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Immunoselection or depletion methods can be
applied to ensure that resulting antibodies are specific for
antigenic determinants presented by the complex of polypeptides,
distinct from those presented by the individual components
themselves. Polyclonal serum, or hybridomas may be prepared. In
appropriate situations, the binding reagent is either labeled as
described above, e.g., fluorescence or otherwise, or immobilized to
a substrate for panning methods. Immunoselection, immunodepletion,
and related techniques are available to prepare selective reagents,
as desired, e.g., for the complex between the two subunits.
VI. IL-12 p40 and IL-B30 Coprecipitate
[0198] A mouse IL-12 p40-Ig fusion construct was prepared in an
expression vector. The Ig domain binds to Protein A, and can
precipitate that polypeptide. An IL-B30Etag (epitope tagged with a
FLAG motif at the N terminus) construct was also prepared, which
polypeptide is immunoprecipitable with the M2 antibody. The
expression constructs were transfected into 293 T cells, either
with the IL-12 p40-Ig construct alone, the IL-B30Etag construct
alone, or both together. Cells were labeled with .sup.35S
methionine. With the IL-12 p40 construct alone, no soluble protein
was detected in the cell supernatant using Protein A. Likewise,
with the FLAG-IL-B30 construct, no soluble protein was detected in
the cell supernatant using the M2 antibody. However, with
cotransfection of the two expression constructs, the cell
supernatant produced a soluble complex which was precipitable with
either the Protein A reagent or the M2 antibody. PAGE analysis of
the complex revealed that the Protein A precipitated complex was
made of polypeptides corresponding to the two expected polypeptides
IL-12 p40-Ig fusion and the FLAG-IL-B30 polypeptides.
Correspondingly, the complex precipitated with the M2 antibody was
made up of the FLAG-IL-B30 polypeptide and the IL-12 p40-Ig fusion
protein.
[0199] Similar experiments with a human IL-12 p40 expression
construct and a human FLAG-IL-B30 construct provided the expected
results. Transfection with the FLAG-IL-B30 construct resulted in no
significant soluble protein. Cotransfection of both expression
vectors into primate cells resulted in effective secretion of a
complex which was immunoprecipitable with the M2 antibody. PAGE
analysis of the resulting complex confirmed that the complex was
made up of the FLAG-IL-B30 polypeptide and the IL-12 p40
polypeptide.
VII. Receptor Identification
[0200] The IL-12 receptor is made up of the IL-12 receptor subunits
.beta.1 and .beta.2. A fusion construct of p40/IL-B30 binds to
cells expressing the receptor subunit .beta.1.
[0201] A homodimer of the IL-12 p40 subunits can block the binding
of IL-12 to the mouse subunit .beta.1, but not to the subunit
.beta.2. The p40 subunit is a component of the p40/IL-B30 complex,
so it was tested whether the IL-12 receptor subunit .beta.1 could
be a component of the receptor for a fusion construct of
p40/IL-B30. Antibodies to the IL-12 receptor subunit .beta.1 block
binding of the fusion construct to cells expressing the receptor
subunit .beta.1. Antibodies against the p40/p70 complex, mainly
recognizing the p40 subunit, can block the effect of the p40/IL-B30
composition, suggesting that the p40 component is important in
receptor interaction. These observations suggest that the receptor
subunit .beta.1 binds to the p40/IL-B30 fusion construct. Similar
experiments testing involvement of the common gp130 subunit shared
among related receptors suggest that the gp130 is not a relevant
subunit of the receptor for p40/IL-B30.
[0202] Having identified one subunit of the receptor, expression
cloning efforts have been initiated. Cells expressing this one
subunit but showing no binding will be used to expression clone an
additional subunit. Other receptor subunit .beta.2 homologs are
being screened. Alternatively, libraries from appropriate cells can
be used in standard expression cloning methods.
VIII. Evaluation of Breadth of Biological Functions
[0203] Biological activities of p40/IL-B30 complex are tested
based, e.g., on the sequence and structural homology between IL-B30
and IL-6 and G-CSF. Initially, assays that had shown biological
activities of IL-6 or G-CSF were examined. Assays were performed on
either recombinant complex or fusion construct. Fusion construct
consisted of a construct with the IL-12 p40 signal sequence linked
to an N terminal FLAG epitope fused to the mature IL-12 p40
sequence fused to a ser/gly rich linker sequence of appropriate
length fused to the mature sequence of IL-B30. This construct both
expresses well, is secreted, and the epitope tag allows both
purification and localization. Both mouse and human sequence forms
were generated. Adenovirus expression constructs of both separate
polypeptides and fusion proteins are also made available.
[0204] Target cell types include lympoid, myeloid, mast, pre-B,
pre-T, and fibroblast-endothelial cell types. For example,
macrophage/monocyte cells will be evaluated for cell surface marker
changes, e.g., MHC class II, B7, CD40, and related families;
cytokine and chemokine production; and antigen presentation
capacity. CD4+ T cells, both naive CD45Rb.sup.hi and memory
CD45Rb.sup.low T cells, will be assayed, e.g., for growth and
activation markers, and for effector functions, e.g., cytokine and
chemokine production. Cytotoxic CD4+, CD8+ and NK cells will be
evaluated for effects on generation and function. Effects on
antibody production will be tested, e.g., on splenic and MLN B
cells. Dendritic cells will be evaluated for generation,
maturation, and function, including factor production. Apoptosis
assays are also being developed.
[0205] Long term bone marrow cultures will be tested for effects on
modulation of stroma cells and stem cell generation and
differentiation (Dexter cultures), for modulation of stromal cells
and B cell progenitor generation and differentiation
(Whitlock-Witte cultures), and for evaluation of potential to
regulate primitive myeloid and B lymphoid populations.
[0206] A. Effects on Proliferation of Cells
[0207] The effect on proliferation of various cell types are
evaluated with various concentrations of cytokine. A dose response
analysis is performed, in combinations with the related cytokines
IL-6, G-CSF, etc. A cytosensor machine may be used, which detects
cell metabolism and growth (Molecular Devices, Sunnyvale,
Calif.).
[0208] Human p40/IL-B30 fusion protein enhanced proliferation of
human PHA blasts stimulated with anti-CD3 or both anti-CD3 and
anti-CD28. The anti-CD3 stimulation appears to be essential. Human
p40/IL-B30 fusion protein also enhanced proliferation of activated
Th1 or Th2 cell clones, but not resting Th1 or Th2 cell clones.
[0209] Either mouse or human fusion protein worked on mouse target
cells. Fusion protein supported proliferation of CD4+
CD45Rb.sup.low CD62L.sup.low CD44.sup.hi cells (memory/activated T
cells) when stimulated with anti-CD3. Stimulation by fusion protein
is not enhanced by anti-CD28 costimulation. This is not grossly
dependent on presence of IL-2. This suggests that p40/IL-B30 may be
an important factor for expanding a population of cells with a
memory phenotype and/or generating or maintaining immunologic
memory. This cytokine seems to selectively support activated memory
cells with a Th1 phenotype, e.g., cells which produce IFN.gamma.,
but no IL-4 or IL-5.
[0210] B. Effects on Differentiation of Naive T Cells
[0211] Human cord blood cells were collected and naive CD4+ T cells
were isolated. These were cultured, e.g., for 2 weeks, in the
presence of anti-CD3 and IL-2 and with irradiated fibroblasts
expressing CD32, CD58, and CD80, thereby activating and
proliferating T cells. The T cell culture was evaluated for effects
of various cytokines on proliferation or differentiation.
Individual cells were evaluated for cytokine production by FACS
analysis. The p40/IL-B30 fusion protein supported the proliferation
and differentiation of T cells producing IFN.gamma. and no IL-4, a
cytokine expression profile characteristic of Th1 cells.
[0212] C. Effects on the Expression of Cell Surface Molecules
[0213] Monocytes are purified by negative selection from peripheral
blood mononuclear cells of normal healthy donors. Briefly,
3.times.10.sup.8 ficoll banded mononuclear cells are incubated on
ice with a cocktail of monoclonal antibodies (Becton-Dickinson;
Mountain View, Calif.) consisting, e.g., of 200 .mu.l of .alpha.CD2
(Leu-5A), 200 .mu.l of .alpha.CD3 (Leu-4), 100 .mu.l of .alpha.CD8
(Leu 2a), 100 .mu.l of .alpha.CD19 (Leu-12), 100 .mu.l of
.alpha.CD20 (Leu-16), 100 .mu.l of .alpha.CD56 (Leu-19), 100 .mu.l
of .alpha.CD67 (IOM 67; Immunotech, Westbrook, Me.), and
anti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody
bound cells are washed and then incubated with sheep anti-mouse IgG
coupled magnetic beads (Dynal, Oslo, Norway) at a bead to cell
ratio of 20:1. Antibody bound cells are separated from monocytes by
application of a magnetic field. Subsequently, human monocytes are
cultured in Yssel's medium (Gemini Bioproducts, Calabasas, Calif.)
containing 1% human AB serum in the absence or presence of IL-B30,
IL-6, G-CSF or combinations.
[0214] Analyses of the expression of cell surface molecules can be
performed by direct immunofluorescence. For example,
2.times.10.sup.5 purified human monocytes are incubated in
phosphate buffered saline (PBS) containing 1% human serum on ice
for 20 minutes. Cells are pelleted at 200.times.g. Cells are
resuspended in 20 ml PE or FITC labeled mAb. Following an
additional 20 minute incubation on ice, cells are washed in PBS
containing 1% human serum followed by two washes in PBS alone.
Cells are fixed in PBS containing 1% paraformaldehyde and analyzed
on FACScan flow cytometer (Becton Dickinson; Mountain View,
Calif.). Exemplary mAbs are used, e.g.: CD11b (anti-mac1), CD11c (a
gp150/95), CD14 (Leu-M3), CD54 (Leu 54), CD80 (anti-BB1/B7), HLA-DR
(L243) from Becton-Dickinson and CD86 (FUN 1; Pharmingen), CD64
(32.2; Medarex), CD40 (mAb89; Schering-Plough France).
[0215] D. Effects on Cytokine Production by Human Cells
[0216] Human monocytes are isolated as described and cultured in
Yssel's medium (Gemini Bioproducts, Calabasas, Calif.) containing
1% human AB serum in the absence or presence of IL-B30 (1/100
dilution baculovirus expressed material). In addition, monocytes
are stimulated with LPS (E. coli 0127:B8 Difco) in the absence or
presence of IL-B30 and the concentration of cytokines (IL-1.beta.,
IL-6, TNF.alpha., GM-CSF, and IL-10) in the cell culture
supernatant determined by ELISA.
[0217] For intracytoplasmic staining for cytokines, monocytes are
cultured (1 million/ml) in Yssel's medium in the absence or
presence of IL-B30 and LPS (E. coli 0127:B8 Difco) and 10 mg/ml
Brefeldin A (Epicentre technologies Madison Wis.) for 12 hrs. Cells
are washed in PBS and incubated in 2% formaldehyde/PBS solution for
20 minutes at RT. Subsequently cells are washed, resuspended in
permeabilization buffer (0.5% saponin (Sigma) in PBS/BSA
(0.5%)/Azide (1 mM)) and incubated for 20 minutes at RT. Cells
(2.times.10.sup.5) are centrifuged and resuspended in 20 ml
directly conjugated anti-cytokine mAbs diluted 1:10 in
permeabilization buffer for 20 minutes at RT. The following
antibodies can be used: IL-1.alpha.-PE (364-3B3-14); IL-6-PE
(MQ2-13A5); TNF.alpha.-PE (MAb11); GM-CSF-PE (BVD2-21C11); and
IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.). Subsequently,
cells are washed twice in permeabilization buffer and once in
PBS/BSA/Azide and analyzed on FACScan flow cytometer (Becton
Dickinson; Mountain View, Calif.).
[0218] Human PHA blasts produced IFN.gamma. in response to
contacting with human p40/IL-B30 fusion construct. The effects were
synergistic with IL-2. Fusion product enhanced IFN.gamma.
production by activated, but not resting T cells, resting Th1 cell
clones, or resting Th2 cell clones.
[0219] E. Effects on Proliferation of Human Peripheral Blood
Mononuclear Cells (PBMC)
[0220] Total PBMC are isolated from buffy coats of normal healthy
donors by centrifugation through ficoll-hypaque as described (Boyum
et al.). PBMC are cultured in 200 .mu.l Yssel's medium (Gemini
Bioproducts, Calabasas, Calif.) containing 1% human AB serum in 96
well plates (Falcon, Becton-Dickinson, N.J.) in the absence or
presence of IL-B30. Cells are cultured in medium alone or in
combination with 100 U/ml IL-2 (R&D Systems) for 120 hours.
3H-Thymidine (0.1 mCi) is added during the last six hours of
culture and 3H-Thymidine incorporation determined by liquid
scintillation counting.
[0221] The native, recombinant, and fusion proteins would be tested
for agonist and antagonist activity in many other biological assay
systems, e.g., on T-cells, B-cells, NK, macrophages, dendritic
cells, hematopoietic progenitors, etc. Because of the IL-6 and
G-CSF structural relationship, assays related to those activities
should be analyzed
[0222] p40/IL-B30 is evaluated for agonist or antagonist activity
on transfected cells expressing IL-6 or G-CSF receptor and
controls. See, e.g., Ho et al., (1993) Proc. Natl. Acad. Sci. USA
90, 11267-11271; Ho et al., (1995) Mol. Cell. Biol. 15:5043-5053;
and Liu et al., (1994). J. Immunol. 152:1821-1829.
[0223] p40/IL-B30 is evaluated for effect in macrophage/dendritic
cell activation and antigen presentation assays, T cell cytokine
production and proliferation in response to antigen or allogenic
stimulus. See, e.g., de Waal Malefyt et al., (1991) J. Exp. Med.
174:1209-1220; de Waal Malefyt et al., (1991) J. Exp. Med.
174:915-924; Fiorentino et al., (1991) J. Immunol. 147, 3815-3822;
Fiorentino et al., (1991) J. Immunol. 146:3444-3451; and Groux et
al., (1996) J. Exp. Med. 184:19-29.
[0224] p40/IL-B30 will also be evaluated for effects on NK cell
stimulation. Assays may be based, e.g., on Hsu et al., (1992)
Internat. Immunol. 4:563-569; and Schwarz et al., (1994) J.
Immunother. 16:95-104.
[0225] B cell growth and differentiation effects will be analyzed,
e.g., by the methodology described, e.g., in Defrance et al.,
(1992). J. Exp. Med. 175:671-682; Rousset et al., (1992) Proc.
Natl. Acad. Sci. USA 89:1890-1893; including IgG2 and IgA2 switch
factor assays.
IX. Generation and Analysis of Genetically Altered Animals
[0226] Transgenic mice can be generated by standard methods. Such
animals are useful to determine the effects of overexpression of
the genes, in specific tissues, or completely throughout the
organism. Such may provide interesting insight into development of
the animal or particular tissues in various stages. Moreover, the
effect on various responses to biological stress can be evaluated.
See, e.g., Hogan et al., (1995) Manipulating the Mouse Embryo: A
Laboratory Manual (2d ed.) Cold Spring Harbor Laboratory Press.
[0227] Adenovirus techniques are available for expression of the
genes in various cells and organs. See, e.g., Hitt et al., (1997)
Adv. Pharmacol. 40:137-195; and literature from Quantum
Biotechnologies, Montreal, Canada. Animals may be useful to
determine the effects of the genes on various developmental or
physiologically functional animal systems.
[0228] A 0.5 kb cDNA encoding for IL-B30 was cloned as an EcoRI
fragment into an expression vector containing the CMV enhancer
.beta.-actin promoter and the rabbit .beta.-globin polyadenylation
signal, previously described by Niwa et al., (1991) Gene
108:193-200. Separation of the transgene from vector sequence was
accomplished by zonal sucrose gradient centrifugation as described
by Mann et al., (1993) "Factor Influencing Production Frequency of
Transgenic Mice", Methods in Enzymology 225: 771-781. Fractions
containing the transgene were pooled, microcentrifugation through
Microcon-100 filters and washed 5 times with microinjection buffer
(5 mM Tris-HCl, pH 7.4, 5 mM NaCl, 0.1 mM EDTA).
[0229] The transgene was resuspended in microinjection buffer (5 mM
Tris-HCl, pH 7.4, 5 mM NaCl, 0.1 mM EDTA) to a final concentration
of 1-5 ng/ml, microinjected into ([C57BL/6J.times.DBA/2]F.sub.1;
The Jackson Laboratory) eggs, which were then transferred into
oviducts of ICR (Sprague-Dawley) foster mothers, according to
published procedures by Hogan et al., (1994) Manipulation of the
Mouse Embryo, Plainview, N.Y., Cold Spring Harbor Laboratory Press.
By 10 days of life, a piece of tail from the resulting animals was
clipped for DNA analysis. Identification of transgenic founders was
carried out by polymerase chain reaction (PCR) analysis, as
previously described by Lira et al., (1990) Proc. Natl. Acad. Sci.
USA, 87: 7215-7219. Identification of the IL-B30 transgenic mice
was accomplished by amplification of mouse tail DNA. As an internal
control for the amplification reaction primers for the endogenous
LDL gene were used. The primers amplify a 200 bp segment of the
IL-B30 transgene and 397 bp segment of the LDL gene. PCR conditions
were: 95.degree. C., 30 seconds; 60.degree. C., 30 seconds;
72.degree. C., 60 seconds for 30 cycles. Transgenic animals were
kept under pathogen-free conditions.
Analysis of IL-B30 Transgenic Mice
[0230] RNA was extracted from tissues using RNA STAT-60, following
specifications from the manufacturer (TEL-TEST, Inc. Friendswood,
Tex.). Total RNA (20 mg) was denatured and blotted onto Biotrans
membrane (ICN Biomedicals, Costa Mesa, Calif.). Transgene
expression was assessed by hybridization to randomly labeled L-30
cDNA (Stratagene, La Jolla, Calif.). Acute phase liver gene
expression was assessed by hybridizing total RNA with randomly
labeled PCR segments of the murine hemopexin gene, of the murine
alpha-1-acid glycoprotein, and of the murine haptoglobin gene.
[0231] ELISA kits were purchased from commercial sources and run
according to the manufacturer's instructions. ELISA kits for murine
IL-2 (sensitivity <3 pg/ml), murine IL-1b (sensitivity <3
pg/ml), murine IFN-gamma (sensitivity <2 pg/ml) and murine
TNF-alpha (sensitivity <5.1 pg/ml) were purchased from R & D
systems (Minneapolis, Minn.). Murine IL-6 ELISA kits (sensitivity
<8 pg/ml) were purchased from Biosource International
(Camarillo, Calif.). Murine IL-1.alpha. ELISA kits (sensitivity
<6 pg/ml) were purchased from Endogen (Cambridge, Mass.).
[0232] ELISA assays for serum immunoglobulin levels were run using
antibody pairs purchased from PharMingen (San Diego, Calif.)
following the manufacturer's guidelines. Anti-mouse IgM (clone
11/41), anti-mouse IgA (clone R5-140), anti-mouse IgG1 (clone
A85-3), anti-mouse IgG2a (clone R11-89) and anti-mouse IgG2b (clone
R9-91) were used as capture antibodies. Purified mouse IgM (clone
G155-228), IgA (clone M18-254), IgG1 (clone 107.3), IgG2a (clone
G155-178) and IgG2b (clone 49.2) were used to generate standard
curves. Biotin anti-mouse IgM (clone R6-60.2), biotin anti-mouse
IgA (clone R5-140), biotin anti-mouse IgG1 (clone A85-1), biotin
anti-mouse IgG2a (clone R19-15) and biotin anti-mouse IgG2b (clone
R12-3) were used as detection antibodies.
[0233] Levels of IGF-1 in mouse serum were determined using a
commercially available radioimmunoassay for human IGF-1 that also
recognizes murine IGF-1 after serum samples were acid-ethanol
extracted according to instructions provided by the manufacturer
(Nichols Institute, San Juan Capistrano, Calif.).
[0234] After sacrifice, tissues were either snap frozen with
freezing media for cryosection, or fixed by immersion in 10%
phosphate-buffered formalin. Formalin fixed tissues were routinely
processed at 5 mm, and were stained with hematoxylin and eosin (H
& E). For immunostaining, snap frozen sections were fixed with
acetone and air dried.
[0235] Blood samples were collected from the infra-orbital sinus
into sterile, evacuated tubes with added EDTA (Vacutainer Systems,
Becton Dickinson, Rutherford, N.J.). Hematologic values were
determined with an automated system (Abbot Cell-Dyn 3500, Abbot
Park, Ill.). Platelet counts were performed manually when the
instrument was unable to provide accurate platelet counts due to
excessive clumping or excessively large platelets. Blood smears
were stained with Modified Wright-Giemsa stain (Hema-Tek Stain
Pack, Bayer Corp., Elkhart, Ind.) using an automated stainer (Bayer
Hema-Tek 2000, Elkhart, Ind.) and examined manually for immature
cells and platelet, red blood cell, and white blood cell
morphology.
Bone Marrow Transfer
[0236] The femur and tibia were cleaned of muscle and bone marrow
was expelled by flushing the bone with PBS. Bone marrow cells were
washed once, and injected i.v. into recipient mice which had been
lethally irradiated (1000 RAD).
Phenotype of IL-B30 Transgenic Mice
[0237] To analyze the biological function of IL-B30, the gene was
expressed under the control of the CMV enhancer/actin promoter,
described by Niwa et al., (1991) Gene 108:193-200, in transgenic
mice. This enhancer/promoter cassette directs high levels of
transgene expression primarily to skeletal muscle and pancreas, but
the transgene can be expressed in virtually all organs and cells.
See Lira et al., (1990) Proc. Natl. Acad. Sci. USA 87:
7215-7219.
[0238] IL-B30 transgenic mice were runted compared to control
littermates. The rate of body weight gain in IL-B30 transgenic mice
varied widely but was clearly lower than that found in control
littermates. Northern blot analysis, of RNA extracted from either
muscle or skin of IL-B30 transgenic mice and control littermates
hybridized to IL-B30 cDNA, revealed that IL-B30 mRNA was detected
in both muscle and skin of all IL-B30 transgenic mice, whereas no
IL-B30 mRNA was detected in control littermates. This demonstrated
that stunted growth was always associated with expression of
IL-B30.
[0239] Of the IL-B30 transgenic mice obtained, 25% survived to
adulthood and were affected by expression of IL-B30 as exhibited by
impaired growth, a swollen abdomen, ruffled fur, infertility and
sudden death. Thus, transgenic expression of IL-B30 caused a
phenotype that prevented the generation of IL-B30 transgenic
progeny. The results presented here are derived from the
preliminary analysis of IL-B30 transgenic founder mice.
Histological Analysis of IL-B30 Transgenic Mice
[0240] Microscopical examination of tissues collected from IL-B30
transgenic mice revealed minimal to moderate inflammation in
multiple sites, including the lung, skin, esophagus, small
intestine and liver (bile ducts), large intestine, and pancreas.
Inflammatory infiltrates consisted of neutrophils, lymphocytes,
and/or macrophages. Inflammation in the skin was associated with
acanthosis and/or ulceration in some mice. In the lungs,
peribronchial mononuclear cell infiltrates were sometimes
prominent, alveolar walls contained increased numbers of
leukocytes, and the epithelium lining airways was hyperplastic.
Minimal periportal mononuclear cell infiltrates were also common in
the liver. The cortex of lymph nodes was sometimes sparsely
cellular and lacked follicular development.
[0241] Extramedullary hematopoiesis (EMH) was observed in the
liver, spleen, and lymph nodes. The EMH was especially marked in
the spleen. The spleens from three transgenic mice and one control
mouse were examined after immunohistological staining for T cells
(anti-CD3), B cells (anti-B220), and macrophages (anti-F4/80). In
the transgenic mice the CD3-, B220-, and F4/80-positive cells were
present in their normal locations. However, the white pulp was
separated by the EMH in the red pulp, and the positively staining
cells within the red pulp were interspersed with hematopoietic
cells that stained with less intensity, or did not stain
positively, with the various antibodies. These observations
demonstrate that transgenic expression of IL-B30 induces systemic
inflammation that is associated with EMH.
[0242] To analyze the effect of IL-B30 on leukocyte and platelet
counts in the peripheral blood, a complete blood analysis was
performed. The number of neutrophils in the blood of IL-B30
transgenic mice was increased 3- to 11-fold over the highest
neutrophil count in control littermates. Increases in peripheral
blood neutrophils are typical of inflammation and correlate with
the infiltration of neutrophils observed in various tissues.
Accordingly, the myeloid (granulocytic)/erythroid ratio was
increased in the bone marrow.
[0243] In addition, the number of circulating platelets was
increased up to 3-fold in IL-B30 transgenic mice over control
littermates. An increased number of platelets could originate from
either an increased number of megakaryocytes, or from an increase
in production of platelets by megakaryocytes. To test either
possibility, the peripheral blood, bone marrow and spleen from
IL-B30 transgenic mice were analyzed microscopically. In the
peripheral blood, platelets of bizarre morphology, including
elongated and spindle-shaped platelets, were frequently detected.
In bone marrow and spleen of some mice, megakaryocytes were
enlarged due to increased amounts of cytoplasm. In contrast, the
number of megakaryocytes in bone marrow and spleen was not
increased. This suggests that IL-B30 induces an increase in the
number of platelets by accelerating their production by
megakaryocytes.
[0244] All IL-B30 transgenic mice examined also suffered from mild
to moderate microcytic hypochromic anemia with schistocytes and
varying degrees of regeneration evident. The hematocrit values were
lower than the control mean by 36 to 70%. The presence of
microcytic hypochromic anemia suggests a defect in hemoglobin
production.
Cytokine Profile of IL-B30 Transgenic Mice
[0245] To test whether the systemic inflammation seen in IL-B30
transgenic mice correlated with altered expression of
pro-inflammatory cytokines, we determined the concentrations of
IL-1, TNF.alpha., IL-6, and IFN.gamma. in the peripheral blood. In
all IL-B30 transgenic mice tested, the levels of TNF.alpha. and
IFN.gamma. were increased. In addition, the level of IL-1 was
increased in 25% of IL-B30 transgenic mice tested. Concentrations
of IL-1 and TNF.alpha. found in IL-B30 transgenic mice reached
levels associated with the induction of an acute inflammatory
response by LPS. Surprisingly, no IL-6 was detected in the
peripheral blood of IL-B30 transgenic mice, even though expression
of IL-6 is highly induced under inflammatory conditions (Reinecker
et al., (1993) Clin. Exp. Immunology 94: 174-181; Stevens et al.,
(1992) Dig. Dis. Sci. 37: 818-826) and can be induced directly by
TNF.alpha., IL-1 and IFN.gamma. (Helle et al., (1988) Eur. J.
Immunol. 18: 957-959.
Acute Phase Genes in the Livers of IL-B30 Transgenic Mice
[0246] The body reacts to inflammation with an acute phase response
characterized by the expression of defined plasma proteins in the
liver. Since IL-B30 transgenic mice exhibit a phenotype
characteristic of systemic inflammation, we examined the expression
of acute phase genes in the livers of IL-B30 transgenic mice and
control littermates. The acute phase liver genes alpha-1-acid
glycoprotein, haptoglobin and hemopexin were highly expressed in
all IL-B30 transgenic mice tested, while no expression of these
genes was detected in control littermates. This demonstrates that
acute phase liver genes are constitutively expressed in IL-B30
transgenic animals.
Serum Immunoglobulins of IL-B30 Transgenic Mice
[0247] During an immune response, some cytokines induce B cell
differentiation and subsequent immunoglobulin synthesis. To test
whether immunoglobulin synthesis was altered in IL-B30 transgenic
mice, the concentrations of immunoglobulin isotypes in the
peripheral blood were determined. In 2 of 7 IL-B30 transgenic mice,
the concentration of IgA was increased 6- to 9-fold when compared
to control littermates. Furthermore, the concentrations of IgG1,
IgG2a and IgG2b were increased 2.5 to 6-fold in all IL-B30
transgenic mice tested when compared to control littermates. In
contrast, no significant increase in IgM or IgE titers could be
detected in any of the IL-B30 transgenic mice tested. In fact, 4 of
7 IL-B30 transgenic mice displayed markedly decreased levels of IgM
synthesis. In summary, a subset of IL-B30 transgenic mice displayed
a 6- to 9-fold increase in the concentrations of immunoglobulin
isotypes IgA and IgG, whereas no significant increase was detected
in the concentrations of immunoglobulin isotypes IgM and IgE.
Serum IGF-1 Levels in IL-B30 Transgenic Mice
[0248] Chronic inflammatory conditions (Kirschner and Sutton,
(1986) Gastroenterology 91: 830-836; Laursen et al., (1995) Arch.
Dis. Child. 72: 494-497) or overexpression of cytokines in
transgenic animals (De Benedetti et al., (1994) J. Clin. Invest.
93: 2114-2119) can cause growth impairment that is associated with
a decrease of insulin-like growth factor-1 (IGF-1). To test whether
stunted growth of IL-B30 transgenic mice could be traced to reduced
levels of IGF-1, serum samples of transgenic mice were assayed for
IGF-1. In all IL-B30 transgenic mice tested, the amount of IGF-1 in
the serum was 12 to 14% of the level found in age-matched control
littermates. This suggests that transgenic expression of IL-B30, as
well as the subsequent inflammatory response produced, results in
the reduction of IGF-1 in IL-B30 transgenic mice, and could
consequently be the cause of impaired growth and infertility (Gay
et al., (1997) Endocrinology 137(7): 2937-2947).
Expression of Biologically Active IL-B30 in Hematopoietic Cells
[0249] Cytokines are secreted proteins that regulate the immune
system locally or mediate long-range effects. To test whether
IL-B30 functions as a cytokine and can induce distant multi-organ
inflammation and an acute phase liver response, we transferred
IL-B30 transgenic bone marrow into lethally irradiated wildtype
recipient mice.
[0250] Bone marrow recipients were monitored weekly for the
induction of an acute phase response. Increased concentrations of
the acute phase protein SAA could be detected in IL-B30 bone marrow
recipients as early as 35 days post transfer and levels of SAA
increased over time. Concurrent with increasing concentrations of
SAA in the peripheral blood the health of IL-B30 bone marrow
recipients deteriorated as judged by the appearance of ruffled fur
and inflamed skin around the snout and throat. In contrast,
recipients of wildtype bone marrow did not have elevated levels of
SAA in blood, nor did they appear sick.
[0251] Animals were terminated when they appeared severely sick.
Expression of IL-B30 could be detected in the bone, marrow and
spleen of recipients of IL-B30 transgenic bone marrow, but not in
organs of recipients of wildtype bone marrow. As in IL-B30
transgenic donors skin, lung, liver, and the gastrointestinal tract
were inflamed in recipients of IL-B30 transgenic bone marrow, but
not in wildtype bone marrow recipients. Again acute phase liver
genes (hemopexin, AGP-1) were highly expressed in IL-B30 transgenic
bone marrow recipients, but no IL-6 could be detected in blood
serum. These results suggest that IL-B30 is a true cytokine with
long-ranging properties.
[0252] Transgenic expression of IL-B30 induces a striking phenotype
characterized by runting, systemic inflammation, infertility and
death of transgenic animals. IL-B30 transgenic animals have
systemic inflammation with infiltration of inflammatory cells into
lung, liver, skin, and the digestive tract.
[0253] Overexpression of IL-B30 in vivo caused a phenotype of
impaired growth and inflammation--that was strikingly similar to
that of several models of transgenic expression of IL-6. Similar to
the effect of transgenic expression of IL-6, or after
administration of recombinant IL-6 to mice, neutrophil infiltration
and anemia were observed in animals as a result of transgenic
expression of IL-B30. As in IL-6 transgenic animals, impaired
growth of IL-B30 transgenic founders was linked to decreased levels
of IGF-1 that might be related to the systemic inflammation
observed in these animals.
[0254] This phenotype of IL-B30 transgenic animals could be caused
by upregulated IL-6 expression either as a direct effect of IL-B30
overexpression, or by IL-B30 mediated upregulation of IL-1 and
TNF.alpha. expression. IL-1 and TNF.alpha. are known inducers of
IL-6 and increased concentrations of TNF.alpha. and IL-1 were found
in the peripheral blood of IL-B30 transgenic mice.
[0255] However, no IL-6 could be detected in blood of IL-B30
transgenic suggesting that the phenotype of IL-B30 animals is
directly linked to overexpression of this novel cytokine and, as
had been implied by their sequence homologies, that IL-B30 has
biological activities similar to IL-6.
[0256] IL-6 is a pleiotropic cytokine that among a wide variety of
functions induces thrombocytosis, acute-phase protein synthesis,
and B cell differentiation.
[0257] Indeed, IL-B30 transgenic animals express constitutively
acute phase liver genes like AGP-1, haptoglobin, hemopexin and
serum amyloid A protein. A similar phenotype has been shown in mice
as an effect of transgenic overexpression of IL-6, or after
administration of recombinant IL-6. In addition, transgenic
expression of IL-B30 resulted in thrombocytosis that was unusual in
that many of the platelets had bizarre morphology (elongated
appearance, large size, and/or spindle shapes). We suspect that
IL-B30 and/or other upregulated cytokines have an effect on normal
platelet production. This suggests again that IL-B30 shares a
biological activity with IL-6 and its relatives.
[0258] IL-6 has also been identified as a B cell differentiation
factor. Transgenic overexpression of IL-6 causes plasmocytoma and
IL-6 deficient mice show a reduced IgG response. While we saw
increases in IgG and IgA production in some IL-B30 transgenic mice,
this observation was not consistent between different founders.
Thus further analysis is needed to characterize IL-B30 further as a
potential B cell differentiation factor.
[0259] In IL-B30 transgenic mice increases in circulating
neutrophils were consistent with the inflammation evident in
various tissues, however, the changes in red blood cell parameters
are not as easily explained. IL-1, TNF-alpha, and IFN-gamma are
mediators of a syndrome commonly called anemia of chronic disease
(ACD), which generally presents as a normocytic, normochromic,
nonregenerative (or minimally regenerative) anemia and is seen in a
variety of chronic inflammatory diseases. Anemia of Chronic Disease
may also present as microcytic, hypochromic in some human patients.
The syndrome is due to altered iron metabolism and diminished
response to erythropoietin. The microcytic hypochromic anemia
observed in the IL-B30 mice may be due to ACD, as suggested by the
increases in peripheral cytokine concentrations. However, the most
common cause of microcytic hypochromic anemia is iron deficiency,
which is more consistent with the partial bone marrow response
(regeneration) and thrombocytosis seen in the IL-B30 mice. Further
investigation, including measurement of serum ferritin, iron, and
total iron binding capacity, which would allow differentiation of
ACD from iron deficiency anemia, was not undertaken due to the
difficulties in obtaining adequate blood from affected mice.
[0260] IL-1 and TNF-alpha are known inducers of IL-6 expression and
IL-6 expression is usually upregulated during an inflammatory
response. Therefore it is surprising that IL-6 could not be
detected in the peripheral blood of IL-B30 transgenic animals. This
suggests that IL-B30 has a negative effect on IL-6 expression by a
yet unidentified mechanism. Indeed the absence of IL-6 in IL-B30
transgenic animals might explain the high levels of IL-1 and
TNF-alpha observed in these animals since IL-6 has a negative
effect on the concentrations of circulating IL-1 and TNF-alpha in
mice. The high concentrations of circulating TNF-alpha observed in
IL-B30 transgenic mice could also be an result of the increased
concentrations of IFN-gamma. IFN-gamma is produced by IL-2
activated T cells or IL-4-activated B cells, and induces the
expression of TNF-alpha in monocytes and macrophages. It remains to
be determined whether expression of IFN-gamma is mediated directly
by IL-B30 or by other cytokines induced by IL-B30. In summary, our
results suggest that IL-B30 shares a wide variety of biological
activities with IL-6. It remains to be seen whether these
biological activities are mediated by a common receptor, a signal
transduction element or transcription factor shared with IL-6.
These issues will hopefully be clarified by ongoing experiments
using genetic and biochemical approaches.
[0261] All references cited herein are incorporated herein by
reference to the same extent as if each individual publication or
patent application was specifically and individually indicated to
be incorporated by reference in its entirety for all purposes.
[0262] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled.
Sequence CWU 1
1
361570DNAUnknownDescription of Unknown Organism surmised Homo
sapiens 1atg ctg ggg agc aga gct gta atg ctg ctg ttg ctg ctg ccc
tgg aca 48 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro
Trp Thr -20 -15 -10 gct cag ggc aga gct gtg cct ggg ggc agc agc cct
gcc tgg act cag 96 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro
Ala Trp Thr Gln-5 -1 1 5 10 tgc cag cag ctt tca cag aag ctc tgc aca
ctg gcc tgg agt gca cat 144 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr
Leu Ala Trp Ser Ala His 15 20 25 cca cta gtg gga cac atg gat cta
aga gaa gag gga gat gaa gag act 192 Pro Leu Val Gly His Met Asp Leu
Arg Glu Glu Gly Asp Glu Glu Thr 30 35 40 aca aat gat gtt ccc cat
atc cag tgt gga gat ggc tgt gac ccc caa 240 Thr Asn Asp Val Pro His
Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln 45 50 55 gga ctc agg gac
aac agt cag ttc tgc ttg caa agg atc cac cag ggt 288 Gly Leu Arg Asp
Asn Ser Gln Phe Cys Leu Gln Arg Ile His Gln Gly60 65 70 75ctg att
ttt tat gag aag ctg cta gga tcg gat att ttc aca ggg gag 336 Leu Ile
Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85 90
cct tct ctg ctc cct gat agc cct gtg gcg cag ctt cat gcc tcc cta 384
Pro Ser Leu Leu Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 95
100 105 ctg ggc ctc agc caa ctc ctg cag cct gag ggt cac cac tgg gag
act 432 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu
Thr 110 115 120 cag cag att cca agc ctc agt ccc agc cag cca tgg cag
cgt ctc ctt 480 Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln
Arg Leu Leu 125 130 135 ctc cgc ttc aaa atc ctt cgc agc ctc cag gcc
ttt gtg gct gta gcc 528 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala
Phe Val Ala Val Ala140 145 150 155gcc cgg gtc ttt gcc cat gga gca
gca acc ctg agt ccc taa 570 Ala Arg Val Phe Ala His Gly Ala Ala Thr
Leu Ser Pro 160 165 2189PRTUnknownsurmised Homo sapiens 2Met Leu
Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro Trp Thr -20 -15 -10
Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala Trp Thr Gln -5
-1 1 5 10 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp Ser
Ala His 15 20 25 Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly
Asp Glu Glu Thr 30 35 40 Thr Asn Asp Val Pro His Ile Gln Cys Gly
Asp Gly Cys Asp Pro Gln 45 50 55 Gly Leu Arg Asp Asn Ser Gln Phe
Cys Leu Gln Arg Ile His Gln Gly 60 65 70 75 Leu Ile Phe Tyr Glu Lys
Leu Leu Gly Ser Asp Ile Phe Thr Gly Glu 80 85 90 Pro Ser Leu Leu
Pro Asp Ser Pro Val Ala Gln Leu His Ala Ser Leu 95 100 105 Leu Gly
Leu Ser Gln Leu Leu Gln Pro Glu Gly His His Trp Glu Thr 110 115 120
Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp Gln Arg Leu Leu 125
130 135 Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe Val Ala Val
Ala 140 145 150 155 Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser
Pro 160 165 31203DNAUnknownDescription of Unknown Organism surmised
Mus sp. 3cgcttagaag tcggactaca gagttagact cagaaccaaa ggaggtggat
agggggtcca 60caggcctggt gcagatcaca gagccagcca gatctgagaa gcagggaaca
ag atg ctg 118 Met Leu -20gat tgc aga gca gta ata atg cta tgg ctg
ttg ccc tgg gtc act cag 166 Asp Cys Arg Ala Val Ile Met Leu Trp Leu
Leu Pro Trp Val Thr Gln -15 -10 -5 ggc ctg gct gtg cct agg agt agc
agt cct gac tgg gct cag tgc cag 214 Gly Leu Ala Val Pro Arg Ser Ser
Ser Pro Asp Trp Ala Gln Cys Gln -1 1 5 10 cag ctc tct cgg aat ctc
tgc atg cta gcc tgg aac gca cat gca cca 262 Gln Leu Ser Arg Asn Leu
Cys Met Leu Ala Trp Asn Ala His Ala Pro 15 20 25 gcg gga cat atg
aat cta cta aga gaa gaa gag gat gaa gag act aaa 310 Ala Gly His Met
Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu Thr Lys30 35 40 45aat aat
gtg ccc cgt atc cag tgt gaa gat ggt tgt gac cca caa gga 358 Asn Asn
Val Pro Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro Gln Gly 50 55 60
ctc aag gac aac agc cag ttc tgc ttg caa agg atc cgc caa ggt ctg 406
Leu Lys Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile Arg Gln Gly Leu 65
70 75 gct ttt tat aag cac ctg ctt gac tct gac atc ttc aaa ggg gag
cct 454 Ala Phe Tyr Lys His Leu Leu Asp Ser Asp Ile Phe Lys Gly Glu
Pro 80 85 90 gct cta ctc cct gat agc ccc atg gag caa ctt cac acc
tcc cta cta 502 Ala Leu Leu Pro Asp Ser Pro Met Glu Gln Leu His Thr
Ser Leu Leu 95 100 105 gga ctc agc caa ctc ctc cag cca gag gat cac
ccc cgg gag acc caa 550 Gly Leu Ser Gln Leu Leu Gln Pro Glu Asp His
Pro Arg Glu Thr Gln110 115 120 125cag atg ccc agc ctg agt tct agt
cag cag tgg cag cgc ccc ctt ctc 598 Gln Met Pro Ser Leu Ser Ser Ser
Gln Gln Trp Gln Arg Pro Leu Leu 130 135 140 cgt tcc aag atc ctt cga
agc ctc cag gcc ttt ttg gcc ata gct gcc 646 Arg Ser Lys Ile Leu Arg
Ser Leu Gln Ala Phe Leu Ala Ile Ala Ala 145 150 155 cgg gtc ttt gcc
cac gga gca gca act ctg act gag ccc tta gtg cca 694 Arg Val Phe Ala
His Gly Ala Ala Thr Leu Thr Glu Pro Leu Val Pro 160 165 170 aca gct
taaggatgcc caggttccca tggctaccat gataagacta atctatcagc 750 Thr Ala
175 ccagacatct accagttaat taacccatta ggacttgtgc tgttcttgtt
tcgtttgttt 810tgcgtgaagg gcaaggacac cattattaaa gagaaaagaa
acaaacccca gagcaggcag 870ctggctagag aaaggagctg gagaagaaga
ataaagtctc gagcccttgg ccttggaagc 930gggcaagcag ctgcgtggcc
tgaggggaag ggggcggtgg catcgagaaa ctgtgagaaa 990acccagagca
tcagaaaaag tgagcccagg ctttggccat tatctgtaag aaaaacaaga
1050aaaggggaac attatacttt cctgggtggc tcagggaaat gtgcagatgc
acagtactcc 1110agacagcagc tctgtacctg cctgctctgt ccctcagttc
taacagaatc tagtcactaa 1170gaactaacag gactaccaat acgaactgac aaa
12034196PRTUnknownsurmised Mus sp. 4Met Leu Asp Cys Arg Ala Val Ile
Met Leu Trp Leu Leu Pro Trp Val -20 -15 -10 Thr Gln Gly Leu Ala Val
Pro Arg Ser Ser Ser Pro Asp Trp Ala Gln -5 -1 1 5 10 Cys Gln Gln
Leu Ser Arg Asn Leu Cys Met Leu Ala Trp Asn Ala His 15 20 25 Ala
Pro Ala Gly His Met Asn Leu Leu Arg Glu Glu Glu Asp Glu Glu 30 35
40 Thr Lys Asn Asn Val Pro Arg Ile Gln Cys Glu Asp Gly Cys Asp Pro
45 50 55 Gln Gly Leu Lys Asp Asn Ser Gln Phe Cys Leu Gln Arg Ile
Arg Gln 60 65 70 75 Gly Leu Ala Phe Tyr Lys His Leu Leu Asp Ser Asp
Ile Phe Lys Gly 80 85 90 Glu Pro Ala Leu Leu Pro Asp Ser Pro Met
Glu Gln Leu His Thr Ser 95 100 105 Leu Leu Gly Leu Ser Gln Leu Leu
Gln Pro Glu Asp His Pro Arg Glu 110 115 120 Thr Gln Gln Met Pro Ser
Leu Ser Ser Ser Gln Gln Trp Gln Arg Pro 125 130 135 Leu Leu Arg Ser
Lys Ile Leu Arg Ser Leu Gln Ala Phe Leu Ala Ile 140 145 150 155 Ala
Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Thr Glu Pro Leu 160 165
170 Val Pro Thr Ala 175 5102PRTUnknownDescription of Unknown
Organism surmised Sus sp. 5Ser Cys Leu Gln Arg Ile His Gln Gly Leu
Val Phe Tyr Glu Lys Leu 1 5 10 15 Leu Gly Ser Asp Ile Phe Thr Gly
Glu Pro Ser Leu His Pro Asp Gly 20 25 30 Ser Val Gly Gln Leu His
Ala Ser Leu Leu Gly Leu Arg Gln Leu Leu 35 40 45 Gln Pro Glu Gly
His His Trp Glu Thr Glu Gln Thr Pro Ser Pro Ser 50 55 60 Pro Ser
Gln Pro Trp Gln Arg Leu Leu Leu Arg Leu Lys Ile Leu Arg 65 70 75
80Ser Leu Gln Ala Phe Val Ala Val Ala Ala Arg Val Phe Ala His Gly
85 90 95 Ala Ala Thr Leu Ser Gln 100 6306PRTHomo sapiens 6Ile Trp
Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr1 5 10 15Pro
Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu 20 25
30Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly
35 40 45Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala
Gly 50 55 60Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser
Leu Leu65 70 75 80Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr
Asp Ile Leu Lys 85 90 95Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu
Arg Cys Glu Ala Lys 100 105 110Asn Tyr Ser Gly Arg Phe Thr Cys Trp
Trp Leu Thr Thr Ile Ser Thr 115 120 125Asp Leu Thr Phe Ser Val Lys
Ser Ser Arg Gly Ser Ser Asp Pro Gln 130 135 140Gly Val Thr Cys Gly
Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly145 150 155 160Asp Asn
Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala 165 170
175Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala
180 185 190Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe
Ile Arg 195 200 205Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
Leu Lys Pro Leu 210 215 220Lys Asn Ser Arg Gln Val Glu Val Ser Trp
Glu Tyr Pro Asp Thr Trp225 230 235 240Ser Thr Pro His Ser Tyr Phe
Ser Leu Thr Phe Cys Val Gln Val Gln 245 250 255Gly Lys Ser Lys Arg
Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr 260 265 270Ser Ala Thr
Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala 275 280 285Gln
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro 290 295
300Cys Ser30571840DNAMus musculus 7gcacatcaga ccaggcagct cgcagcaaag
caagatgtgt cctcagaagc taaccatctc 60ctggtttgcc atcgttttgc tggtgtctcc
actcatggcc atgtgggagc tggagaaaga 120cgtttatgtt gtagaggtgg
actggactcc cgatgcccct ggagaaacag tgaacctcac 180ctgtgacacg
cctgaagaag atgacatcac ctggacctca gaccagagac atggagtcat
240aggctctgga aagaccctga ccatcactgt caaagagttt ctagatgctg
gccagtacac 300ctgccacaaa ggaggcgaga ctctgagcca ctcacatctg
ctgctccaca agaaggaaaa 360tggaatttgg tccactgaaa ttttaaaaaa
tttcaaaaac aagactttcc tgaagtgtga 420agcaccaaat tactccggac
ggttcacgtg ctcatggctg gtgcaaagaa acatggactt 480gaagttcaac
atcaagagca gtagcagttc ccctgactct cgggcagtga catgtggaat
540ggcgtctctg tctgcagaga aggtcacact ggaccaaagg gactatgaga
agtattcagt 600gtcctgccag gaggatgtca cctgcccaac tgccgaggag
accctgccca ttgaactggc 660gttggaagca cggcagcaga ataaatatga
gaactacagc accagcttct tcatcaggga 720catcatcaaa ccagacccgc
ccaagaactt gcagatgaag cctttgaaga actcacaggt 780ggaggtcagc
tgggagtacc ctgactcctg gagcactccc cattcctact tctccctcaa
840gttctttgtt cgaatccagc gcaagaaaga aaagatgaag gagacagagg
aggggtgtaa 900ccagaaaggt gcgttcctcg tagagaagac atctaccgaa
gtccaatgca aaggcgggaa 960tgtctgcgtg caagctcagg atcgctatta
caattcctca tgcagcaagt gggcatgtgt 1020tccctgcagg gtccgatcct
aggatgcaac gttggaaagg aaagaaaagt ggaagacatt 1080aaggaagaaa
aatttaaact caggatggaa gagtccccca aaagctgtct tctgcttggt
1140tggctttttc cagttttcct aagttcatca tgacaccttt gctgatttct
acatgtaaat 1200gttaaatgcc cgcagagcca gggagctaat gtatgcatag
atattctagc attccacttg 1260gccttatgct gttgaaatat ttaagtaatt
tatgtattta ttaatttatt tctgcatttc 1320acatttgtat accaagatgt
attgaatatt tcatgtgctc gtggcctgat ccactgggac 1380caggccctat
tatgcaaatt gtgagcttgt tatcttcttc aacagctctt caatcagggc
1440tgcgtaggta cattagcttt tgtgacaacc aataagaaca taatattctg
acacaagcag 1500tgttacatat ttgtgaccag taaagacata ggtggtattt
ggagacatga agaagctgta 1560aagttgactc tgaagagttt agcactagtt
tcaacaccaa gaaagacttt ttagaagtga 1620tattgataag aaaccagggc
cttctttaga agggtaccta aatttaaaag aattttgaaa 1680ggctgggtat
cggtggtata tgcttttaat tccagcactc aggagaccaa ggcaggcaga
1740tctctgtgag tttgaggaca gcctggtgta cagagggagt tccagcacag
ccagtgccac 1800acagaaattc tgtctcaaaa acaattaaaa aaaaaaaaaa
18408335PRTMus musculus 8Met Cys Pro Gln Lys Leu Thr Ile Ser Trp
Phe Ala Ile Val Leu Leu1 5 10 15Val Ser Pro Leu Met Ala Met Trp Glu
Leu Glu Lys Asp Val Tyr Val 20 25 30Val Glu Val Asp Trp Thr Pro Asp
Ala Pro Gly Glu Thr Val Asn Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu
Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60Arg His Gly Val Ile Gly
Ser Gly Lys Thr Leu Thr Ile Thr Val Lys65 70 75 80Glu Phe Leu Asp
Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr 85 90 95Leu Ser His
Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110Ser
Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115 120
125Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln
130 135 140Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser
Ser Pro145 150 155 160Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser
Leu Ser Ala Glu Lys 165 170 175Val Thr Leu Asp Gln Arg Asp Tyr Glu
Lys Tyr Ser Val Ser Cys Gln 180 185 190Glu Asp Val Thr Cys Pro Thr
Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205Ala Leu Glu Ala Arg
Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220Phe Phe Ile
Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln225 230 235
240Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro
245 250 255Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe
Phe Val 260 265 270Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr
Glu Glu Gly Cys 275 280 285Asn Gln Lys Gly Ala Phe Leu Val Glu Lys
Thr Ser Thr Glu Val Gln 290 295 300Cys Lys Gly Gly Asn Val Cys Val
Gln Ala Gln Asp Arg Tyr Tyr Asn305 310 315 320Ser Ser Cys Ser Lys
Trp Ala Cys Val Pro Cys Arg Val Arg Ser 325 330 33591008DNARattus
norvegicus 9atgtgtcatc agaagttaac cttctcctgg tttgccatgg ttttgctggt
gtctccactc 60atggccatgt gggagctgga gaaagatgtt tatgttgtag aggtggactg
gcgccccgat 120gcccctggag aaacggtgac cctcacctgt gacagtcctg
aagaagatga catcacctgg 180acctcagacc agagacgtgg agtcataggc
tctggaaaga ccctgaccat cactgtcaga 240gagtttctag atgctggcca
atacacctgc cacagaggag gcgagactct gagccactca 300catctgctgc
tccacaagaa ggaaaatgga atttggtcca ccgagatttt aaaaaatttc
360aaaaataaga ctttcctgaa gtgtgaagca ccaaactact ccggacggtt
cacctgctca 420tggctggtgc acagaaacac ggacttgaag tttaacatca
agagcagcag cagttcccct 480gagtctcggg cggtgacatg tggacgagca
tctctgtctg cagagaaggt cacactgaac 540caaagggact acgagaagta
ctcagtggcg tgccaggagg acgtcacctg cccaactgcc 600gaggagaccc
tgcccattga actggtggtg gaggcccagc agcagaataa atatgagaac
660tacagcacca gcttcttcat cagggacatc atcaaaccgg acccacccaa
gaacctgcag 720gtgaaacctt tgaagaactc tcaggtggag gtcagctggg
agtaccctga
ctcctggagc 780actccccatt cctacttctc cctcaagttc ttcgtccgca
tccagcgcaa gaaagaaaag 840acgaaggaga cagaggagga gtgtaaccag
aaaggtgcgt tcctcgtaga gaagacctct 900gccgaagtcc aatgcaaagg
ggcgaatatc tgcgtgcaag cgcaggaccg ctactacaat 960tcatcatgca
gcaaatggac atgtgtaccc tgcaggggcc gatcctaa 100810335PRTRattus
norvegicus 10Met Cys His Gln Lys Leu Thr Phe Ser Trp Phe Ala Met
Val Leu Leu1 5 10 15Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys
Asp Val Tyr Val 20 25 30Val Glu Val Asp Trp Arg Pro Asp Ala Pro Gly
Glu Thr Val Thr Leu 35 40 45Thr Cys Asp Ser Pro Glu Glu Asp Asp Ile
Thr Trp Thr Ser Asp Gln 50 55 60Arg Arg Gly Val Ile Gly Ser Gly Lys
Thr Leu Thr Ile Thr Val Arg65 70 75 80Glu Phe Leu Asp Ala Gly Gln
Tyr Thr Cys His Arg Gly Gly Glu Thr 85 90 95Leu Ser His Ser His Leu
Leu Leu His Lys Lys Glu Asn Gly Ile Trp 100 105 110Ser Thr Glu Ile
Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys 115 120 125Glu Ala
Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val His 130 135
140Arg Asn Thr Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser
Pro145 150 155 160Glu Ser Arg Ala Val Thr Cys Gly Arg Ala Ser Leu
Ser Ala Glu Lys 165 170 175Val Thr Leu Asn Gln Arg Asp Tyr Glu Lys
Tyr Ser Val Ala Cys Gln 180 185 190Glu Asp Val Thr Cys Pro Thr Ala
Glu Glu Thr Leu Pro Ile Glu Leu 195 200 205Val Val Glu Ala Gln Gln
Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser 210 215 220Phe Phe Ile Arg
Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln225 230 235 240Val
Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro 245 250
255Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val
260 265 270Arg Ile Gln Arg Lys Lys Glu Lys Thr Lys Glu Thr Glu Glu
Glu Cys 275 280 285Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser
Ala Glu Val Gln 290 295 300Cys Lys Gly Ala Asn Ile Cys Val Gln Ala
Gln Asp Arg Tyr Tyr Asn305 310 315 320Ser Ser Cys Ser Lys Trp Thr
Cys Val Pro Cys Arg Gly Arg Ser 325 330 33511821DNARattus
norvegicus 11ctcgcagcag agcaagatgt gtcatcagaa gttaaccttc tcctggtttg
ccatggtttt 60gctggtgtct ccactcatgg ccatgtggga gctggagaaa gatgtttatg
ttgtagaggt 120ggactggcgc cccgatgccc ctggagaaac ggtgaccctc
acctgtgaca gtcctgaaga 180agatgacatc acctggacct cagaccagag
acgtggagtc ataggctctg gaaagaccct 240gaccatcact gtcagagagt
ttctagatgc tggccaatac acctgccaca gaggaggcga 300gactctgagc
cactcacatc tgctgctcca caagaaggaa aatggaattt ggtccaccga
360gattttaaaa aatttcaaaa ataagacttt cctgaagaga gaagcaccaa
actactccgg 420acggttcacc tgctcatggc tggtgcacag aaacacggac
ttgaagttta acatcaagag 480cagcagcagt tcccctgagt ctcgggcggt
gacatgtgga gcagcatctc tgtctgcaga 540gaaggtcaca ctgaaccaaa
gggactacga gaagtactca gtggcgtgcc aggaggacgt 600cacctgccca
actgccgagg agaccctgcc cattgaactg gtggtggagg cccagcagca
660gaataaatat gagaactaca gcaccagctt cttcatcagg gacatcatca
aaccggaccc 720acccaagaac ctgcaggtga aacctttgaa gaactctcag
gtggaggtca gctgggagta 780ccctgactcc tggagcactc cccattccta
cttctccctc a 82112268PRTRattus norvegicus 12Met Cys His Gln Lys Leu
Thr Phe Ser Trp Phe Ala Met Val Leu Leu1 5 10 15Val Ser Pro Leu Met
Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val 20 25 30Val Glu Val Asp
Trp Arg Pro Asp Ala Pro Gly Glu Thr Val Thr Leu 35 40 45Thr Cys Asp
Ser Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60Arg Arg
Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Arg65 70 75
80Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Arg Gly Gly Glu Thr
85 90 95Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile
Trp 100 105 110Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe
Leu Lys Arg 115 120 125Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys
Ser Trp Leu Val His 130 135 140Arg Asn Thr Asp Leu Lys Phe Asn Ile
Lys Ser Ser Ser Ser Ser Pro145 150 155 160Glu Ser Arg Ala Val Thr
Cys Gly Ala Ala Ser Leu Ser Ala Glu Lys 165 170 175Val Thr Leu Asn
Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ala Cys Gln 180 185 190Glu Asp
Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu 195 200
205Val Val Glu Ala Gln Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser
210 215 220Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
Leu Gln225 230 235 240Val Lys Pro Leu Lys Asn Ser Gln Val Glu Val
Ser Trp Glu Tyr Pro 245 250 255Asp Ser Trp Ser Thr Pro His Ser Tyr
Phe Ser Leu 260 26513990DNAFelis catus 13atgcatcctc agcagctggt
catcgcctgg ttttccctgg ttttgctggc acctcccctc 60atggccatat gggaactgga
gaaaaacgtt tatgttgtag agttggactg gcaccctgat 120gcccccggag
aaatggtggt cctcacctgt gacacgcctg aagaagatga catcacctgg
180acctctgacc agagcagtga agtcctaggc tctggtaaaa ctctgaccat
ccaagtcaaa 240gaatttgcag atgctggcca gtatacctgt cataaaggag
gcgaggttct gagccattcg 300ttcctcctga tacacaaaaa ggaagatgga
atttggtcca ctgatatctt aagggaacag 360aaagaatcca aaaataagat
ctttctaaaa tgtgaggcaa agaattattc tggacgtttc 420acctgctggt
ggctgacggc aatcagtacc gatttgaaat tcactgtcaa aagcagcaga
480ggctcctctg acccccaagg ggtgacttgt ggagcagcga cactctcagc
agagaaggtc 540agagtggaca acagggatta taagaagtac acagtggagt
gtcaggaggg cagtgcctgc 600ccggctgccg aggagagcct acccattgaa
gtcgtggtgg acgctattca caagctcaag 660tacgaaaact acaccagcag
cttcttcatc agggacatca tcaaaccgga cccacccaag 720aacctgcaac
tgaagccatt aaaaaattct cggcatgtgg aagtgagctg ggaataccct
780gacacctgga gcaccccaca ttcctacttc tccttaacat ttggcgtaca
ggtccagggc 840aagaacaaca gagaaaagaa agacagactc tccgtggaca
agacctcagc caaggtcgtg 900tgccacaagg atgccaagat ccgcgtgcaa
gccagagacc gctactatag ctcatcctgg 960agcaactggg catccgtgtc
ctgcagttag 99014329PRTFelis catus 14Met His Pro Gln Gln Leu Val Ile
Ala Trp Phe Ser Leu Val Leu Leu1 5 10 15Ala Pro Pro Leu Met Ala Ile
Trp Glu Leu Glu Lys Asn Val Tyr Val 20 25 30Val Glu Leu Asp Trp His
Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45Thr Cys Asp Thr Pro
Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50 55 60Ser Ser Glu Val
Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe
Ala Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95Leu
Ser His Ser Phe Leu Leu Ile His Lys Lys Glu Asp Gly Ile Trp 100 105
110Ser Thr Asp Ile Leu Arg Glu Gln Lys Glu Ser Lys Asn Lys Ile Phe
115 120 125Leu Lys Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys
Trp Trp 130 135 140Leu Thr Ala Ile Ser Thr Asp Leu Lys Phe Thr Val
Lys Ser Ser Arg145 150 155 160Gly Ser Ser Asp Pro Gln Gly Val Thr
Cys Gly Ala Ala Thr Leu Ser 165 170 175Ala Glu Lys Val Arg Val Asp
Asn Arg Asp Tyr Lys Lys Tyr Thr Val 180 185 190Glu Cys Gln Glu Gly
Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro 195 200 205Ile Glu Val
Val Val Asp Ala Ile His Lys Leu Lys Tyr Glu Asn Tyr 210 215 220Thr
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys225 230
235 240Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg His Val Glu Val
Ser 245 250 255Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr
Phe Ser Leu 260 265 270Thr Phe Gly Val Gln Val Gln Gly Lys Asn Asn
Arg Glu Lys Lys Asp 275 280 285Arg Leu Ser Val Asp Lys Thr Ser Ala
Lys Val Val Cys His Lys Asp 290 295 300Ala Lys Ile Arg Val Gln Ala
Arg Asp Arg Tyr Tyr Ser Ser Ser Trp305 310 315 320Ser Asn Trp Ala
Ser Val Ser Cys Ser 325151006DNAFelis catus 15atgcatcctc agcagttggt
catcgcctgg ctttccctgg ttttgctggc acctcccctc 60atggccatat gggaactgga
gaaaaacgtt tatgttgtag agttggactg gcaccctgat 120gcccccggag
aaatggtggt cctcacctgc aatactcctg aagaagatga catcacctgg
180acctctgacc agagcagtga agtcctaggc tctggtaaaa ctctgaccat
ccaagtcaaa 240gaatttgcag atgctggcca gtatacctgt cataaaggag
gcgaggttct gagccattcg 300ttcctcctga tacacaaaaa ggaagatgga
atttggtcca ctgatatctt aagggaacag 360aaagaatcca aaaataagat
ctttctaaaa tgtgaggcaa agaattattc tggacgtttc 420acctgctggt
ggctgacggc aatcagtacc gatttgaaat tcactgtcaa aagcagcaga
480ggctcctctg acccccaaga ggtgacttgt ggagcagcga cactctcagc
agagaaggtc 540agagtggaca acagggatta taagaagtac acagtggagt
gtcaggaggg cagtgcctgc 600ccggctgccg aggagagcct acccattgaa
gtcgtggtgg acgctattca caagctcaag 660tacgaaaact acaccagcag
cttcttcatc agggacatca tcaaaccgga cccacccaag 720aacctgcaac
tgaagccatt aaaaaattct cggcatgtgg aagtgagctg ggaataccct
780gacacctgga gcaccccaca ttcctacttc tccttaacat ttggcgtaca
ggtccagggc 840aagaacaaca gagaaaagaa agacagactc tccgtggaca
agacctcagc caaggtcgtg 900tgccacaagg atgccaagat ccgcgtgcaa
gccagagacc gctactatag ctcatcctgg 960agcaactggg catccgtgtc
ctgcagttag gttccacccc caggat 100616329PRTFelis catus 16Met His Pro
Gln Gln Leu Val Ile Ala Trp Leu Ser Leu Val Leu Leu1 5 10 15Ala Pro
Pro Leu Met Ala Ile Trp Glu Leu Glu Lys Asn Val Tyr Val 20 25 30Val
Glu Leu Asp Trp His Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40
45Thr Cys Asn Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln
50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val
Lys65 70 75 80Glu Phe Ala Asp Ala Gly Gln Tyr Thr Cys His Lys Gly
Gly Glu Val 85 90 95Leu Ser His Ser Phe Leu Leu Ile His Lys Lys Glu
Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Arg Glu Gln Lys Glu
Ser Lys Asn Lys Ile Phe 115 120 125Leu Lys Cys Glu Ala Lys Asn Tyr
Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140Leu Thr Ala Ile Ser Thr
Asp Leu Lys Phe Thr Val Lys Ser Ser Arg145 150 155 160Gly Ser Ser
Asp Pro Gln Glu Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175Ala
Glu Lys Val Arg Val Asp Asn Arg Asp Tyr Lys Lys Tyr Thr Val 180 185
190Glu Cys Gln Glu Gly Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro
195 200 205Ile Glu Val Val Val Asp Ala Ile His Lys Leu Lys Tyr Glu
Asn Tyr 210 215 220Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro
Asp Pro Pro Lys225 230 235 240Asn Leu Gln Leu Lys Pro Leu Lys Asn
Ser Arg His Val Glu Val Ser 245 250 255Trp Glu Tyr Pro Asp Thr Trp
Ser Thr Pro His Ser Tyr Phe Ser Leu 260 265 270Thr Phe Gly Val Gln
Val Gln Gly Lys Asn Asn Arg Glu Lys Lys Asp 275 280 285Arg Leu Ser
Val Asp Lys Thr Ser Ala Lys Val Val Cys His Lys Asp 290 295 300Ala
Lys Ile Arg Val Gln Ala Arg Asp Arg Tyr Tyr Ser Ser Ser Trp305 310
315 320Ser Asn Trp Ala Ser Val Ser Cys Ser 325171058DNAEquus
caballus 17atgtgtcacc agtggttggt cctctcctgg ttttccctgg ttttgctggc
gtctcccctc 60atggccatat gggaactgga gaaagatgtg tatgttgtag aattggattg
gtaccctgat 120gcccctggag aaatggtggt cctcacctgc aatacccctg
aagaagaagg catcacctgg 180acctcggccc agagcaatga ggtcttaggc
tctggcaaaa ccttgaccat ccaagtcaaa 240gagtttggag atgctggctg
gtacacctgt cacaaaggag gcgaggttct gagccattct 300cacctgctgc
ttcacaagaa ggaagatgga atttggtcca ctgacatttt aaaagaccag
360aaagaatcca aaaataagac ctttctaaaa tgtgaggcaa agaattattc
cggacgtttc 420acatgctggt ggctgacagc aatcagtact gatttgaaat
tcagtgtcaa aagcagcaga 480ggttcctctg acccccgagg ggtgacgtgt
ggagcagcga cactctccgc agagagggtc 540agcgtggacg acagggagta
taagaagtac acggtggagt gtcaggaggg cagtgcctgc 600ccggccgccg
aggagagcct gcccattgag atcgtggtgg atgctgttca caagctcaag
660tatgaaaact acaccagcgg cttcttcatc agggacatca tcaaaccaga
cccacccaag 720aacctgcagc tgaagccatt aaagaattct cggcaggtgg
aggtcagctg ggagtacccc 780gagacctgga gcaccccaca ttcctacttc
tccctgacat tctctattca ggtccagggc 840aagaacaaga aggaaaggaa
agacagactc ttcatggatg agacttcagc cacagtcaca 900tgccacaagg
atggccagat ccgtgtccaa gccagggacc gctactacag ctcatcctgg
960agcgaatggg catccgtatc ctgcagttag ggatgcagac tcaggcagcc
caggccagac 1020ctgaacactc agtgtaccca ggttctaacc tcagtatg
105818329PRTEquus caballus 18Met Cys His Gln Trp Leu Val Leu Ser
Trp Phe Ser Leu Val Leu Leu1 5 10 15Ala Ser Pro Leu Met Ala Ile Trp
Glu Leu Glu Lys Asp Val Tyr Val 20 25 30Val Glu Leu Asp Trp Tyr Pro
Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45Thr Cys Asn Thr Pro Glu
Glu Glu Gly Ile Thr Trp Thr Ser Ala Gln 50 55 60Ser Asn Glu Val Leu
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly
Asp Ala Gly Trp Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95Leu Ser
His Ser His Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105
110Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Ser Lys Asn Lys Thr Phe
115 120 125Leu Lys Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys
Trp Trp 130 135 140Leu Thr Ala Ile Ser Thr Asp Leu Lys Phe Ser Val
Lys Ser Ser Arg145 150 155 160Gly Ser Ser Asp Pro Arg Gly Val Thr
Cys Gly Ala Ala Thr Leu Ser 165 170 175Ala Glu Arg Val Ser Val Asp
Asp Arg Glu Tyr Lys Lys Tyr Thr Val 180 185 190Glu Cys Gln Glu Gly
Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro 195 200 205Ile Glu Ile
Val Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr 210 215 220Thr
Ser Gly Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys225 230
235 240Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val
Ser 245 250 255Trp Glu Tyr Pro Glu Thr Trp Ser Thr Pro His Ser Tyr
Phe Ser Leu 260 265 270Thr Phe Ser Ile Gln Val Gln Gly Lys Asn Lys
Lys Glu Arg Lys Asp 275 280 285Arg Leu Phe Met Asp Glu Thr Ser Ala
Thr Val Thr Cys His Lys Asp 290 295 300Gly Gln Ile Arg Val Gln Ala
Arg Asp Arg Tyr Tyr Ser Ser Ser Trp305 310 315 320Ser Glu Trp Ala
Ser Val Ser Cys Ser 325191399DNAHomo sapiens 19ctgtttcagg
gccattggac tctccgtcct gcccagagca agatgtgtca ccagcagttg 60gtcatctctt
ggttttccct ggtttttctg gcatctcccc tcgtggccat atgggaactg
120aagaaagatg tttatgtcgt agaattggat tggtatccgg atgcccctgg
agaaatggtg 180gtcctcacct gtgacacccc tgaagaagat ggtatcacct
ggaccttgga ccagagcagt 240gaggtcttag gctctggcaa aaccctgacc
atccaagtca aagagtttgg agatgctggc 300cagtacacct gtcacaaagg
aggcgaggtt ctaagccatt cgctcctgct gcttcacaaa 360aaggaagatg
gaatttggtc cactgatatt ttaaaggacc agaaagaacc caaaaataag
420acctttctaa gatgcgaggc caagaattat tctggacgtt tcacctgctg
gtggctgacg 480acaatcagta ctgatttgac attcagtgtc aaaagcagca
gaggctcttc tgacccccaa 540ggggtgacgt gcggagctgc tacactctct
gcagagagag tcagagggga caacaaggag 600tatgagtact cagtggagtg
ccaggaggac agtgcctgcc cagctgctga ggagagtctg 660cccattgagg
tcatggtgga tgccgttcac aagctcaagt atgaaaacta caccagcagc
720ttcttcatca gggacatcat caaacctgac ccacccaaga acttgcagct
gaagccatta 780aagaattctc ggcaggtgga ggtcagctgg gagtaccctg
acacctggag tactccacat 840tcctacttct ccctgacatt ctgcgttcag
gtccagggca agagcaagag agaaaagaaa 900gatagagtct tcacggacaa
gacctcagcc acggtcatct gccgcaaaaa tgccagcatt 960agcgtgcggg
cccaggaccg ctactatagc tcatcttgga gcgaatgggc atctgtgccc
1020tgcagttagg ttctgatcca ggatgaaaat ttggaggaaa agtggaagat
attaagcaaa 1080atgtttaaag acacaacgga atagacccaa aaagataatt
tctatctgat ttgctttaaa 1140acgttttttt aggatcacaa tgatatcttt
gctgtatttg tatagttaga tgctaaatgc 1200tcattgaaac aatcagctaa
tttatgtata gattttccag ctctcaagtt gccatgggcc 1260ttcatgctat
ttaaatattt aagtaattta tgtatttatt agtatattac tgttatttaa
1320cgtttgtctg ccaggatgta tggaatgttt catactctta tgacctgatc
catcaggatc 1380agtccctatt atgcaaaat 139920328PRTHomo sapiens 20Met
Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu1 5 10
15Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val
Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu
Asp Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile
Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His
Lys Gly Gly Glu Val 85 90 95Leu Ser His Ser Leu Leu Leu Leu His Lys
Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Lys Asp Gln
Lys Glu Pro Lys Asn Lys Thr Phe 115 120 125Leu Arg Cys Glu Ala Lys
Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140Leu Thr Thr Ile
Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg145 150 155 160Gly
Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170
175Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu
Pro Ile 195 200 205Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr
Glu Asn Tyr Thr 210 215 220Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
Pro Asp Pro Pro Lys Asn225 230 235 240Leu Gln Leu Lys Pro Leu Lys
Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255Glu Tyr Pro Asp Thr
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270Phe Cys Val
Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285Val
Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295
300Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp
Ser305 310 315 320Glu Trp Ala Ser Val Pro Cys Ser 325211012DNACapra
hircus 21atgcaccctc agcagttggt cgtttcctgg ttttccctgg ttttgctggc
atctcccatc 60gtggccatat gggaactgga gaaaaatgtt tatgttgtag aattggattg
gtatcctaat 120gctcctggag aaacagtggt cctcacgtgt gacactcctg
aagaagacgg catcacctgg 180acctcagacc agagcagtga ggtcctgggc
tctggcaaaa ccttgaccat ccaagtcaaa 240gagtttggag atgctgggca
gtacacctgt cacaaaggag gcgaggttct gagtcgttca 300ctcctcctgc
ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggatcag
360aaagaaccca aagctaagag ttttttaaaa tgtgaggcaa aggattattc
tggacacttc 420acctgctcgt ggctgacagc aatcagtact aatctgaaat
tcagtgtcaa aagcagcaga 480ggctcctctg acccccgagg ggtgacgtgc
ggagcagcgt cactctcagc agagaaggtc 540agcatggacc acagggagta
taacaagtac acagtggagt gtcaggaggg cagtgcctgc 600ccggccgccg
aggagagcct gcccattgag gtcgtgatgg aagctgtgca caagctcaag
660tatgaaaact acaccagcag cttcttcatc agggacatca tcaaaccaga
cccacccaag 720aacctgcaac tgagaccact aaagaattct cggcaggtgg
aggtcagctg ggagtaccct 780gacacgtgga gcaccccaca ttcctacttc
tccctgacgt tttgtgttca ggtccaggga 840aagaacaaga gagaaaagaa
actcttcacg gaccaaacct cagccaaagt cacatgccac 900aaggatgcca
acatccgtgt gcaagcccgg gaccgctact acagctcatt ctggagtgaa
960tgggcatctg tgtcctgcag ttaggttcta acctcagtat gaaacctcag ag
101222327PRTCapra hircus 22Met His Pro Gln Gln Leu Val Val Ser Trp
Phe Ser Leu Val Leu Leu1 5 10 15Ala Ser Pro Ile Val Ala Ile Trp Glu
Leu Glu Lys Asn Val Tyr Val 20 25 30Val Glu Leu Asp Trp Tyr Pro Asn
Ala Pro Gly Glu Thr Val Val Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu
Asp Gly Ile Thr Trp Thr Ser Asp Gln 50 55 60Ser Ser Glu Val Leu Gly
Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly Asp
Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90 95Leu Ser Arg
Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105 110Ser
Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Ala Lys Ser Phe 115 120
125Leu Lys Cys Glu Ala Lys Asp Tyr Ser Gly His Phe Thr Cys Ser Trp
130 135 140Leu Thr Ala Ile Ser Thr Asn Leu Lys Phe Ser Val Lys Ser
Ser Arg145 150 155 160Gly Ser Ser Asp Pro Arg Gly Val Thr Cys Gly
Ala Ala Ser Leu Ser 165 170 175Ala Glu Lys Val Ser Met Asp His Arg
Glu Tyr Asn Lys Tyr Thr Val 180 185 190Glu Cys Gln Glu Gly Ser Ala
Cys Pro Ala Ala Glu Glu Ser Leu Pro 195 200 205Ile Glu Val Val Met
Glu Ala Val His Lys Leu Lys Tyr Glu Asn Tyr 210 215 220Thr Ser Ser
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys225 230 235
240Asn Leu Gln Leu Arg Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser
245 250 255Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe
Ser Leu 260 265 270Thr Phe Cys Val Gln Val Gln Gly Lys Asn Lys Arg
Glu Lys Lys Leu 275 280 285Phe Thr Asp Gln Thr Ser Ala Lys Val Thr
Cys His Lys Asp Ala Asn 290 295 300Ile Arg Val Gln Ala Arg Asp Arg
Tyr Tyr Ser Ser Phe Trp Ser Glu305 310 315 320Trp Ala Ser Val Ser
Cys Ser 325231080DNAMacaca mulatta 23gcccagagca agatgtgtca
ccagcagctg gtcatctctt ggttttccct ggtttttctg 60gcatctcccc tcatggccat
atgggaactg aagaaagacg tttatgttgt agaattggac 120tggtacccgg
atgcccctgg agaaatggtg gtcctcacct gtgacacccc tgaagaagat
180ggtatcacct ggaccttgga ccagagtggt gaggtcttag gctctggcaa
aaccctgacc 240atccaagtca aagagtttgg agatgctggc cagtacacct
gtcacaaagg aggcgaggct 300ctaagccatt cactcctgct gcttcacaaa
aaggaagatg gaatttggtc cactgatgtt 360ttaaaggacc agaaagaacc
caaaaataag acctttctaa gatgtgaggc caaaaattat 420tctggacgtt
tcacctgctg gtggctgacg acaatcagta ctgatctgac attcagtgtc
480aaaagcagca gaggctcttc taacccccaa ggggtgacgt gtggagccgt
tacactctct 540gcagagaggg tcagagggga caataaggag tatgagtact
cagtggagtg ccaggaggac 600agtgcctgcc cagccgctga ggagaggctg
cccattgagg tcatggtgga tgccattcac 660aagctcaagt atgaaaacta
caccagcagc ttcttcatca gggacatcat caaacccgac 720ccacccaaga
acttgcagct gaagccatta aagaattctc ggcaggtgga ggtcagctgg
780gagtaccctg acacctggag tactccacat tcctacttct ccctgacatt
ctgcatccag 840gtccagggca agagcaagag agaaaagaaa gatagaatct
tcacagacaa gacctcagcc 900acggtcatct gccgcaaaaa tgccagcttt
agcgtgcagg cccaggaccg ctactatagc 960tcatcttgga gcgaatgggc
atctgtgccc tgcagttagg ttgtgatccc aggatgaaaa 1020attggaggaa
aagtagaaga tattaaccaa aacgtttaaa gacacaacgg aatagaccca
108024328PRTMacaca mulatta 24Met Cys His Gln Gln Leu Val Ile Ser
Trp Phe Ser Leu Val Phe Leu1 5 10 15Ala Ser Pro Leu Met Ala Ile Trp
Glu Leu Lys Lys Asp Val Tyr Val 20 25 30Val Glu Leu Asp Trp Tyr Pro
Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45Thr Cys Asp Thr Pro Glu
Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60Ser Gly Glu Val Leu
Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly
Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Ala 85 90 95Leu Ser
His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp 100 105
110Ser Thr Asp Val Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
115 120 125Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys
Trp Trp 130 135 140Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val
Lys Ser Ser Arg145 150 155 160Gly Ser Ser Asn Pro Gln Gly Val Thr
Cys Gly Ala Val Thr Leu Ser 165 170 175Ala Glu Arg Val Arg Gly Asp
Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185 190Cys Gln Glu Asp Ser
Ala Cys Pro Ala Ala Glu Glu Arg Leu Pro Ile 195 200 205Glu Val Met
Val Asp Ala Ile His Lys Leu Lys Tyr Glu Asn Tyr Thr 210 215 220Ser
Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn225 230
235 240Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser
Trp 245 250 255Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe
Ser Leu Thr 260 265 270Phe Cys Ile Gln Val Gln Gly Lys Ser Lys Arg
Glu Lys Lys Asp Arg 275 280 285Ile Phe Thr Asp Lys Thr Ser Ala Thr
Val Ile Cys Arg Lys Asn Ala 290 295 300Ser Phe Ser Val Gln Ala Gln
Asp Arg Tyr Tyr Ser Ser Ser Trp Ser305 310 315 320Glu Trp Ala Ser
Val Pro Cys Ser 325251012DNABos taurus 25atgcaccctc agcagttggt
cgtttcctgg ttttccctgg ttttgctggc atctcccatc 60gtggccatgt gggaactgga
gaaaaatgtt tatgttgtag aattggattg gtatcctgat 120gctcctggag
aaacagtggt cctcacatgt gacactcctg aagaagatgg catcacctgg
180acctcagacc agagcagtga ggtcttgggc tctggcaaaa ccttgaccat
ccaagtcaaa 240gagtttggag atgctgggca gtacacctgt cacaaaggag
gcgaggctct gagtcgttca 300ctcctcctgc tgcacaaaaa ggaagatgga
atttggtcca ctgatatttt aaaggatcag 360aaagaaccca aagctaagag
ttttttaaaa tgtgaggcaa aggattattc tggacacttc 420acctgctggt
ggctgacagc aatcagtact gatttgaaat tcagtgtcaa aagcagcaga
480ggctcctctg acccccgagg ggtgacgtgc ggagcagcgt tgctctcagc
agagaaggtc 540agcttggagc acagggagta taacaagtac acagtggagt
gtcaggaggg cagcgcctgc 600ccagccgctg aggagagcct gcttattgag
gtcgtggtag aagctgtgca caagctcaag 660tatgaaaact acaccagcag
cttcttcatc agggacatca tcaaaccaga cccacccaag 720aacctgcaac
tgagaccatt aaagaattct cggcaggtgg aggtcagctg ggagtaccct
780gacacgtgga gcaccccgca ttcctacttc tccctgacgt tttgtgttca
ggtccaggga 840aagaacaaga gagaaaagaa actcttcatg gaccaaacct
cagccaaagt cacatgccac 900aaggatgcca acgtccgcgt gcaagcccgg
gaccgctact acagctcatt ctggagtgaa 960tgggcatctg tgtcctgcag
ttaggttcta acctcagtat gaaacctcag ag 101226327PRTBos taurus 26Met
His Pro Gln Gln Leu Val Val Ser Trp Phe Ser Leu Val Leu Leu1 5 10
15Ala Ser Pro Ile Val Ala Met Trp Glu Leu Glu Lys Asn Val Tyr Val
20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Thr Val Val
Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Ser
Asp Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile
Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His
Lys Gly Gly Glu Ala 85 90 95Leu Ser Arg Ser Leu Leu Leu Leu His Lys
Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Lys Asp Gln
Lys Glu Pro Lys Ala Lys Ser Phe 115 120 125Leu Lys Cys Glu Ala Lys
Asp Tyr Ser Gly His Phe Thr Cys Trp Trp 130 135 140Leu Thr Ala Ile
Ser Thr Asp Leu Lys Phe Ser Val Lys Ser Ser Arg145 150 155 160Gly
Ser Ser Asp Pro Arg Gly Val Thr Cys Gly Ala Ala Leu Leu Ser 165 170
175Ala Glu Lys Val Ser Leu Glu His Arg Glu Tyr Asn Lys Tyr Thr Val
180 185 190Glu Cys Gln Glu Gly Ser Ala Cys Pro Ala Ala Glu Glu Ser
Leu Leu 195 200 205Ile Glu Val Val Val Glu Ala Val His Lys Leu Lys
Tyr Glu Asn Tyr 210 215 220Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile
Lys Pro Asp Pro Pro Lys225 230 235 240Asn Leu Gln Leu Arg Pro Leu
Lys Asn Ser Arg Gln Val Glu Val Ser 245 250 255Trp Glu Tyr Pro Asp
Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu 260 265 270Thr Phe Cys
Val Gln Val Gln Gly Lys Asn Lys Arg Glu Lys Lys Leu 275 280 285Phe
Met Asp Gln Thr Ser Ala Lys Val Thr Cys His Lys Asp Ala Asn 290 295
300Val Arg Val Gln Ala Arg Asp Arg Tyr Tyr Ser Ser Phe Trp Ser
Glu305 310 315 320Trp Ala Ser Val Ser Cys Ser 32527993DNACervus
elaphus 27atgcaccctc agcagttggt cgtttcctgg ttttccctgg ttttgctgac
atctcccatt 60gtggccatat gggaactgga gaaaaatgtt tatgttgtag aattggattg
gtatcctgat 120gctcctggag aaacggtggt cctcaggtgt gacactcctg
aagaagacgg tatcacctgg 180acctcagacc agagcagtga ggtcttgggc
tctggcaaaa ccttgaccgt ccaagtcaaa 240gagtttggag atgctgggca
gtacacctgt cacaaaggag gcgaggttct gagtcgttca 300ctcctcctgc
tgcacaaaaa ggaagatgga atttggtcta ctgatatttt aaaggatcag
360aaagaaccca aagccaagag ttttttaaaa tgtgaggcaa aggattattc
tggacacttc 420acctgctggt ggctgacagc aatcagtact gatttgaaat
tcagtgtcaa aagcagcaga 480ggctcctctg acccccgagg ggtgacgtgc
ggagcagcgt cgctctcaac agagaaggtc 540attgtggacc acagggagta
taagaagtac acagtggagt gtcaagaggg cagcgcctgc 600ccggccgccg
aggagagcct gcccattgag gtcgtagtgg aagctgtgca caagctcaag
660tatgaaaact acaccagcag cttcttcatc agggacatca tcaaaccaga
cccacccaag 720aacctgcaac tgagaccatt aaagaattct cggcaggtgg
aggtcagctg ggagtaccct 780gacacgtgga gcaccccaca ttcctacttc
tccctgacgt tttgtgttca ggtccaggga 840aagaacaaga gagaaaagaa
actcttcatg gaccaaacct cagccaaagt cacgtgtcac 900aaggatgcca
gcatccgcgt gcaagcccgg gaccgctact acaactcatt ctggagtgaa
960tgggcatctg tgtcctgcag ttaggttcta acc 99328327PRTCervus elaphus
28Met His Pro Gln Gln Leu Val Val Ser Trp Phe Ser Leu Val Leu Leu1
5 10 15Thr Ser Pro Ile Val Ala Ile Trp Glu Leu Glu Lys Asn Val Tyr
Val 20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Thr Val
Val Leu 35 40 45Arg Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr
Ser Asp Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr
Val Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys
His Lys Gly Gly Glu Val 85 90 95Leu Ser Arg Ser Leu Leu Leu Leu His
Lys Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Lys Asp
Gln Lys Glu Pro Lys Ala Lys Ser Phe 115 120 125Leu Lys Cys Glu Ala
Lys Asp Tyr Ser Gly His Phe Thr Cys Trp Trp 130 135 140Leu Thr Ala
Ile Ser Thr Asp Leu Lys Phe Ser Val Lys Ser Ser Arg145 150 155
160Gly Ser Ser Asp Pro Arg Gly Val Thr Cys Gly Ala Ala Ser Leu Ser
165 170 175Thr Glu Lys Val Ile Val Asp His Arg Glu Tyr Lys Lys Tyr
Thr Val 180 185 190Glu Cys Gln Glu Gly Ser Ala Cys Pro Ala Ala Glu
Glu Ser Leu Pro 195 200 205Ile Glu Val Val Val Glu Ala Val His Lys
Leu Lys Tyr Glu Asn Tyr 210 215 220Thr Ser Ser Phe Phe Ile Arg Asp
Ile Ile Lys Pro Asp Pro Pro Lys225 230 235 240Asn Leu Gln Leu Arg
Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser 245 250 255Trp Glu Tyr
Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu 260 265 270Thr
Phe Cys Val Gln Val Gln Gly Lys Asn Lys Arg Glu Lys Lys Leu 275 280
285Phe Met Asp Gln Thr Ser Ala Lys Val Thr Cys His Lys Asp Ala Ser
290 295 300Ile Arg Val Gln Ala Arg Asp Arg Tyr Tyr Asn Ser Phe Trp
Ser Glu305 310 315 320Trp Ala Ser Val Ser Cys Ser 32529984DNAOvis
aries 29atgcaccctc agcagttggt cgtttcctgg ttttccctgg ttttgctggc
atcgcccatc 60gtggccatat gggaactgga gaaaaatgtt tatgttgtag aattggattg
gtatcctaat 120gctcctggag aaacagtggt cctcacgtgt gacactcctg
aagaagacgg catcacctgg 180acctcagacc agagcagtga ggtcctgggc
tctggcaaaa ccttgaccat ccaagtcaaa 240gagtttggag atgctgggca
gtacacctgt cacaaaggag gcgaggttct gagtcgttca 300ctcctcctgc
tgcacaaaaa
ggaagatgga atttggtcca ctgatatttt aaaggatcag 360aaagaaccca
aagctaagag ttttttaaaa tgtgaggcaa aggattattc tggacacttc
420acctgctcgt ggctgacagc aatcagtact aatctgaaat tcagtgtcaa
aagcagcaga 480ggctcctctg acccccgagg ggtgacgtgc ggagcagcgt
ccctctcagc agagaaggtc 540agcatggacc acagggagta taacaagtac
acagtggagt gtcaggaggg cagtgcctgc 600ccggccgccg aggagagcct
gcccattgag gtcgtgatgg aagctgtgca caagctcaag 660tatgaaaact
acaccagcag cttcttcatc agggacatca tcaaaccaga cccacccaag
720aacctgcaac tgagaccact aaagaattct cggcaggtgg aagtcagctg
ggagtaccct 780gacacgtgga gcaccccaca ttcctacttc tccctgacgt
tttgtgttca ggtccaggga 840aagaacaaga gagaaaagaa actcttcaca
gaccaaacct cagccaaagt cacatgccac 900aaggatgcca acatccgcgt
gcaagcccgg gaccgctact acagctcatt ctggagtgaa 960tgggcatctg
tgtcctgcag ttag 98430327PRTOvis aries 30Met His Pro Gln Gln Leu Val
Val Ser Trp Phe Ser Leu Val Leu Leu1 5 10 15Ala Ser Pro Ile Val Ala
Ile Trp Glu Leu Glu Lys Asn Val Tyr Val 20 25 30Val Glu Leu Asp Trp
Tyr Pro Asn Ala Pro Gly Glu Thr Val Val Leu 35 40 45Thr Cys Asp Thr
Pro Glu Glu Asp Gly Ile Thr Trp Thr Ser Asp Gln 50 55 60Ser Ser Glu
Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu
Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val 85 90
95Leu Ser Arg Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
100 105 110Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Ala Lys
Ser Phe 115 120 125Leu Lys Cys Glu Ala Lys Asp Tyr Ser Gly His Phe
Thr Cys Ser Trp 130 135 140Leu Thr Ala Ile Ser Thr Asn Leu Lys Phe
Ser Val Lys Ser Ser Arg145 150 155 160Gly Ser Ser Asp Pro Arg Gly
Val Thr Cys Gly Ala Ala Ser Leu Ser 165 170 175Ala Glu Lys Val Ser
Met Asp His Arg Glu Tyr Asn Lys Tyr Thr Val 180 185 190Glu Cys Gln
Glu Gly Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro 195 200 205Ile
Glu Val Val Met Glu Ala Val His Lys Leu Lys Tyr Glu Asn Tyr 210 215
220Thr Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro
Lys225 230 235 240Asn Leu Gln Leu Arg Pro Leu Lys Asn Ser Arg Gln
Val Glu Val Ser 245 250 255Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro
His Ser Tyr Phe Ser Leu 260 265 270Thr Phe Cys Val Gln Val Gln Gly
Lys Asn Lys Arg Glu Lys Lys Leu 275 280 285Phe Thr Asp Gln Thr Ser
Ala Lys Val Thr Cys His Lys Asp Ala Asn 290 295 300Ile Arg Val Gln
Ala Arg Asp Arg Tyr Tyr Ser Ser Phe Trp Ser Glu305 310 315 320Trp
Ala Ser Val Ser Cys Ser 325311015DNACanis familiaris 31atgcatcctc
agcagttggt catctcctgg ttttccctcg ttttgctggc gtcttccctc 60atgaccatat
gggaactgga gaaagatgtt tatgttgtag agttggactg gcaccctgat
120gcccccggag aaatggtggt cctcacctgc catacccctg aagaagatga
catcacttgg 180acctcagcgc agagcagtga agtcctaggt tctggtaaaa
ctctgaccat ccaagtcaaa 240gaatttggag atgctggcca gtatacctgc
cataaaggag gcaaggttct gagccgctca 300ctcctgttga ttcacaaaaa
agaagatgga atttggtcca ctgatatctt aaaggaacag 360aaagaatcca
aaaataagat ctttctgaaa tgtgaggcaa agaattattc tggacgtttc
420acatgctggt ggctgacggc aatcagtact gatttgaaat tcagtgtcaa
aagtagcaga 480ggcttctctg acccccaagg ggtgacatgt ggagcagtga
cactttcagc agagagggtc 540agagtggaca acagggatta taagaagtac
acagtggagt gtcaggaagg cagtgcctgc 600ccctctgccg aggagagcct
acccatcgag gtcgtggtgg atgctattca caagctcaag 660tatgaaaact
acaccagcag cttcttcatc agagacatca tcaaaccaga cccacccaca
720aacctgcagc tgaagccatt gaaaaattct cggcacgtgg aggtcagctg
ggaatacccc 780gacacctgga gcaccccaca ttcctacttc tccctgacat
tttgcgtaca ggcccagggc 840aagaacaata gagaaaagaa agatagactc
tgcgtggaca agacctcagc caaggtcgtg 900tgccacaagg atgccaagat
ccgcgtgcaa gcccgagacc gctactatag ttcatcctgg 960agcgactggg
catctgtgtc ctgcagttag gttccacccc caggatgaat cttgg 101532329PRTCanis
familiaris 32Met His Pro Gln Gln Leu Val Ile Ser Trp Phe Ser Leu
Val Leu Leu1 5 10 15Ala Ser Ser Leu Met Thr Ile Trp Glu Leu Glu Lys
Asp Val Tyr Val 20 25 30Val Glu Leu Asp Trp His Pro Asp Ala Pro Gly
Glu Met Val Val Leu 35 40 45Thr Cys His Thr Pro Glu Glu Asp Asp Ile
Thr Trp Thr Ser Ala Gln 50 55 60Ser Ser Glu Val Leu Gly Ser Gly Lys
Thr Leu Thr Ile Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln
Tyr Thr Cys His Lys Gly Gly Lys Val 85 90 95Leu Ser Arg Ser Leu Leu
Leu Ile His Lys Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile
Leu Lys Glu Gln Lys Glu Ser Lys Asn Lys Ile Phe 115 120 125Leu Lys
Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp 130 135
140Leu Thr Ala Ile Ser Thr Asp Leu Lys Phe Ser Val Lys Ser Ser
Arg145 150 155 160Gly Phe Ser Asp Pro Gln Gly Val Thr Cys Gly Ala
Val Thr Leu Ser 165 170 175Ala Glu Arg Val Arg Val Asp Asn Arg Asp
Tyr Lys Lys Tyr Thr Val 180 185 190Glu Cys Gln Glu Gly Ser Ala Cys
Pro Ser Ala Glu Glu Ser Leu Pro 195 200 205Ile Glu Val Val Val Asp
Ala Ile His Lys Leu Lys Tyr Glu Asn Tyr 210 215 220Thr Ser Ser Phe
Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Thr225 230 235 240Asn
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg His Val Glu Val Ser 245 250
255Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu
260 265 270Thr Phe Cys Val Gln Ala Gln Gly Lys Asn Asn Arg Glu Lys
Lys Asp 275 280 285Arg Leu Cys Val Asp Lys Thr Ser Ala Lys Val Val
Cys His Lys Asp 290 295 300Ala Lys Ile Arg Val Gln Ala Arg Asp Arg
Tyr Tyr Ser Ser Ser Trp305 310 315 320Ser Asp Trp Ala Ser Val Ser
Cys Ser 325331005DNACercocebus torquatus 33agagcaagat gtgtcaccag
cagctggtca tctcttggtt ttccctggtt tttctggcat 60ctcccctcat ggccatatgg
gaactgaaga aagacgttta tgttgtagaa ttggactggt 120acccggatgc
ccctggagaa atggtggtcc tcacctgtga cacccctgaa gaagatggta
180tcacctggac cttggaccag agtggtgagg tcttaggctc tggcaaaacc
ctgaccatcc 240aagtcaaaga gtttggagat gctggccagt acacctgtca
caaaggaggc gaggctttga 300gccattcact cctgctgcct cacaaaaagg
aagatggaat ttggtccact gatattttaa 360aggaccagaa agaacccaaa
aatgagacct ttctaagatg cgaggccaaa aattattctg 420gacgtatcac
ctgctggtgg ctgtcgacaa tcagtactga tctgacattc agtatcataa
480gcagcagagg ctcttctaac ccccaagggg tgacgtgtgg agccgctaca
ctctctgcag 540agagggtcag aggggacaat aaggagtatg agtactcagt
ggagtgccag gaggacagtg 600cctgcccagc cgctgaggag aggctgccca
ttgaggtcat ggtggatgcc attcacaagc 660tcaagtatga aaactacacc
agcagcttct tcatcaggga catcatcaaa cccgacccac 720ccaagaactt
gcagctgaag ccattaaaga attctcggca ggtggaggtc agctgggagt
780accctgacac ctggagtact ccacattcct acttctccct gacattctgc
attcaggtcc 840agggcaagag caagagagaa aagaaagata gaatcttcac
agacaagacc tcagccacgg 900tcatctgccg caaaaatgcc agctttagcg
tgcaggccca ggaccgctac tatagctcat 960cttggaacga atggacatct
gtgccctgca gttaggttct gatcc 100534328PRTCercocebus torquatus 34Met
Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu1 5 10
15Ala Ser Pro Leu Met Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
20 25 30Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val
Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu
Asp Gln 50 55 60Ser Gly Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile
Gln Val Lys65 70 75 80Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His
Lys Gly Gly Glu Ala 85 90 95Leu Ser His Ser Leu Leu Leu Pro His Lys
Lys Glu Asp Gly Ile Trp 100 105 110Ser Thr Asp Ile Leu Lys Asp Gln
Lys Glu Pro Lys Asn Glu Thr Phe 115 120 125Leu Arg Cys Glu Ala Lys
Asn Tyr Ser Gly Arg Ile Thr Cys Trp Trp 130 135 140Leu Ser Thr Ile
Ser Thr Asp Leu Thr Phe Ser Ile Ile Ser Ser Arg145 150 155 160Gly
Ser Ser Asn Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170
175Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Arg Leu
Pro Ile 195 200 205Glu Val Met Val Asp Ala Ile His Lys Leu Lys Tyr
Glu Asn Tyr Thr 210 215 220Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys
Pro Asp Pro Pro Lys Asn225 230 235 240Leu Gln Leu Lys Pro Leu Lys
Asn Ser Arg Gln Val Glu Val Ser Trp 245 250 255Glu Tyr Pro Asp Thr
Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270Phe Cys Ile
Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285Ile
Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295
300Ser Phe Ser Val Gln Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp
Asn305 310 315 320Glu Trp Thr Ser Val Pro Cys Ser
32535984DNAMarmota monax 35atgtgtcttc agcagttggt catctcctgg
gtctccctgg tttggctggc atctcccctc 60ttggccatat gggaactgga gaaaaatgtc
tacgtggtgg agttggattg gcaccctgac 120acacctggag aaaaggtggt
cctcacctgt gacactcctg aagaagacgg catcacctgg 180acctcagagc
agagcagtga ggtcttaggc tccggcaaaa ccctgaccat tctagtcaaa
240gagtttgaag acgctggcca ctacacctgc cgcagaggag gtgaagttct
gagccagatg 300ctcctgctgc ttcacaaaaa tgaagatggg atttggtcca
ctgatattct gaagaaaaaa 360gaacctgaaa ataagaacct tgtaacatgc
gaggcaaaga attactctgg acgttttacc 420tgctggtggc tgacggcaat
cagtactgat gtgaacttca gtgtcaagag ccacagaggc 480tcctctgacc
ctcaaggggt gacgtgtgga gaagcaactc tctctgcaga gagggtcaaa
540atagagcaga gggagtacaa gaagtactcg gtgcagtgcc aggaggacaa
tgcctgcccc 600accgctgagg agaccctgcc catcacagtg gtggtggacg
cagttcacaa gctcaagtac 660gaaaactaca tcagcagctt cttcatcaga
gacatcatca aacctgaccc acccaagaac 720ctaaagatga agccatccaa
gactcctcag caggtggagg tcacctggga gtacccggac 780agctggagca
ccccgcactc ctacttctcc ctgacattct ctgtgcaggt ccagggcaaa
840aagaagaaaa ggagcaatac tctccacgtg gataagacct cagtcacagt
gacctgccag 900aagggtgcca aggtcagcgt gcaagcccgg gaccgatact
acaactcatc gtggagtgaa 960tgggcaacta tgtcctgccc ttag
98436327PRTMarmota monax 36Met Cys Leu Gln Gln Leu Val Ile Ser Trp
Val Ser Leu Val Trp Leu1 5 10 15Ala Ser Pro Leu Leu Ala Ile Trp Glu
Leu Glu Lys Asn Val Tyr Val 20 25 30Val Glu Leu Asp Trp His Pro Asp
Thr Pro Gly Glu Lys Val Val Leu 35 40 45Thr Cys Asp Thr Pro Glu Glu
Asp Gly Ile Thr Trp Thr Ser Glu Gln 50 55 60Ser Ser Glu Val Leu Gly
Ser Gly Lys Thr Leu Thr Ile Leu Val Lys65 70 75 80Glu Phe Glu Asp
Ala Gly His Tyr Thr Cys Arg Arg Gly Gly Glu Val 85 90 95Leu Ser Gln
Met Leu Leu Leu Leu His Lys Asn Glu Asp Gly Ile Trp 100 105 110Ser
Thr Asp Ile Leu Lys Lys Lys Glu Pro Glu Asn Lys Asn Leu Val 115 120
125Thr Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu
130 135 140Thr Ala Ile Ser Thr Asp Val Asn Phe Ser Val Lys Ser His
Arg Gly145 150 155 160Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Glu
Ala Thr Leu Ser Ala 165 170 175Glu Arg Val Lys Ile Glu Gln Arg Glu
Tyr Lys Lys Tyr Ser Val Gln 180 185 190Cys Gln Glu Asp Asn Ala Cys
Pro Thr Ala Glu Glu Thr Leu Pro Ile 195 200 205Thr Val Val Val Asp
Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Ile 210 215 220Ser Ser Phe
Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn225 230 235
240Leu Lys Met Lys Pro Ser Lys Thr Pro Gln Gln Val Glu Val Thr Trp
245 250 255Glu Tyr Pro Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser
Leu Thr 260 265 270Phe Ser Val Gln Val Gln Gly Lys Lys Lys Lys Arg
Ser Asn Thr Leu 275 280 285His Val Asp Lys Thr Ser Val Thr Val Thr
Cys Gln Lys Gly Ala Lys 290 295 300Val Ser Val Gln Ala Arg Asp Arg
Tyr Tyr Asn Ser Ser Trp Ser Glu305 310 315 320Trp Ala Thr Met Ser
Cys Pro 325
* * * * *