U.S. patent application number 09/363993 was filed with the patent office on 2002-05-09 for mammalian cytokine; related reagents.
Invention is credited to FICKENSCHER, HELMUT, FLECKENSTEIN, BERNHARD, KNAPPE, ANDREA.
Application Number | 20020054877 09/363993 |
Document ID | / |
Family ID | 26702382 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020054877 |
Kind Code |
A1 |
KNAPPE, ANDREA ; et
al. |
May 9, 2002 |
MAMMALIAN CYTOKINE; RELATED REAGENTS
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: |
KNAPPE, ANDREA; (ERLANGEN,
DE) ; FICKENSCHER, HELMUT; (ERLANGEN, DE) ;
FLECKENSTEIN, BERNHARD; (WIESENTHAU, DE) |
Correspondence
Address: |
DNAX RESEARCH INSTITUTE
LEGAL DEPARTMENT
901 CALIFORNIA AVENUE
PALO ALTO
CA
94304
US
|
Family ID: |
26702382 |
Appl. No.: |
09/363993 |
Filed: |
July 29, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09363993 |
Jul 29, 1999 |
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08934959 |
Sep 22, 1997 |
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5989867 |
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60027368 |
Sep 23, 1996 |
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Current U.S.
Class: |
424/154.1 ;
424/184.1; 424/192.1; 424/198.1; 435/320.1; 435/325; 435/7.1;
435/810; 530/351; 530/387.9; 536/23.5 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/5428 20130101 |
Class at
Publication: |
424/154.1 ;
530/351; 424/184.1; 424/198.1; 424/192.1; 530/387.9; 435/320.1;
435/810; 435/7.1; 435/325; 536/23.5 |
International
Class: |
G01N 033/53; C07H
021/04; C12P 021/02; A61K 039/395; A61K 039/00; A61K 039/38; C12N
015/09; C12N 015/00; C12N 015/63; C12N 015/70; C12N 015/74; C12N
005/00; C12N 005/02; C07K 001/00; C07K 014/00; C07K 017/00; C07K
016/00; C12P 021/08; C12N 001/00 |
Claims
What is claimed is:
1. A substantially pure or recombinant IL-XX protein.
2. An antigenic protein or peptide fragment of the IL-XX of claim
1.
3. A peptide of claim 2, selected from the group consisting of: a)
a full length natural protein or peptide from a mammal, including a
primate; b) a full length natural protein or peptide comprising at
least one polypeptide segment of SEQ ID NO: 2; c) a full length
protein or peptide which exhibits a post-translational modification
pattern distinct from natural IL-XX; and d) a protein or peptide
which exhibits a plurality of immunological activities of
IL-10.
4. A fusion protein comprising sequence of a protein or peptide of
claim 2.
5. A composition comprising a protein or peptide of claim 2, and a
pharmaceutically acceptable carrier.
6. An antibody which specifically binds a protein or peptide of
claim 2.
7. An antibody of claim 6, wherein: a) said IL-XX is a mammalian
protein, including a primate; b) said antibody is raised against a
purified peptide sequence from SEQ ID NO; 2; c) said antibody is a
monoclonal antibody; or d) said antibody is labeled.
8. A method of purifying an IL-XX protein or peptide from other
materials in a mixture comprising contacting said mixture to an
antibody of claim 6, and separating bound IL-XX from other
materials.
9. An isolated or recombinant expression vector capable of encoding
a protein or peptide of claim 1.
10. The vector of claim 9, wherein said nucleic acid: a) encodes a
sequence of SEQ ID NO: 2; b) comprises a sequence of SEQ ID NO: 1;
or c) encodes a sequence from an extracellular domain of a natural
IL-XX; or d) encodes a sequence from an intracellular domain of a
natural IL-XX.
11. A kit comprising: a) a substantially pure IL-XX or fragment of
claim 1; b) an antibody or receptor which specifically binds an
IL-XX; or c) a nucleic acid encoding an IL-XX or peptide.
12. A method for detecting in a sample for the presence of an IL-XX
nucleic acid, protein, or antibody, comprising testing said sample
with a kit of claim 11.
13. A method of modulating the physiology of a cell comprising
contacting said cell with: a) a substantially pure IL-XX or
fragment of claim 1; b) an antibody or binding partner which
specifically binds an IL-XX; or c) a nucleic acid encoding an IL-XX
or peptide.
14. The method of claim 13, wherein said cell is a T cell and said
modulating of physiology is activation of said T cell.
15. A method of claim 13, wherein said cell is in a tissue and/or
in an organism.
16. A method of making IL-XX comprising expressing a vector of
claim 9.
17. A cell, tissue, organ, or organism comprising a vector of claim
9.
18. A recombinant nucleic acid comprising sequence at least about
70% identity over a stretch of at least about 30 nucleotides to an
IL-XX nucleic acid sequence of SEQ ID NO: 1.
19. A nucleic acid of claim 19, further encoding a polypeptide
comprising at least about 60% identity over a stretch of at least
about 20 amino acids to an IL-XX sequence of SEQ ID NO: 2.
20. A method of treating a patient having an abnormal immune
response by administering to said patient an effective dose of: a)
an antibody or binding partner which binds specifically to an
IL-XX; b) a substantially pure IL-XX protein or peptide thereof; or
c) a nucleic acid encoding an IL-XX peptide.
Description
[0001] This filing is a conversion of a provisional patent
application U.S. Ser. No. 60/027,368, filed Sep. 23, 1996, which is
incorporated herein by reference, to a regular utility Patent
Application.
FIELD OF THE INVENTION
[0002] The present invention pertains to compositions related to
proteins which function in controlling biology and physiology of
mammalian cells, e.g., cells of a mammalian immune system. In
particular, it provides purified genes, proteins, antibodies, and
related reagents 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". 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.
[0005] Lymphokines apparently mediate cellular activities in a
variety of ways. They have been shown to support the proliferation,
growth, and 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] Another important cell lineage is the mast cell (which has
not been positively identified in all mammalian species), which is
a granule-containing connective tissue cell located proximal to
capillaries throughout the body. These cells are found in
especially high concentrations in the lungs, skin, and
gastrointestinal and genitourinary tracts. Mast cells play a
central role in allergy-related disorders, particularly anaphylaxis
as follows: when selected antigens crosslink one class of
immunoglobulins bound to receptors on the mast cell surface, the
mast cell degranulates and releases mediators, e.g., histamine,
serotonin, heparin, and prostaglandins, which cause allergic
reactions, e.g., anaphylaxis.
[0008] Research to better understand and treat various immune
disorders has been hampered by the general inability to maintain
cells of the immune system in vitro. Immunologists have discovered
that culturing these cells can be accomplished through the use of
T-cell and other cell supernatants, which contain various growth
factors, including many of the lymphokines.
[0009] The gene encoding IL-10, originally designated Cytokine
Synthesis Inhibitiory Factor (CSIF), was isolated in the 1980's.
See, e.g., Mosmann, et al., U.S. Pat. No. 5,231,012. Since then,
much has been learned of the biology and physiology mediated by the
cytokine. See, e.g., de Vries and de Waal Malefyt (1995)
Interleukin-10 Landes Co., Austin, Tex.
[0010] From the foregoing, it is evident that the discovery and
development of new lymphokines, e.g., related to IL-10, could
contribute to new therapies for a wide range of degenerative or
abnormal conditions which directly or indirectly involve the immune
system and/or hematopoietic cells. In particular, the discovery and
development of lymphokines which enhance or potentiate the
beneficial activities of known lymphokines would be highly
advantageous. The present invention provides new interleukin
compositions and related compounds, and methods for their use.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to mammalian, e.g.,
rodent, canine, feline, primate, interleukin-XX (IL-XX) and its
biological activities. It includes nucleic acids coding for
polypeptides themselves and methods for their production and use.
The nucleic acids of the invention are characterized, in part, by
their homology to cloned complementary DNA (cDNA) sequences
enclosed herein, and/or by functional assays for IL-10-like
activities applied to the polypeptides, which are typically encoded
by these nucleic acids. Methods for modulating or intervening in
the control of an immune response are provided.
[0012] The present invention is based, in part, upon the discovery
of a new cytokine exhibiting high sequence similarity to cellular
IL-10. In particular, it provides a gene encoding a protein whose
mature size is about 150 amino acids, which is expressed in virally
transformed cells, and certain tissues, e.g., kidney, and possibly
lung and liver. Functional equivalents exhibiting significant
sequence homology will be available from other mammalian, e.g.,
mouse and rat, and non-mammalian species.
[0013] More particularly, the present invention provides a
substantially pure or recombinant IL-XX protein or peptide fragment
thereof. Various embodiments include an antigenic protein or
peptide selected from a protein or peptide from a warm blooded
animal selected from the group of birds and mammals, including a
primate; a protein or peptide comprising at least one polypeptide
segment of SEQ ID NO: 2; a protein or peptide which exhibits a
post-translational modification pattern distinct from natural
IL-XX; or a protein or peptide which is capable of co-stimulating a
T cell with another signal. The protein or peptide can comprise a
fusion protein. Another embodiment is a composition comprising an
IL-XX protein or peptide and a pharmaceutically acceptable
carrier.
[0014] The invention also embraces an antibody which specifically
binds a IL-XX protein or peptide, e.g., wherein the IL-XX is a
mammalian protein, including a primate; the antibody is raised
against a purified IL-XX peptide sequence of SEQ ID NO: 2; the
antibody is a monoclonal antibody; or the antibody is labeled. The
antibodies also make available a method of purifying an IL-XX
protein or peptide from other materials in a mixture comprising
contacting the mixture to an anti-IL-XX antibody, and separating
bound IL-XX from other materials.
[0015] Another aspect of the invention is an isolated or
recombinant nucleic acid capable of encoding a full length or
mature IL-XX protein or peptide, including a nucleic acid which
encodes a sequence of SEQ ID NO: 2; which includes a sequence of
SEQ ID NO: 1; or which encodes a sequence from a natural IL-XX.
Such nucleic acid embodiments also include an expression or
replicating vector.
[0016] The invention also provides a kit containing a substantially
pure IL-XX or fragment; an antibody or receptor which specifically
binds an IL-XX; or a nucleic acid, or its complement, encoding an
IL-XX or peptide. This kit also provides methods for detecting in a
sample the presence of a nucleic acid, protein, or antibody,
comprising testing said sample with such a kit.
[0017] The invention also supplies methods of modulating the
physiology of a cell comprising contacting said cell with a
substantially pure IL-XX or fragment; an antibody or binding
partner which specifically binds an IL-XX; or a nucleic acid
encoding an IL-XX or peptide. Certain preferred embodiments include
a method where the cell is a T cell and the modulating of
physiology is activation of the T cell or apoptosis of the T cell;
or where the cell is in a tissue and/or in an organism.
[0018] Also provided are a method of expressing an IL-XX peptide by
expressing a nucleic acid encoding an IL-XX polypeptide. The
invention also provides a cell, tissue, organ, or organism
comprising a nucleic acid encoding an IL-XX peptide.
[0019] The invention also provides a recombinant nucleic acid
comprising sequence at least about 70% identity over a stretch of
at least about 30 nucleotides to an IL-XX nucleic acid sequence of
SEQ ID NO: 1, useful, e.g., as a probe or PCR primer for a related
gene. Another embodiment further encodes a polypeptide comprising
at least about 60% identity over a stretch of at least about 20
amino acids to an IL-XX sequence of SEQ ID NO: 2.
[0020] The invention further provides a method of treating a
patient having an abnormal immune response by administering an
effective dose of an antibody or binding partner specific for
IL-XX; an IL-XX protein or polypeptide; or a nucleic acid encoding
an IL-XX peptide. The abnormal immune response is characterized by
a T cell immune deficiency; chronic inflammation; or tissue
rejection.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0021] 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.
[0022] OUTLINE
[0023] I. General
[0024] II. Purified IL-XX
[0025] A. physical properties
[0026] B. biological properties
[0027] III. Physical Variants
[0028] A. sequence variants, fragments
[0029] B. post-translational variants
[0030] 1. glycosylation
[0031] 2. others
[0032] IV. Functional Variants
[0033] A. analogs, fragments
[0034] 1. agonists
[0035] 2. antagonists
[0036] B. mimetics
[0037] 1. protein
[0038] 2. chemicals
[0039] C. species variants
[0040] V. Antibodies
[0041] A. polyclonal
[0042] B. monoclonal
[0043] C. fragments, binding compositions
[0044] VI. Nucleic Acids
[0045] A. matural isolates; methods
[0046] B. synthetic genes
[0047] C. methods to isolate
[0048] VII. Making IL-XX, mimetics
[0049] A. recombinant methods
[0050] B. synthetic methods
[0051] C. natural purification
[0052] VIII. Uses
[0053] A. diagnostic
[0054] B. therapeutic
[0055] IX. Kits
[0056] A. nucleic acid reagents
[0057] B. protein reagents
[0058] C. antibody reagents
[0059] X. Isolating the IL-XX receptor
[0060] I. General
[0061] The present invention provides amino acid sequences and DNA
sequences encoding various mammalian proteins which are cytokines,
e.g., which are secreted molecules which can mediate a signal
between immune or other cells. See, e.g., Paul (1994) Fundamental
Immunology, Raven Press, N.Y. The full length cytokines, and
fragments, or antagonists will be useful in physiological
modulation of cells expressing a receptor. It is likely that IL-XX
has either stimulatory or inhibitory effects on T-cells, B-cells,
natural killer (NK) cells, macrophages, dentritic 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.
[0062] A cDNA encoding IL-XX was isolated from a virally infected
cell. The IL-XX cDNA contains a stretch of 510 bp in length and
contained one large open reading frame encoding a small soluble
cytokine-like protein. Structural features include an N-terminal
leader sequence of about 21 amino acids, though the natural
cleavage site may vary with cell, and may be on either side by a
few residues. See Table 1 and SEQ. ID. NO: 1 and 2. IL-XX exhibits
structural motifs characteristic of a member of the short chain
cytokines. Compare, e.g., IL-XX, cellular IL-10s from mouse and
human, EBV viral IL-10, and the Equine herpesvirus IL-10. See Table
2. Table 3 represents nucleotide sequences which encode the protein
sequence.
1TABLE 1 Human IL-XX nucleotide and predicted amino-acid sequence.
Predicted leader sequence ends after about 21 amino acids, though
natural boundaries may be different, also depending upon cell type.
The standard domain boundaries to helix A correspond to residues
about 16-39; .alpha.1 from about 47-55; helix B from about 81-100;
.alpha.2 from about 110-123; and helix D from about 125-150. See
SEQ ID NO: 1 and 2. CTGTGAGTGA CACACGCTGA GTGGGGTGAA GGGAA ATG CTG
GTG AAT TTC ATT 53 Met Leu Val Asn Phe Ile -21 -20 TTG AGG TGT GGG
TTG CTG TTA GTC ACT CTG TCT CTT GCC ATT GCC AAG 101 Leu Arg Cys Gly
Leu Leu Leu Val Thr Leu Ser Leu Ala Ile Ala Lys -15 -10 -5 1 CAC
AAG CAA TCT TCC TTC ACC AAA AGT TGT TAC CCA AGG GGA ACA TTG 149 His
Lys GLn Ser Ser Phe Thr Lys Ser cys Tyr Pro Arg Gly Thr Leu 5 10 15
TCC CAA GCT GTT GAC CCT CTC TAT ATC AAA GCA GCA TCG CTC AAA GCA 197
Ser GLn Ala VaL Asp Ala Leu Tyr Ile Lys Ala Ala Trp Leu Lys Ala 20
25 30 ACG ATT GCA GAA GAC CGC ATA AAA AAT ATA CGA TTA TTA AAA AAG
AAA 245 Thr Ile Pro Glu Asp Arg Ile Lys Asn Ile Arg Leu Leu Lys Lys
Lys 35 40 45 ACA AAA AAG CAG TTT ATG AAA AAC TGT CAA TTT CAA GAA
CAG CTT CTG 293 Thr Lys Lys Gln Phe Met Lys Asn Cys Gln Phe Gln Glu
Gln Leu Leu 50 55 60 65 TCC TTC TTC ATG GAA GAC GTT TTT GGT CAA CTG
CAA TTG CAA GGC TGC 341 Ser Phe Phe Met Glu Asp Val Phe Gly Gln Leu
Gln Leu Gln Gly Cys 70 75 80 AAG AAA ATA CGC TTT GTG GAG GAC TTT
CAT AGO CTT AGG CAG AAA TTG 389 Lys Lys Ile Arg Phe Val Glu Asp Phe
His Ser Leu Arg Gln Lys Leu 85 90 95 AGC CAC TGT ATT TCC TGT GCT
TCA TCA GCT AGA GAG ATG AAA TCC ATT 437 Ser His Cys Ile Ser Cys Ala
Ser Ser Ala Arg Glu Met Lys Ser Ile 100 105 110 ACC AGG ATG AAA AGA
ATA TTT TAT AGG ATT GGA AAC AAA GGA ATC TAC 485 Thr Arg Met Lys Arg
Ile Phe Tyr Arg Ile Gly Asn Lys Gly Ile Tyr 115 120 125 AAA GCC ATC
AGT GAA CTG GAT ATT CTT CTT TCC TGG ATT AAA AAA TTA 533 Lys Ala Ile
Ser Glu Leu Asp Ile Leu Leu Ser Trp Ile Lys Lys Leu 130 135 140 145
TTG GAA AGC AGT CAG TAAACCAAAG CCAAGTACAT TGATTTTACA GTTATTTTGA 588
Leu Glu Ser Ser Gln 150 AATACAATAA GAACTGCTAG AAATATGTTT ATAACAGTCT
ATTTCTTTTA AAAACTTTTT 648 AACATAATAC TGACGGCATG TTAGGTGATT
CAGAATAGAC AAGAAGGATT TAGTAAATTA 708 ACGTTTTGCA TATAAGTTGT
CACTAATTTG CACATTTTCT GTGTTTTCAA ATAATGTTTC 768 CATTCTGAAC
ATGTTTTGTC ATTCACAAGT ACATTGTGTC AACTTAATTT AAAGTATGTA 828
ACCTGAATTA ACTCGTGTAA TATTTGTGTG TGGAGTGGGA TGTGGGGGGT GGAGGGGGAA
888 TGACAQATTT CTGGAATGCA ATGTAATGTT ACTGACACTT AAATAGATGT
TATGTATATG 948 ATTCTCTGTT TAAGTGTTTQ AAAATTGTTA ATTATGCCCA
GTGTGAACTT AGTACTTAAC 1008 ACATTTTGAT TTTAATTAAA TAAATTGGGT
TTCCTTCTCA AAAAAAAAAAAAAAAAAAAAA 1068 AAAAAAAA 1076
[0063]
2TABLE 2 Comparison of various IL-10 embodiments compared to IL-XX.
First group is signal sequences, which are not aligned. See SEQ ID
NO:2-6. MFRASLLCCLVLLAGVWA Equine Herpes Virus (EHV)
MERRLVVTLQCLVLLYLAPECGG Epstein Barr Virus (EBV) MPGSALLCCLLLLTGMRT
moIL-10 MHSSALLCCLVLLTGVRA huIL-10 MLVNFILRCGLLLVTLSLATA huIL-XX
DNKYDSESGDDCPTLPTSLPHMLHELRAAFSRVKTFFQMKDQL EHV
TDQCDNFPQMLRDLRDAFSRVKTFFQTKDEV EBV
SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQL moIL-10
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQL huIL-10 KHKQSSFTKSC
YPRCTLSQAVDALYIKAAWLKATIPEDRIK huIL-XX
DNMLLDGSLLEDFKGYLGCQALSEMIQFYLEEVMPQAENHSTDQ EHV
DNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPE EBV
DNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPE moIL-10
DNLLLKESLLEDFKGYLCCQALSEMTQFYLEEVMPQAENQDPD huIL-10 NTRLLKKKTKKQFM
KNCQFQEQLLSFFI4EDVEGQLQLQG huIL-XX EKDKVNSLCEKLKTLRVRLRRCHRFLPCENK
EHV AKDHVNSLGENLKTLRLRLRRCHRFLPCENK EBV
IKEHLNSLGEKLKTLRMRLRRCHRFLPCENK moIL-10
IKAHVNSLGENLKTLRLRLRRCHRFLPCENK huIL-10 CKKIRFVEDFHTLRQKLSHCIS CASS
huTL-XX SKAVEQVKSAFSKLQEKGVYKAMSEFDTFTNYTEAYMTTKMKN EHV
SKAVEQIKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTIKAR EBV
SKAVEQVKSDFNKLQDQOVYKAMNEFDTFTNCTEAYMMTKMKS mIL-10
SKAVEQVKNAFNKLQEKGTYKAMSEFDTFTNYTEAYMTMKIRN huIL-10
AREMKSITRMKRTFYRTGNKGTYKATSELDTLLSWTKKLLESSQ huIL-XX
[0064]
3TABLE 3 Reverse Translation of the amino acid sequence of human
IL-XX, e.g., those nucleotide sequences which encode said protein.
See SEQ ID NO: 7. ATG YTN GTN AAY TTY ATH YTN MGN TGY GGN YTN YTN
YTN GTN ACN YTN WSN YTN GCN ATh GCN (signal) AAR CAY CAR WSN WSN
TTY ACN AAR WSN TGY TAY CCN MGN GGN ACN YTN WSN CAR GCN GTN GAY GCN
YTN TAY ATH AAR GCN GCN TGG YTN AAR GCN ACN ATh CCN GAR GAY NGN ATH
AAR AAY ATH NGN YTN YTN AAR AAR AAR ACN AAR AAR CAR TTY ATO AAR AAY
TGY CAR TTY CAR GAR CAR YTN YTN WSN TTY TTY ATG GAR GAY GTN TTY GGN
CAR YTN CAR YTN CAR GGN TGY AAR AAR ATH NGN TTY GTN GAR GAY TTY CAY
ACN YTN NGN CAR AAR YTN WSN CAY TGY ATH WSN TGY GCN WSN WSN GCN NGN
GAR ATG AAR WSN ATH ACN NGN ATG AAR NGN ATH TTY TAY NGN ATE GGN AAY
AAR GGN ATH TAY AAR GCN ATH WSN GAR YTN GAY ATE YTN YTN WSN TGG ATE
AAR AAR YTN YTN GAR WSN WSN CAR
[0065] By Northern analysis, it is clear that IL-XX is expressed in
virus transformed T cell lines from primates, including humans. RT
PCR has indicated that IL-XX is also expressed in PHA activated
PBMC, and in Jurkat and SupTil cell lines. Hybridization to mRNA
indicates expression in human kidney, and is detected in lung and
liver tissue. The transcript size is about 1.0-1.2 kb, and the gene
has been mapped to human chromosome 12q15. Transcripts for IL-XX
have not been detected by Northern analysis in PHA activated PBMC,
Jurkat cells, owl monkey kidney (OMK) cells, and human herpes
infected OMK cells; and by RT PCR in HeLa cells, and the EBV-free B
cell line BJA-B.
[0066] The structural homology of IL-XX to the related IL-10
proteins suggests similar function of this molecule. IL-XX, as a
small chain cytokine, likely mediates immune functions via a
receptor of the class of cytokine receptors, possibly even sharing
parts or all of the functional IL-10 receptor complex.
[0067] IL-XX agonists, or antagonists, may also act as functional
or receptor antagonists, e.g., which block IL-10 binding to its
receptor, or mediating the opposite actions. Thus, IL-XX, or its
antagonists, may be useful in the treatment of abnormal immune
disorders, e.g., T cell immune deficencies, chronic inflammation,
or tissue rejection.
[0068] The natural antigens are capable of mediating various
biochemical responses which lead to biological or physiological
responses in target cells. The embodiment charcterized herein is
from human, but other primate, or other species counterparts are
expected to exist in nature. Additional sequences for proteins in
other mammalian species, e.g., primates, canines, felines, and
rodents, should also be available. See below. The descriptions
below are directed, for exemplary purposes, to a human IL-XX, but
are likewise applicable to related embodiments from other
species.
[0069] The human IL-XX protein exhibits structural features
characteristic of short chain cytokines.
[0070] II. Purified IL-XX
[0071] Human IL-XX amino acid sequence is shown in SEQ ID NO: 2.
These amino acid sequences, provided amino to carboxy, are
important in providing sequence information in the cytokine
allowing for distinguishing the protein antigen from other proteins
and exemplifying numerous variants. Moreover, the peptide sequences
allow preparation of peptides to generate antibodies to recognize
such segments, and nucleotide sequences allow preparation of
oligonucleotide probes, both of which are strategies for detection
or isolation, e.g., cloning, of genes encoding such sequences.
[0072] As used herein, the term "human IL-XX" shall encompass, when
used in a protein context, a protein having amino acid sequence
shown in SEQ ID NO: 2, or a significant fragment of such a protein,
or another highly homologous protein derived from human, as
distinguished from human IL-10. Binding components, e.g.,
antibodies, typically bind to an IL-XX 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. Homologous proteins would be found in mammalian species other
than human, e.g., other primates or rodents. Non-mammalian species
should also possess structurally or functionally related genes and
proteins, e.g., birds or amphibians.
[0073] The term "polypeptide" as used herein includes a significant
fragment or segment, and encompasses a stretch of amino acid
residues of at least about 8 amino acids, generally at least about
12 amino acids, typically at least about 16 amino acids, preferably
at least about 20 amino acids, and, in particularly preferred
embodiments, at least about 30 or more amino acids, e.g., 35, 40,
45, 50, etc. Such fragments may have ends which begin and/or end at
virtually all positions, e.g., beginning at residues 1, 2, 3, etc.,
and ending at, e.g., 150, 149, 148, etc., in all combinations.
Particularly interesting peptides have ends corresponding to
structural domain boundaries, e.g., helices A, B, C, and/or D. See
Table 1. Note that the sequence of IL-XX exhibits particular
identity to cellular IL-10 in the region from residue 126-137, and
the other regions exhibit greater extents of IL-XX specific
sequence. Seemingly important residues are those shared among all
of the four entities in Table 3.
[0074] The term "binding composition" refers to molecules that bind
with specificity to IL-XX, e.g., in an antibody-antigen
interaction. It also includes compounds, e.g., proteins, which
specifically associate with IL-XX, 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.)
(1990) Goodman & Gilman's: The Pharmacological Bases of
Therapeutics (8th ed.), Pergamon Press.
[0075] Substantially pure 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.
[0076] 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.
[0077] The size and structure of the polypeptide should generally
be in a substantially stable state, and usually not in a denatured
state. The polypeptide may be associated with other polypeptides in
a quaternary structure, e.g., to confer solubility, or associated
with lipids or detergents.
[0078] The solvent and electrolytes will usually be a biologically
compatible buffer, of a type used for preservation of biological
activities, and will usually approximate a physiological aqueous
solvent. Usually the solvent will have a neutral pH, typically
between about 5 and 10, and preferably about 7.5. On some
occasions, one or more detergents will be added, typically a mild
non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS
(3-[3-cholamidopropyl)dimethylammoni- o]-1-propane sulfonate), or a
low enough concentration as to avoid significant disruption of
structural or physiological properties of the protein.
[0079] III. Physical Variants
[0080] This invention also encompasses proteins or peptides having
substantial amino acid sequence identity with the amino acid
sequence of the IL-XX antigen. The variants include species,
polymorphic, or allelic variants.
[0081] 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 in each
respective 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-XX. 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%.
[0082] The isolated IL-XX DNA can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and
inversions of nucleotide stretches. These modifications result in
novel DNA sequences which encode 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-XX"
encompasses a polypeptide otherwise falling within the sequence
identity definition of the IL-XX as set forth above, but having an
amino acid sequence which differs from that of IL-XX 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 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-XX proteins, particularly those found in various warm
blooded animals, e.g., mammals and birds. These descriptions are
generally meant to encompass all IL-XX proteins, not limited to the
particular mouse embodiments specifically discussed.
[0083] IL-XX 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] IV. Functional Variants
[0088] The blocking of physiological response to IL-XXs may result
from the competitive inhibition of binding of the ligand to its
receptor. IL-XX binding to IL-10 receptor may serve to induce
signaling, e.g., send a signal similar to binding by IL-10.
Alternatively, IL-XX binding to IL-10 receptor may block IL-10
signaling. An IL-XX antagonist would be expected to have the
opposite effect as IL-XX.
[0089] In vitro assays of the present invention will often use
isolated protein, soluble fragments comprising receptor binding
segments of these proteins, or fragments attached to solid phase
substrates. These assays will also allow for the diagnostic
determination of the effects of either binding segment mutations
and modifications, or cytokine mutations and modifications, e.g.,
IL-XX analogues.
[0090] This invention also contemplates the use of competitive drug
screening assays, e.g., where neutralizing antibodies to the
cytokine, or receptor binding fragments compete with a test
compound.
[0091] "Derivatives" of IL-XX antigens include amino acid sequence
mutants from naturally occuring 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-XX 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.
[0092] 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.
[0093] Fusion polypeptides between IL-XXs and other homologous or
heterologous proteins are also provided. Many cytokine receptors or
other surface proteins are multimeric, e.g., homodimeric entities,
and a repeat construct may have various advantages, including
lessened susceptibility to proteolytic cleavage. Typical examples
are fusions of a reporter polypeptide, e.g., luciferase, with a
segment or domain of a protein, e.g., a receptor-binding segment,
so that the presence or location of the fused ligand may be easily
determined. See, e.g., Dull, et al., U.S. Pat. No. 4,859,609. Other
gene fusion partners include bacterial .beta.-galactosidase, trpE,
Protein A, .beta.-lactamase, alpha amylase, alcohol dehydrogenase,
yeast alpha mating factor, and detection or purification tags such
as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al.
(1988) Science 241:812-816.
[0094] 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.
[0095] This invention also contemplates the use of derivatives of
IL-XX 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-XX
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-XX antibodies or an alternative binding
composition. The IL-XX proteins can also be labeled with a
detectable group, e.g., for use in diagnostic assays. Purification
of IL-XX may be effected by an immobilized antibody or
complementary binding partner, e.g., binding portion of a
receptor.
[0096] A solubilized IL-XX 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-XX antigens can also be used as a
reagent to detect antibodies generated in response to the presence
of elevated levels of the cytokine, which may be diagnostic of an
abnormal or specific physiological or disease condition. This
invention contemplates antibodies raised against amino acid
sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, or
fragments of proteins containing it. In particular, this invention
contemplates antibodies having binding affinity to or being raised
against specific domains, e.g., helices A, B, C, or D.
[0097] 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-XXs are widespread in species
variants, e.g., rodents, lagomorphs, carnivores, artiodactyla,
perissodactyla, and primates.
[0098] 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.
[0099] The isolated genes will allow transformation of cells
lacking expression of an IL-XX, 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-XX in
comparison to untransformed control cells.
[0100] 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.
[0101] 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-XX 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.
[0102] Further study of the expression and control of IL-XX 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.
[0103] Structural studies of the IL-XX 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.
[0104] V. Antibodies
[0105] Antibodies can be raised to various epitopes of the IL-XX
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-XXs in either their active forms or in their inactive forms,
including native or denatured versions. Anti-idiotypic antibodies
are also contemplated.
[0106] 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-XXs, or screened for
agonistic or antagonistic activity, e.g., mediated through a
receptor. Antibodies may be agonsitic 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.
[0107] 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-XX 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] Other suitable techniques involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to
selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial
Library of the Immunoglobulin Repertoire in Phage Lambda," Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-546. The
polypeptides and antibodies of the present invention may be used
with or without modification, including chimeric or humanized
antibodies. Frequently, the polypeptides and antibodies will be
labeled by joining, either covalently or non-covalently, a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and are reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent moieties, chemiluminescent moieties,
magnetic particles, and the like. Patents, teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced, see Cabilly, U.S. Pat. No.
4,816,567; Moore, et al., U.S. Pat. No. 4,642,334; and Queen, et
al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033.
[0112] 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.
[0113] Antibodies raised against each IL-XX 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.
[0114] VI. Nucleic Acids
[0115] The described peptide sequences and the related reagents are
useful in detecting, isolating, or identifying a DNA clone encoding
IL-XX, e.g., from a natural source. Typically, it will be useful in
isolating a gene from 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-XX from the same, e.g., polymorphic variants, or
other species. A number of different approaches should be available
to successfully isolate a suitable nucleic acid clone.
[0116] The purified protein or defined peptides 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.
[0117] For example, the specific binding composition could be used
for screening of an expression library made from a cell line which
expresses an IL-XX. 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.
[0118] The peptide segments can also be used to predict appropriate
oligonucleotides to screen a library. The genetic code can be used
to select appropriate oligonucleotides useful as probes for
screening. See, e.g., SEQ ID NO: 1 or 3. 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.
[0119] This invention contemplates use of isolated DNA or fragments
to encode a biologically active corresponding IL-XX polypeptide. In
addition, this invention covers isolated or recombinant DNA which
encodes a biologically active protein or polypeptide and which is
capable of hybridizing under appropriate conditions with the DNA
sequences described herein. Said biologically active protein or
polypeptide can be an intact antigen, or fragment, and have an
amino acid sequence disclosed in, e.g., SEQ ID NO: 2. Further, this
invention covers the use of isolated or recombinant DNA, or
fragments thereof, which encode proteins which exhibit high
identity to an IL-XX or which was isolated using cDNA encoding an
IL-XX as a probe. 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.
[0120] An "isolated" nucleic acid is a nucleic acid, e.g., an RNA,
DNA, or a mixed polymer, which is substantially separated from
other components which naturally accompany a native sequence, e.g.,
ribosomes, polymerases, and/or flanking genomic sequences from the
originating species. The term embraces a nucleic acid sequence
which has been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and chemically
synthesized analogs or analogs biologically synthesized by
heterologous systems. A substantially pure molecule includes
isolated forms of the molecule. Generally, the nucleic acid will be
in a vector or fragment less than about 50 kb, usually less than
about 30 kb, typically less than about 10 kb, and preferably less
than about 6 kb.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] A DNA which codes for an IL-XX 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 are likely
homologs in other species, including primates, rodents, canines,
felines, and birds. Various IL-XX 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-XX proteins are of
particular interest.
[0127] 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
(1987)(ed.) Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical
Oncology 10:180-199.
[0128] 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-XX, e.g., in SEQ
ID NO: 1 or 3. 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.
[0129] Stringent conditions, in referring to homology in the
hybridization context, will be stringent combined conditions of
salt, temperature, organic solvents, and other parameters,
typically those controlled in hybridization reactions. Stringent
temperature conditions will usually include temperatures in excess
of about 30.degree. C., usually in excess of about 37.degree. C.,
typically in excess of about 55.degree. C., preferably in excess of
about 70.degree. C. Stringent salt conditions will ordinarily be
less than about 1000 mm, usually less than about 400 mM, typically
less than about 250 mM, preferably less than about 150 mM,
including about 100, 50, or even 20 mM. However, the combination of
parameters is much more important than the measure of any single
parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol.
31:349-370.
[0130] IL-XX 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.
[0131] VII. Making IL-XX; Mimetics
[0132] DNA which encodes the IL-XX 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-XX; including naturally occuring
embodiments.
[0133] This DNA can be expressed in a wide variety of host cells
for the synthesis of a full-length IL-XX 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.
[0134] 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.
(1988)(eds.) Vectors: A Survey of Molecular Cloning Vectors and
Their Uses, Buttersworth, Boston, Mass.
[0135] 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.
[0136] 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.
[0137] It will often be desired to express an IL-XX 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.
[0138] The IL-XX, 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.
[0139] Now that the IL-XX has been characterized, fragments or
derivatives thereof can be prepared by conventional processes for
synthesizing peptides. These include processes such as are
described in Stewart and Young (1984) Solid Phase Peptide
Synthesis, Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis,
Springer-Verlag, New York; Bodanszky (1984) The Principles of
Peptide Synthesis, Springer-Verlag, New York; and Villafranca (ed.)
(1991) Techniques in Protein Chemistry II, Academic Press, San
Diego, Calif.
[0140] VIII. Uses
[0141] The present invention provides reagents which will find use
in diagnostic applications as described elsewhere herein, e.g., in
IL-XX mediated conditions, or below in the description of kits for
diagnosis.
[0142] This invention also provides reagents with significant
therapeutic potential. The IL-XX (naturally occurring or
recombinant), fragments thereof, and antibodies thereto, along with
compounds identified as having binding affinity to IL-XX, should be
useful in the treatment of conditions associated with abnormal
physiology or development, including inflammatory conditions,
either acute or chronic. In particular, modulation of physiology of
lymphoid cells will be achieved by appropriate therapeutic
treatment using the compositions provided herein. For example, a
disease or disorder associated with abnormal expression or abnormal
signaling by an IL-XX 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 or myeloid
cells, which affect immunological responses, e.g., inflammation
and/or autoimmune disorders.
[0143] In particular, the cytokine should mediate, in various
contexts, cytokine synthesis by the cells, proliferation, etc.
[0144] Conversely, antagonists of IL-XX, such as mutein variants of
a naturally occurring form of IL-XX or blocking antibodies, may
provide a selective and powerful way to block immune responses,
e.g., in situations as inflammatory or autoimmune responses,
including rheumatoid arthritis, systemic lupus erythematosis (SLE),
Hashimoto's autoimmune thyroiditis, as well as acute and chronic
inflammatory responses, e.g., inflammatory bowel disease. See also
Samter, et al. (eds) Immunological Diseases vols. 1 and 2, Little,
Brown and Co. Modulated cytokine release by the naturally occurring
secreted form of IL-XX, which can be produced in large quantities
by recombinant methods, or by blocking antibodies, should be
regulatable by reagents made available herein, e.g., in a
transplantation rejection situation.
[0145] In addition, certain combination compositions would be
useful, e.g., with other modulators of inflammation. Such other
molecules may include steroids, other versions of IL-10, including
cellular species variants, or viral IL-10s, e.g., EBV or EHV, and
all of their respective antagonists.
[0146] Various abnormal conditions are known in each of the cell
types shown to produce IL-XX 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.
[0147] IL-XX antibodies 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.
[0148] Drug screening using IL-XX or fragments thereof can be
performed to identify compounds having binding affinity to or other
relevant biological effects on IL-XX functions, including isolation
of associated components. Subsequent biological assays can then be
utilized to determine if the compound has intrinsic stimulating
activity and is therefore a blocker or antagonist in that it blocks
the activity of the cytokine. Likewise, a compound having intrinsic
stimulating activity can activate the signal pathway and is thus an
agonist in that it simulates the activity of IL-XX. This invention
further contemplates the therapeutic use of blocking antibodies to
IL-XX as antagonists and of stimulatory antibodies as agonists.
This approach should be particularly useful with other IL-XX
species variants.
[0149] In addition, IL-XX may play a role in leukemogenesis or in
viral infections by, e.g., HTLV or herpesviruses. It is induced by
infection with herpesvirus saimiri. The herpesvirus also encodes a
homolog of the cytokine IL-17 (CTLA-8). Thus, the cytokine, or
antagonists, may be useful in anti-tumor therapy. The viral
correlation may suggest that the cytokine may be important in viral
infection or proliferation processes, or oncology processes, e.g.,
oncogenic transformation and proliferative conditions, as cancers
or leukemias. See, e.g., Thorn, et al. Harrison's Principles of
Internal Medicine, McGraw-Hill, N.Y.
[0150] In addition, the cytokine appears to be barely expressed in
kidney cell, and may play a role in that organ's function, e.g.,
ion exchange or blood pressure regulation. The cytokine may also
have water balance functions. The cytokine may have some detectable
expression in kidney.
[0151] 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.
[0152] IL-XX, fragments thereof, and antibodies to it or its
fragments, antagonists, and agonists, may be administered directly
to the host to be treated or, depending on the size of the
compounds, it may be desirable to conjugate them to carrier
proteins such as ovalbumin or serum albumin prior to their
administration. Therapeutic formulations may be administered in
many conventional dosage formulations. While it is possible for the
active ingredient to be administered alone, it is preferable to
present it as a pharmaceutical formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Each carrier should
be both pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and not
injurious to the patient. Formulations include those suitable for
oral, rectal, nasal, topical, or parenteral (including
subcutaneous, intramuscular, intravenous and intradermal)
administration. The formulations may conveniently be presented in
unit dosage form and may be prepared by any methods well known in
the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)
Goodman and Gilman's: The Pharmacological Bases of Therapeutics,
8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences,
17th ed. (1990), Mack Publishing Co., Easton, Pa.; Avis, et al.
(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,
Dekker, New York; Lieberman, et al. (eds.) (1990) Pharmaceutical
Dosage Forms: Tablets, Dekker, New York; and Lieberman, et al.
(eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems,
Dekker, New York. The therapy of this invention may be combined
with or used in association with other agents, e.g., other types of
IL-10s, or their respective antagonists.
[0153] Both the naturally occurring and the recombinant form of the
IL-XXs 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-XX as provided by this invention.
[0154] Other methods can be used to determine the critical residues
in the IL-XX-IL-XX 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. However, residues in the A and D
helices are likely to be most important in receptor
interaction.
[0155] For example, antagonists can normally be found once the
antigen has been structurally defined, e.g., by tertiary structure
data. Testing of potential interacting analogues is now possible
upon the development of highly automated assay methods using a
purified IL-XX. 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-XX molecules, e.g., compounds
which can serve as antagonists for species variants of IL-XX.
[0156] One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing an IL-XX. Cells may be
isolated which express an IL-XX in isolation from other molecules.
Such cells, either in viable or fixed form, can be used for
standard binding partner binding assays. See also, Parce, et al.
(1989) Science 246:243-247; and Owicki, et al. (1990) Proc. Nat'l
Acad. Sci. USA 87:4007-4011, which describe sensitive methods to
detect cellular responses.
[0157] Another technique for drug screening involves an approach
which provides high throughput screening for compounds having
suitable binding affinity to an IL-XX and is described in detail in
Geysen, European Patent Application 84/03564, published on Sep. 13,
1984. First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g., plastic pins
or some other appropriate surface, see Fodor, et al. (1991). Then
all the pins are reacted with solubilized, unpurified or
solubilized, purified IL-XX, and washed. The next step involves
detecting bound IL-XX.
[0158] Rational drug design may also be based upon structural
studies of the molecular shapes of the IL-XX and other effectors or
analogues. Effectors may be other proteins which mediate other
functions in response to binding, or other proteins which normally
interact with IL-XX, 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 madeled,
e.g., against cellular IL-10. For a detailed description of protein
structural determination, see, e.g., Blundell and Johnson (1976)
Protein Crystallography, Academic Press, New York.
[0159] IX. Kits
[0160] This invention also contemplates use of IL-XX proteins,
fragments thereof, peptides, and their fusion products in a variety
of diagnostic kits and methods for detecting the presence of
another IL-XX or binding partner. Typically the kit will have a
compartment containing either a defined IL-XX peptide or gene
segment or a reagent which recognizes one or the other, e.g., IL-XX
fragments or antibodies.
[0161] A kit for determining the binding affinity of a test
compound to an IL-XX would typically comprise a test compound; a
labeled compound, for example a binding partner or antibody having
known binding affinity for IL-XX; a source of IL-XX (naturally
occurring or recombinant); and a means for separating bound from
free labeled compound, such as a solid phase for immobilizing the
molecule. Once compounds are screened, those having suitable
binding affinity to the antigen can be evaluated in suitable
biological assays, as are well known in the art, to determine
whether they act as agonists or antagonists to the IL-XX signaling
pathway. The availability of recombinant IL-XX polypeptides also
provide well defined standards for calibrating such assays.
[0162] A preferred kit for determining the concentration of, e.g.,
an IL-XX in a sample would typically comprise a labeled compound,
e.g., binding partner or antibody, having known binding affinity
for the antigen, a source of cytokine (naturally occurring or
recombinant) and a means for separating the bound from free labeled
compound, e.g., a solid phase for immobilizing the IL-XX.
Compartments containing reagents, and instructions, will normally
be provided.
[0163] Antibodies, including antigen binding fragments, specific
for the IL-XX or fragments are useful in diagnostic applications to
detect the presence of elevated levels of IL-XX 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.
[0164] Anti-idiotypic antibodies may have similar use to diagnose
presence of antibodies against an IL-XX, as such may be diagnostic
of various abnormal states. For example, overproduction of IL-XX
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.
[0165] Frequently, the reagents for diagnostic assays are supplied
in kits, so as to optimize the sensitivity of the assay. For the
subject invention, depending upon the nature of the assay, the
protocol, and the label, either labeled or unlabeled antibody or
binding partner, or labeled IL-XX 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.
[0166] Many of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways. For example,
labeling may be achieved by covalently or non-covalently joining a
moiety which directly or indirectly provides a detectable signal.
In any of these assays, the binding partner, test compound, IL-XX,
or antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels
such as .sup.125I, enzymes (U.S. Pat. No. 3,645,090) such as
peroxidase and alkaline phosphatase, and fluorescent labels (U.S.
Pat. No. 3,940,475) capable of monitoring the change in
fluorescence intensity, wavelength shift, or fluorescence
polarization. Possibilities for indirect labeling include
biotinylation of one constituent followed by binding to avidin
coupled to one of the above label groups.
[0167] There are also numerous methods of separating the bound from
the free IL-XX, or alternatively the bound from the free test
compound. The IL-XX 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.
[0168] 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.
[0169] Another diagnostic aspect of this invention involves use of
oligonucleotide or polynucleotide sequences taken from the sequence
of an IL-XX. These sequences can be used as probes for detecting
levels of the IL-XX message in samples from patients suspected of
having an abnormal condition, e.g., inflammatory or autoimmune.
Since the cytokine may be a marker or mediator for activation, it
may be useful to determine the numbers of activated cells to
determine, e.g., when additional therapy may be called for, e.g.,
in a preventative fashion before the effects become and progress to
significance. The preparation of both RNA and DNA nucleotide
sequences, the labeling of the sequences, and the preferred size of
the sequences has received ample description and discussion in the
literature. See, e.g., Langer-Safer, et al. (1982) Proc. Nat'l.
Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967; and
Wilchek et al. (1988) Anal. Biochem. 171:1-32.
[0170] 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.
[0171] X. Isolating the IL-XX Receptor
[0172] 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-XX 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,
though based on IL-10, the amino-terminus is more likely to
succeed. Such label may be a FLAG epitpe tag, or, e.g., an Ig or Fc
domain. An expression library can be screened for specific binding
of the cytokine, e.g., by cell sorting, or other screening to
detect subpopulations which express such a binding component. See,
e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90:11267-11271;
and Liu, et al. (1994) J. Immunol. 152:1821-29. Alternatively, a
panning method may be used. See, e.g., Seed and Aruffo (1987) Proc.
Nat'l Acad. Sci. USA 84:3365-3369.
[0173] Protein cross-linking techniques with label can be applied
to isolate binding partners of the IL-XX cytookine. This would
allow identification of proteins which specifically interact with
the cytokine, e.g., in a ligand-receptor like manner.
[0174] Early experiments will be performed to determine whether the
known IL-10R is involved in response(s) to IL-XX. It is also quite
possible that the functional IL-10 receptor complex may share many
or all components with an IL-XX receptor complex, either a specific
receptor subunit or an accessory receptor subunit.
[0175] 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
[0176] General Methods
[0177] Some of the standard methods are described or referenced,
e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press;
Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d
ed.), vols 1-3, CSH Press, NY; Ausubel, et al., Biology, Greene
Publishing Associates, Brooklyn, N.Y.; or Ausubel, et al. (1987 and
Supplements) Current Protocols in Molecular Biology, Greene and
Wiley, New York; Innis, et al. (eds.)(1990) PCR Protocols: A Guide
to Methods and Applications, Academic Press, N.Y. Methods for
protein purification include such methods as ammonium sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g., Ausubel, et
al. (1987 and periodic supplements); Deutscher (1990) "Guide to
Protein Purification" in Methods in Enzymology vol. 182, and other
volumes in this series; and manufacturer's literature on use of
protein purification products, e.g., Pharmacia, Piscataway, N.J.,
or Bio-Rad, Richmond, Calif. Combination with recombinant
techniques allow fusion to appropriate segments, e.g., to a FLAG
sequence or an equivalent which can be fused via a
protease-removable sequence. See, e.g., Hochuli (1989) Chemische
Industrie 12:69-70; Hochuli (1990) "Purification of Recombinant
Proteins with Metal Chelate Absorbent" in Setlow (ed.) Genetic
Engineering, Principle and Methods 12:87-98, Plenum Press, N.Y.;
and Crowe, et al. (1992) OIAexpress: The High Level Expression
& Protein Purification System QUIAGEN, Inc., Chatsworth, Calif.
Cell culture techniques are described in Doyle, et al. (eds.)
(1994) Cell and Tissue Culture: Laboratory Procedures, John Wiley
and Sons, NY.
[0178] 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. Fluorescent labeling of appropriate reagents was
performed by standard methods.
Example 1
[0179] Cloning of Human IL-XX
[0180] PBMC were prepared from a healthy human blood donor by
conventional Ficoll gradients, as described, e.g., in Coligan, et
al. Current Protocols in Immunology Greene/Wiley. Cells from this
preparation were stimulated with PHA and cultivated in the presence
of IL-2 for several weeks. See, e.g., Fickenscher and Fleckenstein
pp345-362, "Generation of human T cell lines using lymphotropic
herpesviruses" in Adolph (ed) Methods in Molecular Genetics:
Molecular Virology Techniques Part A Volume 4, Academic Press, San
Diego, Calif. RNA from these PHA-blasts was used later to subtract
the normally occurring cDNAs.
[0181] Another portion of the PBMC preparation was infected with
herpesvirus saimiri C488. See Fickenscher and Fleckenstein ,
pp345-362, above; and Biesinger, et al. (1992) Proc. Nat'l Acad.
Sci. USA 89:3116-3119. The infected cells were cultivated in the
presence of IL-2 until growth transformation was established
(several months). RNA was isolated from the transformed T-cell
line, designated 3C (see Fickenscher, et al. (1996) The
Immunologist 4:41-43), after the cells had been stimulated using 1
ng/ml TPA (Fickenscher, et al.(1996) J. Virol. 70:6012-6019) for
four hours. RNA was isolated according to Chomczynski and Sacchi
(1987) Anal. Biochem. 162:156-159. The subtractive cDNA library was
prepared with a cDNA subtraction kit (from Clontech, Palo Alto,
Calif.).
[0182] PCR products were cloned using a TA cloning kit
(Invitrogen). The resulting cDNA plasmids were sequenced from both
termini on an automated sequencer (Applied Biosystems).
[0183] Plasmid ak155 contains a cDNA fragment of 540 nt. There is a
single large open reading frame found, starting at nucleotide 12,
and ending at nucleotide 524. Termination signals are not found in
this partial cDNA. Using 5' and 3' RACE, the remaining fragments of
the entire cDNA were cloned. The transcript size is approximately
1.0 to 1.2 kb. Genomic structure analysis indicates that introns
exist at or near to between nucleotides 206 and 207, of about 35
nucleotides; between 263 and 264, of about 60 nucleotides; between
398 and 399, of about 1.5 kb; and between 464 and 465, of about 86
nucleotides. The sequences of the short introns have been
determined.
[0184] The sequence derived from plasmid clone ak155 exhibited
distant similarity to IL10s, see Table 3.
Example 2
[0185] Cellular Expression of Human IL-XX
[0186] Because of the sequence similarity to human IL-10,
distribution was investigated for similar type cell types. A probe
specific for cDNA encoding primate IL-XX is labeled, e.g., by
random priming.
[0187] IL-XX/ak155 is strongly transcribed in various T-cell lines
of human and non-human primates, which have been in-vitro
transformed to stable IL-2 dependent growth by herpesvirus saimiri
C488. This expression is analysed by Northern blotting. Owl monkey
kidney cells (OMK) which are a primate permissive system for the
human virus, and virus-infected OMK were negative by Northern
blotting. TPA stimulation did not significantly increase
IL-XX/ak155 transcript levels in virus-transformed T-cells; and
cyclosporin A did not inhibit its expression. Transcription has
been confirmed by RT-PCR from transformed human T-cells in 3C and
CB15 cells (Biesinger, et al. (1992) Proc. Nat'l Acad. Sci. USA
89:3116-3119; Fickenscher and Fleckenstein (1994); and Fickenscher,
et al. (1996) J. Virol.). It is quite notable that IL-XX is so
strongly expressed in herpesvirus saimiri-transfomed T-cells, which
suggests a role in the transformation mechanism. Expression was
also detected in a monkey T cell line 93C488, a cell line from
Saguinus fuscicollis monkeys that produces virus particles.
[0188] By RT-PCR weak transcription was detected in human
PHA-activated PBMC, and in T-cell tumor lines like Jurkat
(Schneider, et al (1977) Int. J. Cancer 19:621-626) and SupTi1
(ATCC CRL-1942; see (1986) Science 232:1123-1127; and (1984) Cancer
Res. 44:5657-5660); and in HTLV-transformed human T cells MT2,
C91PL, and HUT102 (which do produce HTLV-Virions; see Popovic, et
al. (1984) "Biology of Human T-cell leukemia/lymphoma virus" in
Klein (ed.) Advances in Viral Oncology, Vol. 4, Raven Press, NY).
Thus, a low level of ak155 expression seems typical for human T
cells, e.g., leukemia cell lines (Jurkat etc.) and HTLV-transformed
cells. Positive signal was detected in macrophages stimulated with
IFN-.gamma. and/or LPS, but not after treatment with Protein A
expressing cells. No detectable signal was found in HeLa cells,
BJA-B (human B-cell line which does not carry EBV genomes; see
Klein, et al. (1974) Proc. Nat'l Acad. Sci. USA 71:3283-3286),
Tera-2 (human Teratocarcinoma cell line; ATCC HTB106 or CRL-1973),
BCBL-1 (HHV8+; an HHV8 virus, which is a close relative to H.
saimiri, positive EBV negative human B cell line, see Renne, et al.
(1996) Nature Medicine 2:342-346), Kaposi's sarcoma (HHV8+;
clinical sample), cervical carcinoma (HPV16+; clinical sample),
thyroid, or kidney. The negative results from BJA-B and Tera-2 may
suggest a possibility of specific expression in T cells and
macrophages. The HHV8-infected cells like BCBL1 or the tumors did
not express AK155, which means that it is therefore specific fo H.
saimiri. Ak155 transcription was not seen in cervical carcinoma,
which suggests that it does not play a significant role in at least
one cancers condition.
[0189] Using a commercial dot spot mRNA hybridization filter and
standard hybridization conditions, faint expression was detected in
human kidney, and even fainter in human lung and liver. Expression
data from RT-PCR and from the mRNA-dot hybridization should be
confirmed by sensitive Northeren or other means. By Northern
analysis, negative results were obtained from: PHA-activated PBMC,
Jurkat, Owl monkey kidney cells (OMK; ATCC CRL 1556; Daniel, et al.
(1976) In Vitro 12:290), and OMK infected with herpesvirus saimiri
C488. By RT-PCR, Hela cells (epithelial; ATCC CCL-2.1: HeLa229; see
(1985) Am J. Pathol. 119:361-366) and BJA-B (EBV-free B cell line;
see Klein, et al. (1974) Proc. Nat'l Acad. Sci. USA 71:3283-3286)
gave undetectable expression. Dot blots gave undetectable signals
in the following human tissues by mRNA dot spot assay: brain,
amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal
lobe, hippocampus, medulla oblongata, occipital lobe, putamen,
substantia nigra, temporal lobe, thalamus, subthalamic nucleus,
spinal cord, heart, aorta, sceletal muscle, colon, bladder, uterus,
prostate, stomach, testis, ovary, pancreas, pituitary gland,
adrenal gland, thyroid gland, salivary gland, mammary gland, small
intestine, spleen, thymus, peripheral leukocytes, lymph node, bone
marrow, appendix, trachea, placenta, fetal brain, fetal heart,
fetal kidney, fetal liver, fetal spleen, fetal thymus, fetal
lung.
Example 3
[0190] Chromosome Mapping of Human IL-XX
[0191] ak155 is neither transcribed during lytic infection of OMK
cells with herpesvirus saimiri C488, nor can the ak155 sequence be
amplified from purified virus DNA. Chromosome mapping is a standard
technique. See, e.g., BIOS Laboratories (New Haven, Conn.) and
methods for using a mouse somatic cell hybrid panel with PCR. The
gene was mapped to the human chrosome 12q15 region.
Example 4
[0192] Purification of IL-XX Protein
[0193] Multiple transfected cell lines are screened for one which
expresses the cytokine at a high level compared with other cells.
Various cell lines are screened and selected for their favorable
properties in handling. Natural IL-XX can be isolated from natural
sources, or by expression from a transformed cell using an
appropriate expression vector. Early results suggest that the
cytokine, after secretion, rebinds to the cell surface.
Purification of the expressed protein is achieved by standard
procedures, or may be combined with engineered means for effective
purification at high efficiency from cell lysates or supernatants.
FLAG or His6 segments can be used for such purification features.
Alternatively, affinity chromatography may be used with specific
antibodies, see below.
Example 5
[0194] Isolation of Homologous IL-XX Genes
[0195] The IL-XX cDNA can be used as a hybridization probe to
screen a library from a desired source, e.g., a primate cell cDNA
library. Many different species can be screened both for stringency
necessary for easy hybridization, and for presence using a probe.
Appropriate hybridization conditions will be used to select for
clones exhibiting specificity of cross hybridization.
[0196] Screening by hybridization using degenerate probes based
upon the peptide sequences will also allow isolation of appropriate
clones. Alternatively, use of appropriate primers for PCR screening
will yield enrichment of appropriate nucleic acid clones.
[0197] Similar methods are applicable to isolate either species,
polymorphic, or allelic variants. Species variants are isolated
using cross-species hybridization techniques based upon isolation
of a full length isolate or fragment from one species as a
probe.
[0198] Alternatively, antibodies raised against human IL-XX will be
used to screen for cells which express cross-reactive proteins from
an appropriate, e.g., cDNA library. The purified protein or defined
peptides are useful for generating antibodies by standard methods,
as described above. Synthetic peptides or purified protein are
presented to an immune system to generate monoclonal or polyclonal
antibodies. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A
Laboratory Manual Cold Spring Harbor Press. The resulting
antibodies are used for screening, purfication, or diagnosis, as
described.
Example 6
[0199] Preparation of Antibodies Specific for IL-XX
[0200] Synthetic peptides or purified protein are presented to an
immune system to generate monoclonal or polyclonal antibodies. See,
e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;
and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold
Spring Harbor Press. Polyclonal serum, or hybridomas may be
prepared. In appropriate situations, the binding reagent is either
labeled as described above, e.g., fluorescence or otherwise, or
immobilized to a substrate for panning methods.
Example 7
[0201] Evaluation of Breadth of Biological Functions
[0202] The native, recombinant, and fusion proteins would be tested
for agonist and antagonist activity in many biological assay
systems, e.g., on T-cells, B-cells, NK, macrophages, dentritic
cells, hematopoietic progenitors, etc. Because of the IL-10
structural relationship, assays related to IL-10 activity would
analysed.
[0203] IL-XX is evaluated for agonist or antagonist activity on
transfected cells expressing IL-10 receptor and controls. See,
e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA 90, 11267-11271;
Ho, et al. (1995) Mol. Cell. Biol. 15:5043-5053;and Liu, et al.
(1994). J. Immunol. 152:1821-1829.
[0204] Based, in part, upon the structural homology to IL-10, the
IL-XX is evaluated for effect in macrophage/dendritic cell
activation and antigen presentation assays, T cell cytokine
production and proliferation in response to antigen or allogeneic
stimulus. See, e.g., de Waal Malefyt et al. (1991) J. Exp. Med.
174:1209-1220; de Waal Malefyt et al. (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.
[0205] IL-XX 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.
[0206] B cell growth and differentiation effects will be analysed,
e.g., by the methodology described, e.g., in Defrance, et al.
(1992). J. Exp. Med. 175:671-682; Rousset, et al (1992) Proc. Nat'l
Acad. Sci. USA 89:1890-1893; including IgG2 and IgA2 switch factor
assays. Note that, unlike COS7 supernatants, NIH3T3 and COP
supernatants apparently do not interfere with human B cell
assays.
[0207] 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.
[0208] 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
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