U.S. patent application number 11/548108 was filed with the patent office on 2007-02-22 for interferon-like protein zcyto21.
Invention is credited to Brian A. Fox, Teresa Gilbert, Francis J. Grant, Betty A. Haldeman, Scott R. Presnell, Paul O. Sheppard.
Application Number | 20070043209 11/548108 |
Document ID | / |
Family ID | 26910034 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070043209 |
Kind Code |
A1 |
Sheppard; Paul O. ; et
al. |
February 22, 2007 |
INTERFERON-LIKE PROTEIN ZCYTO21
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for Zcyto21, an interferon-like protein, which is most
closely related to interferon-.alpha. at the amino acid sequence
level. The present invention also includes antibodies to the
Zcyto21 polypeptides, and methods of using the polynucleotides and
polypeptides.
Inventors: |
Sheppard; Paul O.; (Granile
Falls, WA) ; Presnell; Scott R.; (Tacoma, WA)
; Fox; Brian A.; (Seattle, WA) ; Gilbert;
Teresa; (Seattle, WA) ; Haldeman; Betty A.;
(Seattle, WA) ; Grant; Francis J.; (Seattle,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Family ID: |
26910034 |
Appl. No.: |
11/548108 |
Filed: |
October 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10927815 |
Aug 27, 2004 |
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11548108 |
Oct 10, 2006 |
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09895834 |
Jun 29, 2001 |
6927040 |
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10927815 |
Aug 27, 2004 |
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60215446 |
Jun 30, 2000 |
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60285424 |
Apr 20, 2001 |
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Current U.S.
Class: |
530/388.23 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 14/47 20130101; C07K 14/52 20130101; A61P 25/28 20180101; C07K
2319/00 20130101 |
Class at
Publication: |
530/388.23 |
International
Class: |
C07K 16/24 20070101
C07K016/24 |
Claims
1. An antibody or antibody fragment that specifically binds with a
polypeptide consisting of an amino acid sequence having at least
95% sequence identity to amino acid residues 20-200 of SEQ ID
NO:2.
2. The antibody of claim 1 wherein the antibody is a
monoclonal.
3. The antibody of claim 1 wherein the antibody is humanized.
4. The antibody of claim 1 wherein the antibody is a
polyclonal.
5. The antibody of claim 1 wherein the antibody fragment is a
F(ab').sub.2, a Fab, or a single chain antibody.
6. The antibody of claim 1 wherein the amino acid sequence consists
of amino acid residues 20-200 of SEQ ID NO:2.
Description
[0001] The present application is a divisional of U.S. patent
application Ser. No. 10/927,815, filed Aug. 27, 2004, which is a
divisional of U.S. patent application Ser. No. 09/895,834, now U.S.
Pat. No. 6,927,040, filed Jun. 29, 2001, which claims the benefit
of U.S. Patent Application Ser. No. 60/215,446, filed Jun. 30,
2000, and 60/285,424, filed Apr. 20, 2001, all of which are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Cellular differentiation of multicellular organisms is
controlled by hormones and polypeptide growth factors. These
diffusable molecules allow cells to communicate with each other and
act in concert to form tissues and organs, and to repair and
regenerate damaged tissue. Examples of hormones and growth factors
include the steroid hormones, parathyroid hormone, follicle
stimulating hormone, the interferons, the interleukins, platelet
derived growth factor, epidermal growth factor, and
granulocyte-macrophage colony stimulating factor, among others.
[0003] Hormones and growth factors influence cellular metabolism by
binding to receptor proteins. Certain receptors are integral
membrane proteins that bind with the hormone or growth factor
outside the cell, and that are linked to signaling pathways within
the cell, such as second messenger systems. Other classes of
receptors are soluble intracellular molecules.
[0004] Cytokines generally stimulate proliferation or
differentiation of cells of the hematopoietic lineage or
participate in the immune and inflammatory response mechanisms of
the body. Examples of cytokines which affect hematopoiesis are
erythropoietin (EPO), which stimulates the development of red blood
cells; thrombopoietin (TPO), which stimulates development of cells
of the megakaryocyte lineage; and granulocyte-colony stimulating
factor (G-CSF), which stimulates development of neutrophils. These
cytokines are useful in restoring normal blood cell levels in
patients suffering from anemia, thrombocytopenia, and neutropenia
or receiving chemotherapy for cancer. Cytokines play important
roles in the regulation of hematopoiesis and immune responses, and
can influence lymphocyte development.
[0005] The human class II cytokine family includes
interferon-.alpha.. (IFN-.alpha.) subtypes, interferon-.beta.,
(IFN-.beta.), interferon-.gamma. (IFN-.gamma.), IL-10, IL-19 (U.S.
Pat. No. 5,985,614), MDA-7 (Jiang et al., Oncogene 11, 2477-2486,
(1995)), IL-20 (Jiang et al., Oncogene 11, 2477-2486, (1995)),
IL-22 (Xie et al., J. Biol. Chem. 275, 31335-31339, (2000)), and
AK-155 (Knappe et al., J. Virol. 74, 3881-3887, (2000)). Most
cytokines bind and transduce signals through either Class I or
Class II cytokine receptors. Members of human class II cytokine
receptor family include interferon-.alpha.R1 (IFN-.DELTA.R1),
interferon-.gamma.-R2 (IFN-.gamma.-R2), interferon-.gamma. R1
(IFN-.gamma. R1), interferon-.gamma.R2 (IFN-.gamma.R2), IL-10R (Liu
et al., J. Immunol. 152, 1821-1829, (1994)), CRF2-4 (Lutfalla et
al. Genomics 16, 366-373, (1993)), IL-20R.beta. (Blumberg et al.,
Cell 104, 9-19, (2001)) (also known as zcytor7 (U.S. Pat. No.
5,945,511) and CRF2-8 (Kotenko et al., Oncogene 19, 2557-2565,
(2000)), IL-20R.beta. (Blumberg et al., ibid, (2001)) (also known
as DIRS1 (PCT WO 99/46379)), IL-22RA1 (IL-22 receptor-.alpha.1,
submitted to HUGO for approval) (also known as IL-22R (Xie et al.,
J. Biol. Chem. 275, 31335-31339, (2000)), zcytor11 (U.S. Pat. No.
5,965,704) and CRF2-9 (Kotenko et al., Oncogene 19, 2557-2565,
(2000)), and tissue factor.
[0006] Class II cytokine receptors are typically heterodimers
composed of two distinct receptor chains, the .alpha. and .beta.
receptor subunits (Stahl et al., Cell 74, 587-590, (1993)). In
general, the .alpha. subunits are the primary cytokine binding
proteins, and the .beta. subunits are required for formation of
high affinity binding sites, as well as for signal transduction. An
exception is the IL-20 receptor in which both subunits are required
for IL-20 binding (Blumberg et al., ibid, (2001)).
[0007] The class II cytokine receptors are identified by a
conserved cytokine-binding domain of about 200 amino acids (D200)
in the extracellular portion of the receptor. This cytokine-binding
domain is comprised of two fibronectin type III (FnIII) domains of
approximately 100 amino acids each (Bazan J. F. Proc. Natl. Acad.
Sci. USA 87, 6934-6938, (1990); Thoreau et al., FEBS Lett. 282,
16-31, (1991)). Each FnIII domain contains conserved Cys, Pro, and
Trp residues that determine a characteristic folding pattern of
seven .beta.-strands similar to the constant domain of
immunoglobulins (Uze et al., J. Interferon Cytokine Res. 15, 3-26,
(1995)). The conserved structural elements of the class II cytokine
receptor family make it possible to identify new members of this
family on the basis of primary amino acid sequence homology.
Previously we have successfully identified two new members of class
II cytokine receptor family, zcytor7 (U.S. Pat. No. 5,945,511)
(also known as IL-20R .alpha. (Blumberg et al., ibid, (2001)) and
zcytor11 (U.S. Pat. No. 5,965,704) (also known as IL-22R (Blumberg
et al., ibid, (2001)), using this approach. Identification of
additional novel members of the class II cytokine receptor family
is of interest because cytokines play a vital role in regulating
biological responses.
[0008] L-22, also known as IL-TIF (IL-10-related T cell-derived
inducible factor) (Dumoutier et al., J. Immunology 164, 1814-1819,
(2000)), is a recently described IL-10 homologue. Mouse IL-22 was
originally identified as a gene induced by IL-9 in T cells and mast
cells in vitro (Dumoutier et al., J. Immunology 164, 1814-1819,
(2000)). Acute phase reactant induction activity was observed in
mouse liver upon IL-22 injection, and IL-22 expression was rapidly
induced after lipopolysaccharide (LPS) injection, suggesting that
IL-22 contributes to the inflammatory response in vivo (Dumoutier
et al., Proc. Natl. Acad. Sci. U.S.A. 97, 10144-10149, (2000)).
[0009] The interleukins are a family of cytokines that mediate
immunological responses, including inflammation. The interleukins
mediate a variety of inflammatory pathologies. Central to an immune
response is the T cell, which produce many cytokines and adaptive
immunity to antigens. Cytokines produced by the T cell have been
classified as type 1 and type 2 (Kelso, A. Immun. Cell Biol.
76:300-317, 1998). Type 1 cytokines include IL-2, IFN-.gamma.,
LT-.alpha., and are involved in inflammatory responses, viral
immunity, intracellular parasite immunity and allograft rejection.
Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are
involved in humoral responses, helminth immunity and allergic
response. Shared cytokines between Type 1 and 2 include IL-3,
GM-CSF and TNF-.alpha.. There is some evidence to suggest that Type
1 and Type 2 producing T cell populations preferentially migrate
into different types of inflamed tissue.
[0010] Of particular interest, from a therapeutic standpoint, are
the interferons (reviews on interferons are provided by De Maeyer
and De Maeyer-Guignard, "Interferons," in The Cytokine Handbook,
3.sup.rd Edition, Thompson (ed.), pages 491-516 (Academic Press
Ltd. 1998), and by Walsh, Biopharmaceuticals: Biochemistry and
Biotechnology, pages 158-188 (John Wiley & Sons 1998)).
Interferons exhibit a variety of biological activities, and are
useful for the treatment of certain autoimmune diseases, particular
cancers, and the enhancement of the immune response against
infectious agents, including viruses, bacteria, fungi, and
protozoa. To date, six forms of interferon have been identified,
which have been classified into two major groups. The so-called
"type I" interferons include interferon-.alpha., interferon-.beta.,
interferon-.omega., interferon-.delta., and interferon-.tau..
Currently, interferon-.gamma. and one subclass of
interferon-.alpha. are the only type II interferons.
[0011] Type I interferons, which are thought to be derived from the
same ancestral gene, have retained sufficient similar structure to
act by the same cell surface receptor. The .alpha.-chain of the
human interferon-.alpha./.beta. receptor comprises an extracellular
N-terminal domain, which has the characteristics of a class II
cytokine receptor. Interferon-.gamma. does not share significant
homology with the type I interferons or with the type II
interferon-.alpha. subtype, but shares a number of biological
activities with the type I interferons.
[0012] In humans, at least 16 non-allelic genes code for different
subtypes of interferon-.alpha., while interferons .beta. and
.omega. are encoded by single genes. Type I interferon genes are
clustered in the short arm of chromosome 9. Unlike typical
structural human genes, interferon-.alpha., interferon-.beta., and
interferon-.omega. lack introns. A single gene for human
interferon-.gamma. is localized on chromosome 12 and contains three
introns. To date, interferon-.tau. has been described only in
cattle and sheep, while interferon-.delta. has been described only
in pigs.
[0013] Clinicians are taking advantage of the multiple activities
of interferons by using the proteins to treat a wide range of
conditions. For example, one form of interferon-.alpha. has been
approved for use in more than 50 countries for the treatment of
medical conditions such as hairy cell leukemia, renal cell
carcinoma, basal cell carcinoma, malignant melanoma, AIDS-related
Kaposi's sarcoma, multiple myeloma, chronic myelogenous leukemia,
non-Hodgkin's lymphoma, laryngeal papillomatosis, mycosis
fungoides, condyloma acuminata, chronic hepatitis B, hepatitis C,
chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.
Food and Drug Administration has approved the use of
interferon-.beta. to treat multiple sclerosis, a chronic disease of
the nervous system. Interferon-.gamma. is used to treat chronic
granulomatous diseases, in which the interferon enhances the
patient's immune response to destroy infectious bacterial, fungal,
and protozoal pathogens. Clinical studies also indicate that
interferon-.gamma. may be useful in the treatment of AIDS,
leishmaniasis, and lepromatous leprosy.
[0014] The demonstrated in vivo activities of the cytokine family
illustrate the enormous clinical potential of, and need for, other
cytokines, cytokine agonists, and cytokine antagonists. The present
invention addresses these needs by providing a new cytokine that
stimulates cells of the hematopoietic cell lineage, as well as
related compositions and methods.
BRIEF DESCRIPTION OF THE FIGURES
[0015] The FIGURE is a Hopp/Woods hydrophilicity profile of the
Zcyto21 protein sequence shown in SEQ ID NO:2. The profile is based
on a sliding six-residue window. Buried G, S, and T residues and
exposed H, Y, and W residues were ignored. These residues are
indicated in the FIGURE by lower case letters.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0017] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0018] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0019] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0020] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0021] The term "complements of a polynucleotide molecule" denotes
a polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to
5'CCCGTGCAT 3'.
[0022] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0023] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0024] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0025] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0026] The term "neoplastic", when referring to cells, indicates
cells undergoing new and abnormal proliferation, particularly in a
tissue where in the proliferation is uncontrolled and progressive,
resulting in a neoplasm. The neoplastic cells can be either
malignant, i.e. invasive and metastatic, or benign.
[0027] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0028] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0029] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0030] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0031] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0032] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0033] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0034] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0035] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0036] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0037] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to 110%.
[0038] All references cited herein are incorporated by reference in
their entirety.
[0039] Zcyto21 gene encodes a polypeptide of 200 amino acids, as
shown in SEQ ID NO:2. The signal sequence for Zcyto21 can be
predicted as comprising amino acid residue 1 (Met) through amino
acid residue 19 (Ala) of SEQ ID NO:2. The mature peptide for
Zcyto21 begins at amino acid residue 20 (Gly).
[0040] The Zcyto21 gene is contained in BAC sequences AC011445, and
AC018477 which have been mapped to human chromosome 19q13.13. This
region of chromosome 19 may also comprise a cluster of
interferon-like genes. A consensus cDNA showing a polynucleotide
sequence of Zcyto21 is shown in SEQ ID NO:6 and the polypeptide it
encodes is shown in SEQ ID NO:7.
[0041] As described below, the present invention provides isolated
polypeptides having an amino acid sequence that is at least 70%, at
least 80%, or at least 90%, 95%, 96%, 97%, 98% or 99% identical to
either amino acid residues 20 to 200 of SEQ ID NO:2 or amino acid
residues 1 to 200 of SEQ ID NO:2. The present invention also
provides isolated polypeptides having an amino acid sequence that
is at least 70%, at least 80%, or at least 90%, 95%, 96%, 97%, 98%
or 99% identical to either amino acid residues 20 to 219 of SEQ ID
NO:9 or amino acid residues 1 to 219 of SEQ ID NO:9. The present
invention also provides isolated polypeptides having an amino acid
sequence that is at least 70%, at least 80%, or at least 90%, 95%,
96%, 97%, 98% or 99% identical to either amino acid residues 20 to
203 of SEQ ID NO:12 or amino acid residues 1 to 203 of SEQ ID
NO:12. The present invention also includes a polypeptide that
further comprises a signal secretory sequence that resides in an
amino-terminal position relative to the first amino acid sequence,
wherein the signal secretory sequence comprises amino acid residues
1 to 19 of the amino acid sequence of SEQ ID NO:2.
[0042] In general, cytokines are predicted to have a four-alpha
helix structure, with helices A, C and D being most important in
ligand-receptor interactions, and are more highly conserved among
members of the family. However, the interferons (INF), and
interferon-alpha and interferon-tau in particular, are
characterized as six helix bundles. INF helix A is equivalent to
helix A of Zcyto21; INF helix B is equivalent to helix C of
Zcyto21; INF helix C is equivalent to helix D of Zcyto21, and INF
helix D is equivalent to helix F of Zcyto21. Thus, the loop between
the AB loop, and CD loop of INF is expanded in Zcyto21 to contain
short helices B and E of Zcyto21.
[0043] Zcyto21 helices are predicted as follows: helix A is defined
by amino acid residues 49 (Ser) to 63 (Leu); helix B by amino acid
residues 76 (Asn) to 84 (Val); helix C by amino acid residues 89
(Val) to 104 (Ala); helix D by amino acid residues 111 (Glu) to 133
(Gln); helix E by amino acid residues 137 (Thr) to 158 (Lys); and
helix F by amino acid residues 163 (Gly) to 189 (Leu); as shown in
SEQ ID NO: 2. The cysteine residues are conserved between Zcyto21,
and INF-.alpha., and may form an intermolecular disulfide bond, in
particular to form homodimers with additional Zcyto21 molecules.
Further analysis of Zcyto21 based on multiple alignments predicts
that cysteines at amino acid residues 34 and 131, and 68 and 164,
(as shown in SEQ ID NO: 2) will form intramolecular disulfide
bonds. The cysteine at residue 190 is free, and may form an
intermolecular disulfide association. The corresponding
polynucleotides encoding the Zcyto21 polypeptide regions, domains,
motifs, residues and sequences described herein are as shown in SEQ
ID NO:1. The degenerate polynucleotide sequence of SEQ ID NO:2 is
shown in SEQ ID NO:3. The degenerate polynucleotide sequence of SEQ
ID NO:9 is shown in SEQ ID NO:10. The degenerate polynucleotide
sequence of SEQ ID NO:12 is shown in SEQ ID NO:13.
[0044] Detailed mutational analysis of murine IL-2 (Zurawski et
al., EMBO J. 12:5113-5119, 1993) shows residues in helices A and C
are important for binding to IL-2R.beta.; critical residues are
Asp.sub.34, Asn.sub.99, and Asn.sub.103. Multiple residues within
murine IL-2 loop A/B and helix B are important for IL-2R.alpha.
binding, while only a single residue, Gln.sub.141 in helix D, is
vital for binding with IL-2R.alpha.. Similarly, helices A and C are
sites of interaction between IL-4 and IL-4R.alpha. (the
structurally similar to IL-2R.alpha.), and residues within helix D
are vital for IL-2R.alpha. interaction (Wang et al., Proc. Natl.
Acad. Sci. USA 94:1657-1662, 1997; Kruse et al., EMBO J.
11:3237-3244, 1992). In particular, the mutation Tyr.sub.124 to Asp
in human IL-4 creates an antagonist, which binds with IL-4R.alpha.
but not IL-2R.alpha. and therefore cannot signal (Kruse et al.
ibid. 1992).
[0045] Four-helical bundle cytokines are also grouped by the length
of their component helices. "Long-helix" form cytokines generally
consist of between 24-30 residue helices, and include IL-6, ciliary
neutrotrophic factor (CNTF), leukemia inhibitory factor (LIF) and
human growth hormone (hGH). "Short-helix" form cytokines generally
consist of between 18-21 residue helices and include IL-2, IL-4 and
GM-CSF. Studies using CNTF and IL-6 demonstrated that a CNTF helix
can be exchanged for the equivalent helix in IL-6, conferring
CTNF-binding properties to the chimera. Thus, it appears that
functional domains of four-helical cytokines are determined on the
basis of structural homology, irrespective of sequence identity,
and can maintain functional integrity in a chimera (Kallen et al.,
J. Biol. Chem. 274:11859-11867, 1999). Therefore, the helical
domains of Zcyto21 will be useful for preparing chimeric fusion
molecules, particularly with other interferons to determine and
modulate receptor binding specificity. Of particular interest are
fusion proteins that combine helical and loop domains from
interferons and cytokines such as INF-.alpha., IL-10, human growth
hormone.
[0046] Zcyto21 mRNA has been identified in tissues of brain, islet,
prostate, testis, pituitary, placenta, ovarian tumor, lung tumor,
rectal tumor and ovarian tumor, as well as an activated immune cell
line (CD3+) and a prostate epithelial cell line, which had been
transformed with human papilloma virus IV (HPVS).
[0047] The present invention provides polynucleotide molecules,
including DNA and RNA molecules, that encode the Zcyto21
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NOs:3, 10, and 13 are degenerate
DNA sequences that encompasses all DNAs that encode the Zcyto21
polypeptide of SEQ ID NOs:2, 9, and 12, respectively. Those skilled
in the art will recognize that the degenerate sequence of SEQ ID
NO:3, for example, also provides all RNA sequences encoding SEQ ID
NO:2 by substituting U for T. Thus, Zcyto21 polypeptide-encoding
polynucleotides comprising nucleotide 1 or 58 to nucleotide 603 of
SEQ ID NO:3 and their RNA equivalents are contemplated by the
present invention. Table 1 sets forth the one-letter codes used
within SEQ ID NO:3 to denote degenerate nucleotide positions.
"Resolutions" are the nucleotides denoted by a code letter.
"Complement" indicates the code for the complementary
nucleotide(s). For example, the code Y denotes either C or T, and
its complement R denotes A or G, with A being complementary to T,
and G being complementary to C. TABLE-US-00001 TABLE 1 Nucleotide
Resolution Complement Resolution A A T T C C G G G G C C T T A A R
A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W
A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
[0048] The degenerate codons used in SEQ ID NOs:3, 10, and 13,
encompassing all possible codons for a given amino acid, are set
forth in Table 2. TABLE-US-00002 TABLE 2 One Amino Letter
Degenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser S AGC AGT
TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro P CCA CCC CCG CCT
CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGN Asn N AAC
AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CAR His H
CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AAR Met
M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTN
Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W
TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X
NNN
[0049] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequence of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0050] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential codon
usage" or "preferential codons" is a term of art referring to
protein translation codons that are most frequently used in cells
of a certain species, thus favoring one or a few representatives of
the possible codons encoding each amino acid (See Table 3). For
example, the amino acid Threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO:3 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0051] As previously noted, the isolated polynucleotides of the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of Zcyto21RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), or by screening
conditioned medium from various cell types for activity on target
cells or tissue. Once the activity or RNA producing cell or tissue
is identified, total RNA can be prepared using guanidinium
isothiocyanate extraction followed by isolation by centrifugation
in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979).
Poly (A)+ RNA is prepared from total RNA using the method of Aviv
and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly(A).sup.+ RNA using
known methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding Zcyto21 polypeptides are then identified
and isolated by, for example, hybridization or PCR.
[0052] A longer clone encoding Zcyto21 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to Zcyto21 receptor fragments, or other specific binding
partners.
[0053] The present invention further provides counterpart
polypeptides and polynucleotides from other species (orthologs).
These species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are Zcyto21
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human Zcyto21 can be cloned using
information and compositions provided by the present invention in
combination with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses Zcyto21 as disclosed herein. Suitable sources of
mRNA can be identified by probing Northern blots with probes
designed from the sequences disclosed herein. A library is then
prepared from mRNA of a positive tissue or cell line. A
Zcyto21-encoding cDNA can then be isolated by a variety of methods,
such as by probing with a complete or partial human cDNA or with
one or more sets of degenerate probes based on the disclosed
sequences. A cDNA can also be cloned using the polymerase chain
reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers
designed from the representative human Zcyto21 sequence disclosed
herein. Within an additional method, the cDNA library can be used
to transform or transfect host cells, and expression of the cDNA of
interest can be detected with an antibody to Zcyto21 polypeptide,
binding studies or activity assays. Similar techniques can also be
applied to the isolation of genomic clones.
[0054] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
Zcyto21 band that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the DNA
sequence shown in SEQ ID NO:1, including those containing silent
mutations and those in which mutations result in amino acid
sequence changes, are within the scope of the present invention, as
are proteins which are allelic variants of SEQ ID NO:2. cDNAs
generated from alternatively spliced mRNAs, which retain the
properties of the Zcyto21 polypeptide, are included within the
scope of the present invention, as are polypeptides encoded by such
cDNAs and mRNAs. Allelic variants and splice variants of these
sequences can be cloned by probing cDNA or genomic libraries from
different individuals or tissues according to standard procedures
known in the art. Examples of alternatively spliced variants are
shown in SEQ ID NO:8 (SEQ ID NO:9 for the corresponding
polypeptide), and in SEQ ID NO:11 (SEQ ID NO:12 for the
corresponding polypeptide). An example of an allelic variant is
shown in SEQ ID NO:4, which corresponds to the polypeptide sequence
as shown in SEQ ID NO:5. There is a polymorphism between the
polypeptide sequence as shown in SEQ ID NO:1 and that shown in SEQ
ID NO:4 at nucleotide number 572. This polymorphism might create an
antagonist of Zcyto21 or a molecule of reduced or altered function,
which might lead to a higher likelihood of disease
susceptibility.
[0055] The present invention also provides reagents, which will
find use in diagnostic applications. For example, the Zcyto21 gene,
a probe comprising Zcyto21 DNA or RNA or a subsequence thereof can
be used to determine if the Zcyto21 gene is present on a human
chromosome, such as chromosome 19, or if a gene mutation has
occurred. Zcyto21 is located at the q13.13 region of chromosome 19.
Detectable chromosomal aberrations at the Zcyto21 gene locus
include, but are not limited to, aneuploidy, gene copy number
changes, loss of heterogeneity (LOH), translocations, insertions,
deletions, restriction site changes and rearrangements. Such
aberrations can be detected using polynucleotides of the present
invention by employing molecular genetic techniques, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, and other
genetic linkage analysis techniques known in the art (Sambrook et
al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65,
1995).
[0056] The precise knowledge of a gene's position can be useful for
a number of purposes, including: 1) determining if a sequence is
part of an existing contig and obtaining additional surrounding
genetic sequences in various forms, such as YACs, BACs or cDNA
clones; 2) providing a possible candidate gene for an inheritable
disease which shows linkage to the same chromosomal region; and 3)
cross-referencing model organisms, such as mouse, which may aid in
determining what function a particular gene might have.
[0057] For example, Delague et al., (Am. J. Hum. Genet. 67:
236-243, 2000) identified that Charcot-Marie-Tooth disease is
localized to 19q13.1-13.3 (Delague et al., Am. J. Hum. Genet. 67:
236-243, 2000).
[0058] A diagnostic could assist physicians in determining the type
of disease and appropriate associated therapy, or assistance in
genetic counseling. As such, the inventive anti-Zcyto21 antibodies,
polynucleotides, and polypeptides can be used for the detection of
Zcyto21 polypeptide, mRNA or anti-Zcyto21 antibodies, thus serving
as markers and can be directly used for detecting or genetic
diseases or cancers, as described herein, using methods known in
the art and described herein. Further, Zcyto21 polynucleotide
probes can be used to detect abnormalities or genotypes associated
with chromosome 19 deletions and translocations associated with
human diseases or other translocations involved with malignant
progression of tumors or other 19q13.13 mutations, which are
expected to be involved in chromosome rearrangements in malignancy;
or in other cancers. Similarly, Zcyto21 polynucleotide probes can
be used to detect abnormalities or genotypes associated with
chromosome 19q13.13 trisomy and chromosome loss associated with
human diseases or spontaneous abortion. Thus, Zcyto21
polynucleotide probes can be used to detect abnormalities or
genotypes associated with these defects.
[0059] In general, the diagnostic methods used in genetic linkage
analysis, to detect a genetic abnormality or aberration in a
patient, are known in the art. Analytical probes will be generally
at least 20 nt in length, although somewhat shorter probes can be
used (e.g., 14-17 nt). PCR primers are at least 5 nt in length,
preferably 15 or more, more preferably 20-30 nt. For gross analysis
of genes, or chromosomal DNA, a Zcyto21 polynucleotide probe may
comprise an entire exon or more. Exons are readily determined by
one of skill in the art by comparing Zcyto21 sequences (SEQ ID
NO:1) with the genomic DNA for Zcyto21. In general, the diagnostic
methods used in genetic linkage analysis, to detect a genetic
abnormality or aberration in a patient, are known in the art. Most
diagnostic methods comprise the steps of (a) obtaining a genetic
sample from a potentially diseased patient, diseased patient or
potential non-diseased carrier of a recessive disease allele; (b)
producing a first reaction product by incubating the genetic sample
with a Zcyto21 polynucleotide probe wherein the polynucleotide will
hybridize to complementary polynucleotide sequence, such as in RFLP
analysis or by incubating the genetic sample with sense and
antisense primers in a PCR reaction under appropriate PCR reaction
conditions; (iii) Visualizing the first reaction product by gel
electrophoresis and/or other known method such as visualizing the
first reaction product with a Zcyto21 polynucleotide probe wherein
the polynucleotide will hybridize to the complementary
polynucleotide sequence of the first reaction; and (iv) comparing
the visualized first reaction product to a second control reaction
product of a genetic sample from wild type patient. A difference
between the first reaction product and the control reaction product
is indicative of a genetic abnormality in the diseased or
potentially diseased patient, or the presence of a heterozygous
recessive carrier phenotype for a non-diseased patient, or the
presence of a genetic defect in a tumor from a diseased patient, or
the presence of a genetic abnormality in a fetus or
pre-implantation embryo. For example, a difference in restriction
fragment pattern, length of PCR products, length of repetitive
sequences at the Zcyto21 genetic locus, and the like, are
indicative of a genetic abnormality, genetic aberration, or allelic
difference in comparison to the normal wild type control. Controls
can be from unaffected family members, or unrelated individuals,
depending on the test and availability of samples. Genetic samples
for use within the present invention include genomic DNA, mRNA, and
cDNA isolated form any tissue or other biological sample from a
patient, such as but not limited to, blood, saliva, semen,
embryonic cells, amniotic fluid, and the like. The polynucleotide
probe or primer can be RNA or DNA, and will comprise a portion of
SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNA equivalent
thereof. Such methods of showing genetic linkage analysis to human
disease phenotypes are well known in the art. For reference to PCR
based methods in diagnostics see see, generally, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
White (ed.), PCR Protocols: Current Methods and Applications
(Humana Press, Inc. 1993), Cotter (ed.), Molecular Diagnosis of
Cancer (Humana Press, Inc. 1996), Hanausek and Walaszek (eds.),
Tumor Marker Protocols (Humana Press, Inc. 1998), Lo (ed.),
Clinical Applications of PCR (Humana Press, Inc. 1998), and Meltzer
(ed.), PCR in Bioanalysis (Humana Press, Inc. 1998)).
[0060] Mutations associated with the Zcyto21 locus can be detected
using nucleic acid molecules of the present invention by employing
standard methods for direct mutation analysis, such as restriction
fragment length polymorphism analysis, short tandem repeat analysis
employing PCR techniques, amplification-refractory mutation system
analysis, single-strand conformation polymorphism detection, RNase
cleavage methods, denaturing gradient gel electrophoresis,
fluorescence-assisted mismatch analysis, and other genetic analysis
techniques known in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular
Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular
Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren
(ed.), Laboratory Protocols for Mutation Detection (Oxford
University Press 1996), Birren et al (eds.), Genome Analysis, Vol
2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),
Dracopoli et al. (eds.), Current Protocols in Human Genetics (John
Wiley & Sons 1998), and Richards and Ward, "Molecular
Diagnostic Testing," in Principles of Molecular Medicine, pages
83-88 (Humana Press, Inc. 1998)). Direct analysis of an Zcyto21
gene for a mutation can be performed using a subject's genomic DNA.
Methods for amplifying genomic DNA, obtained for example from
peripheral blood lymphocytes, are well-known to those of skill in
the art (see, for example, Dracopoli et al (eds.), Current
Protocols in Human Genetics, at pages 7.1.6 to 7.1.7 (John Wiley
& Sons 1998)).
[0061] Within embodiments of the invention, isolated
Zcyto21-encoding nucleic acid molecules can hybridize under
stringent conditions to nucleic acid molecules having the
nucleotide sequence of SEQ ID NO:1, to nucleic acid molecules
having the nucleotide sequence of nucleotides 58 to 603 of SEQ ID
NO:1, or to nucleic acid molecules having a nucleotide sequence
complementary to SEQ ID NO:1. In general, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe.
[0062] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases.
[0063] It is well within the abilities of one skilled in the art to
adapt these conditions for use with a particular polynucleotide
hybrid. The T.sub.m for a specific target sequence is the
temperature (under defined conditions) at which 50% of the target
sequence will hybridize to a perfectly matched probe sequence.
Those conditions which influence the T.sub.m include, the size and
base pair content of the polynucleotide probe, the ionic strength
of the hybridization solution, and the presence of destabilizing
agents in the hybridization solution. Numerous equations for
calculating T.sub.m are known in the art, and are specific for DNA,
RNA and DNA-RNA hybrids and polynucleotide probe sequences of
varying length (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor
Press 1989); Ausubel et al., (eds.), Current Protocols in Molecular
Biology (John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.),
Guide to Molecular Cloning Techniques, (Academic Press, Inc. 1987);
and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below the calculated T.sub.m. This allows for the
maximum rate of hybridization for DNA-DNA and DNA-RNA hybrids.
[0064] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. That is, nucleic acid molecules encoding a variant
Zcyto21 polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0065] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. In other words, nucleic acid
molecules encoding a variant Zcyto21 polypeptide hybridize with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
(or its complement) under highly stringent washing conditions, in
which the wash stringency is equivalent to 0.1.times.-0.2.times.SSC
with 0.1% SDS at 50-65.degree. C., including 0.1.times.SSC with
0.1% SDS at 50.degree. C., or 0.2.times.SSC with 0.1% SDS at
65.degree. C.
[0066] The present invention also provides isolated Zcyto21
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides comprising at least 70%, at least 80%, at least 90%,
at least 95%, or greater than 95%, 96%, 97%, 98%, or 99% sequence
identity to the sequences shown in SEQ ID NO:2, or their orthologs.
The present invention also includes polypeptides that comprise an
amino acid sequence having at least 70%, at least 80%, at least
90%, at least 95% or greater than 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence of amino acid residues 1 to 200
or 20 to 200 of SEQ ID NO:2. The present invention further includes
nucleic acid molecules that encode such polypeptides. Methods for
determining percent identity are described below.
[0067] The present invention also contemplates variant Zcyto21
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and/or a hybridization
assay, as described above. Such Zcyto21 variants include nucleic
acid molecules: (1) that hybridize with a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 (or its complement)
under stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C.; or (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95% or greater than 95%, 96%, 97%, 98%,
or 99% sequence identity to the amino acid sequence of SEQ ID NO:2.
Alternatively, Zcyto21 variants can be characterized as nucleic
acid molecules: (1) that hybridize with a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 (or its complement)
under highly stringent washing conditions, in which the wash
stringency is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS
at 50-65.degree. C.; and (2) that encode a polypeptide having at
least 70%, at least 80%, at least 90%, at least 95% or greater than
95% sequence identity to the amino acid sequence of SEQ ID
NO:2.
[0068] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). Total .times. .times.
number .times. .times. of .times. .times. identical .times. .times.
matches [ length .times. .times. of .times. .times. the .times.
.times. longer .times. .times. sequence .times. .times. plus
.times. .times. the number .times. .times. of .times. .times. gaps
.times. .times. introduced .times. .times. into .times. .times. the
.times. .times. longer sequence .times. .times. in .times. .times.
order .times. .times. to .times. .times. align .times. .times. the
.times. .times. two .times. .times. sequences ] .times. 100
##EQU1## TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W
Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5
E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I
-1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1
2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F
-2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2
-3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0
-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2
-2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2
-1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0
-3 -1 4
[0069] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant Zcyto21. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0070] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then rescored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Preferred
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0071] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as default.
[0072] Variant Zcyto21 polypeptides or polypeptides with
substantially similar sequence identity are characterized as having
one or more amino acid substitutions, deletions or additions. These
changes are preferably of a minor nature, that is conservative
amino acid substitutions (see Table 4) and other substitutions that
do not significantly affect the folding or activity of the
polypeptide; small deletions, typically of one to about 30 amino
acids; and amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides of from about 149 to 230 amino acid
residues that comprise a sequence that is at least 70%, preferably
at least 90%, and more preferably 95%, 96%, 97%, 98%, 99% or more
identical to the corresponding region of SEQ ID NO:2. Polypeptides
comprising affinity tags can further comprise a proteolytic
cleavage site between the Zcyto21 polypeptide and the affinity tag.
Preferred such sites include thrombin cleavage sites and factor Xa
cleavage sites. TABLE-US-00004 TABLE 4 Conservative amino acid
substitutions Basic: arginine lysine histidine Acidic: glutamic
acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine
isoleucine valine Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine methionine
[0073] Determination of amino acid residues that comprise regions
or domains that are critical to maintaining structural integrity
can be determined. Within these regions one can determine specific
residues that will be more or less tolerant of change and maintain
the overall tertiary structure of the molecule. Methods for
analyzing sequence structure include, but are not limited to
alignment of multiple sequences with high amino acid or nucleotide
identity, secondary structure propensities, binary patterns,
complementary packing and buried polar interactions (Barton,
Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al.,
Current Opin. Struct. Biol. 6:3-10, 1996). In general, when
designing modifications to molecules or identifying specific
fragments determination of structure will be accompanied by
evaluating activity of modified molecules.
[0074] Amino acid sequence changes are made in Zcyto21 polypeptides
so as to minimize disruption of higher order structure essential to
biological activity. For example, where the Zcyto21 polypeptide
comprises one or more helices, changes in amino acid residues will
be made so as not to disrupt the helix geometry and other
components of the molecule where changes in conformation abate some
critical function, for example, binding of the molecule to its
binding partners. The effects of amino acid sequence changes can be
predicted by, for example, computer modeling as disclosed above or
determined by analysis of crystal structure (see, e.g., Lapthorn et
al., Nat. Struct. Biol. 2:266-268, 1995). Other techniques that are
well known in the art compare folding of a variant protein to a
standard molecule (e.g., the native protein). For example,
comparison of the cysteine pattern in a variant and standard
molecules can be made. Mass spectrometry and chemical modification
using reduction and alkylation provide methods for determining
cysteine residues which are associated with disulfide bonds or are
free of such associations (Bean et al., Anal. Biochem. 201:216-226,
1992; Gray, Protein Sci. 2:1732-1748, 1993; and Patterson et al.,
Anal. Chem. 66:3727-3732, 1994). It is generally believed that if a
modified molecule does not have the same cysteine pattern as the
standard molecule folding would be affected. Another well known and
accepted method for measuring folding is circular dichrosism (CD).
Measuring and comparing the CD spectra generated by a modified
molecule and standard molecule is routine (Johnson, Proteins
7:205-214, 1990). Crystallography is another well known method for
analyzing folding and structure. Nuclear magnetic resonance (NMR),
digestive peptide mapping and epitope mapping are also known
methods for analyzing folding and structurally similarities between
proteins and polypeptides (Schaanan et al., Science 257:961-964,
1992).
[0075] A Hopp/Woods hydrophilicity profile of the Zcyto21 protein
sequence as shown in SEQ ID NO:2 can be generated (Hopp et al.,
Proc. Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth.
88:1-18, 1986 and Triquier et al., Protein Engineering 11: 153-169,
1998). The profile is based on a sliding six-residue window. Buried
G, S, and T residues and exposed H, Y, and W residues were ignored.
For example, in Zcyto21, hydrophilic regions include residues 155
(Glu) to 160 (Glu); residues 51 (Lys) to 56 (Ala); residues 50
(Phe) to 55 (Asp); residues 140 (Pro) to 145 (Arg); and residues
154 (Gln) to 159 (Lys); as shown in SEQ ID NO: 2.
[0076] Those skilled in the art will recognize that hydrophilicity
or hydrophobicity will be taken into account when designing
modifications in the amino acid sequence of a Zcyto21 polypeptide,
so as not to disrupt the overall structural and biological profile.
Of particular interest for replacement are hydrophobic residues
selected from the group consisting of Val, Leu and Ile or the group
consisting of Met, Gly, Ser, Ala, Tyr and Trp.
[0077] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between INF-.alpha. and other
interferons. Using methods such as "FASTA" analysis described
previously, regions of high similarity are identified within a
family of proteins and used to analyze amino acid sequence for
conserved regions. An alternative approach to identifying a variant
Zcyto21 polynucleotide on the basis of structure is to determine
whether a nucleic acid molecule encoding a potential variant
Zcyto21 gene can hybridize to a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1, as discussed above.
[0078] Other methods of identifying essential amino acids in the
polypeptides of the present invention are procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Natl. Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological or biochemical
activity as disclosed below to identify amino acid residues that
are critical to the activity of the molecule. See also, Hilton et
al, J. Biol. Chem. 271:4699 (1996).
[0079] The present invention also includes functional fragments of
Zcyto21 polypeptides and nucleic acid molecules encoding such
functional fragments. A "functional" Zcyto21 or fragment thereof as
defined herein is characterized by its proliferative or
differentiating activity, by its ability to induce or inhibit
specialized cell functions, or by its ability to bind specifically
to an anti-Zcyto21 antibody or Zcyto21 receptor (either soluble or
immobilized). As previously described herein, Zcyto21 is
characterized by a six-helical-bundle structure comprising: helix A
is defined by amino acid residues 49 (Ser) to 63 (Leu); helix B by
amino acid residues 76 (Asn) to 84 (Val); helix C by amino acid
residues 89 (Val) to 104 (Ala); helix D by amino acid residues 111
(Glu) to 133 (Gln); helix E by amino acid residues 137 (Thr) to 158
(Lys); and helix F by amino acid residues 163 (Gly) to 189 (Leu);
as shown in SEQ ID NO: 2. Thus, the present invention further
provides fusion proteins encompassing: (a) polypeptide molecules
comprising one or more of the helices described above; and (b)
functional fragments comprising one or more of these helices. The
other polypeptide portion of the fusion protein may be contributed
by another helical-bundle cytokine or interferon, such as
INF-.alpha., or by a non-native and/or an unrelated secretory
signal peptide that facilitates secretion of the fusion
protein.
[0080] The Zcyto21 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides can be produced according to conventional techniques
using cells into which have been introduced an expression vector
encoding the polypeptide. As used herein, "cells into which have
been introduced an expression vector" include both cells that have
been directly manipulated by the introduction of exogenous DNA
molecules and progeny thereof that contain the introduced DNA.
Suitable host cells are those cell types that can be transformed or
transfected with exogenous DNA and grown in culture, and include
bacteria, fungal cells, and cultured higher eukaryotic cells.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987.
[0081] In general, a DNA sequence encoding a Zcyto21 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0082] To direct a Zcyto21 polypeptide into the secretory pathway
of a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of
Zcyto21, or may be derived from another secreted protein (e.g.,
t-PA; see, U.S. Pat. No. 5,641,655) or synthesized de novo. The
secretory signal sequence is operably linked to the Zcyto21 DNA
sequence, i.e., the two sequences are joined in the correct reading
frame and positioned to direct the newly synthesized polypeptide
into the secretory pathway of the host cell. Secretory signal
sequences are commonly positioned 5' to the DNA sequence encoding
the polypeptide of interest, although certain signal sequences may
be positioned elsewhere in the DNA sequence of interest (see, e.g.,
Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.
No. 5,143,830).
[0083] Cultured mammalian cells can be used as hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The
production of recombinant polypeptides in cultured mammalian cells
is disclosed, for example, by Levinson et al., U.S. Pat. No.
4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al,
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570
(ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J.
Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1,
ATCC No. CCL 61; or CHO DG44, Chasin et al., Som. Cell. Molec.
Genet. 12:555, 1986) cell lines. Additional suitable cell lines are
known in the art and available from public depositories such as the
American Type Culture Collection, Manassas, Va. In general, strong
transcription promoters are preferred, such as promoters from SV-40
or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other
suitable promoters include those from metallothionein genes (U.S.
Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late
promoter. Expression vectors for use in mammalian cells include
pZP-1 and pZP-9, which have been deposited with the American Type
Culture Collection, Manassas, Va. USA under accession numbers 98669
and 98668, respectively, and derivatives thereof.
[0084] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0085] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected non-293 cells
under conditions where the cells are not rapidly dividing, the
cells can produce proteins for extended periods of time. For
instance, BHK cells are grown to confluence in cell factories, then
exposed to the adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free conditions,
which allows infected cells to survive for several weeks without
significant cell division. In an alternative method, adenovirus
vector-infected 293 cells can be grown as adherent cells or in
suspension culture at relatively high cell density to produce
significant amounts of protein (See Garnier et al., Cytotechnol.
15:145-55, 1994). With either protocol, an expressed, secreted
heterologous protein can be repeatedly isolated from the cell
culture supernatant, lysate, or membrane fractions depending on the
disposition of the expressed protein in the cell. Within the
infected 293 cell production protocol, non-secreted proteins can
also be effectively obtained.
[0086] Insect cells can be infected with recombinant baculovirus,
commonly derived from Autographa californica nuclear polyhedrosis
virus (AcNPV) according to methods known in the art. Within a
preferred method, recombinant baculovirus is produced through the
use of a transposon-based system described by Luckow et al. (J.
Virol. 67:4566-4579, 1993). This system, which utilizes transfer
vectors, is commercially available in kit form (Bac-to-Bac.TM. kit;
Life Technologies, Rockville, Md.). The transfer vector (e.g.,
pFastBac1.TM.; Life Technologies) contains a Tn7 transposon to move
the DNA encoding the protein of interest into a baculovirus genome
maintained in E. coli as a large plasmid called a "bacmid." See,
Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning
et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and
Rapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfer
vectors can include an in-frame fusion with DNA encoding a
polypeptide extension or affinity tag as disclosed above. Using
techniques known in the art, a transfer vector containing a
Zcyto21-encoding sequence is transformed into E. coli host cells,
and the cells are screened for bacmids which contain an interrupted
lacZ gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is isolated, using
common techniques, and used to transfect Spodoptera frugiperda
cells, such as Sf9 cells. Recombinant virus that expresses Zcyto21
protein is subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
[0087] For protein production, the recombinant virus is used to
infect host cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or
Trichoplusia ni (e.g., High Five.TM. cells; Invitrogen, Carlsbad,
Calif.). See, for example, U.S. Pat. No. 5,300,435. Serum-free
media are used to grow and maintain the cells. Suitable media
formulations are known in the art and can be obtained from
commercial suppliers. The cells are grown up from an inoculation
density of approximately 2-5.times.10.sup.5 cells to a density of
1-2.times.10.sup.6 cells, at which time a recombinant viral stock
is added at a multiplicity of infection (MOI) of 0.1 to 10, more
typically near 3. Procedures used are generally known in the
art.
[0088] Other higher eukaryotic cells can also be used as hosts,
including plant cells and avian cells. The use of Agrobacterium
rhizogenes as a vector for expressing genes in plant cells has been
reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,
1987.
[0089] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al, U.S. Pat. No. 5,037,743; and Murray et al.,
U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol
132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; and Raymond et
al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilized
according to the methods of McKnight et al., U.S. Pat. No.
4,935,349. Methods for transforming Acremonium chrysogenum are
disclosed by Sumino et al., U.S. Pat. No. 5,162,228. Methods for
transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No.
4,486,533. Production of recombinant proteins in Pichia methanolica
is disclosed in U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and
5,888,768.
[0090] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a Zcyto21 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0091] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. Liquid cultures are
provided with sufficient aeration by conventional means, such as
shaking of small flasks or sparging of fermentors.
[0092] It is preferred to purify the polypeptides and proteins of
the present invention to .gtoreq.80% purity, more preferably to
.gtoreq.90% purity, even more preferably .gtoreq.95% purity, and
particularly preferred is a pharmaceutically pure state, that is
greater than 99.9% pure with respect to contaminating
macromolecules, particularly other proteins and nucleic acids, and
free of infectious and pyrogenic agents. Preferably, a purified
polypeptide or protein is substantially free of other polypeptides
or proteins, particularly those of animal origin.
[0093] Expressed recombinant Zcyto21 proteins (including chimeric
polypeptides and multimeric proteins) are purified by conventional
protein purification methods, typically by a combination of
chromatographic techniques. See, in general, Affinity
Chromatography: Principles & Methods, Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York,
1994. Proteins comprising a polyhistidine affinity tag (typically
about 6 histidine residues) are purified by affinity chromatography
on a nickel chelate resin. See, for example, Houchuli et al.,
Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag
can be purified by immunoaffinity chromatography according to
conventional procedures. See, for example, Grussenmeyer et al.,
ibid. Maltose binding protein fusions are purified on an amylose
column according to methods known in the art.
[0094] Zcyto21 polypeptides can also be prepared through chemical
synthesis according to methods known in the art, including
exclusive solid phase synthesis, partial solid phase methods,
fragment condensation or classical solution synthesis. See, for
example, Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et
al., Solid Phase Peptide Synthesis (2nd edition), Pierce Chemical
Co., Rockford, Ill., 1984; Bayer and Rapp, Chem. Pept. Prot. 3:3,
1986; and Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, Oxford, 1989. In vitro synthesis is
particularly advantageous for the preparation of smaller
polypeptides.
[0095] Using methods known in the art, Zcyto21 proteins can be
prepared as monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; and may or may not
include an initial methionine amino acid residue.
[0096] Target cells for use in Zcyto21 activity assays include,
without limitation, vascular cells (especially endothelial cells
and smooth muscle cells), hematopoietic (myeloid and lymphoid)
cells, liver cells (including hepatocytes, fenestrated endothelial
cells, Kupffer cells, and Ito cells), fibroblasts (including human
dermal fibroblasts and lung fibroblasts), fetal lung cells,
articular synoviocytes, pericytes, chondrocytes, osteoblasts, and
prostate epithelial cells. Endothelial cells and hematopoietic
cells are derived from a common ancestral cell, the hemangioblast
(Choi et al., Development 125:725-732, 1998).
[0097] Zcyto21 proteins of the present invention are characterized
by their activity, that is, modulation of the proliferation,
differentiation, migration, adhesion, or metabolism of responsive
cell types. Biological activity of Zcyto21 proteins is assayed
using in vitro or in vivo assays designed to detect cell
proliferation, differentiation, migration or adhesion; or changes
in cellular metabolism (e.g., production of other growth factors or
other macromolecules). Many suitable assays are known in the art,
and representative assays are disclosed herein. Assays using
cultured cells are most convenient for screening, such as for
determining the effects of amino acid substitutions, deletions, or
insertions. However, in view of the complexity of developmental
processes (e.g., angiogenesis, wound healing), in vivo assays will
generally be employed to confirm and further characterize
biological activity. Certain in vitro models, such as the
three-dimensional collagen gel matrix model of Pepper et al.
(Biochem. Biophys. Res. Comm. 189:824-831, 1992), are sufficiently
complex to assay histological effects. Assays can be performed
using exogenously produced proteins, or may be carried out in vivo
or in vitro using cells expressing the polypeptide(s) of interest.
Assays can be conducted using Zcyto21 proteins alone or in
combination with other growth factors, such as members of the VEGF
family or hematopoietic cytokines (e.g., EPO, TPO, G-CSF, stem cell
factor). Representative assays are disclosed below.
[0098] Activity of Zcyto21 proteins can be measured in vitro using
cultured cells or in vivo by administering molecules of the claimed
invention to an appropriate animal model. Assays measuring cell
proliferation or differentiation are well known in the art. For
example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990), incorporation of
radiolabelled nucleotides (as disclosed by, e.g., Raines and Ross,
Methods Enzymol. 109:749-773, 1985; Wahl et al., Mol. Cell Biol.
8:5016-5025, 1988; and Cook et al., Analytical Biochem. 179:1-7,
1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in the DNA
of proliferating cells (Porstmann et al., J. Immunol. Methods
82:169-179, 1985), and use of tetrazolium salts (Mosmann, J.
Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res.
48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and
Scudiero et al., Cancer Res. 48:4827-4833, 1988). Differentiation
can be assayed using suitable precursor cells that can be induced
to differentiate into a more mature phenotype. Assays measuring
differentiation include, for example, measuring cell-surface
markers associated with stage-specific expression of a tissue,
enzymatic activity, functional activity or morphological changes
(Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75,
1994; Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161-171,
1989; all incorporated herein by reference).
[0099] Zcyto21 activity may also be detected using assays designed
to measure Zcyto21-induced production of one or more additional
growth factors or other macromolecules. Preferred such assays
include those for determining the presence of hepatocyte growth
factor (HGF), epidermal growth factor (EGF), transforming growth
factor alpha (TGF.alpha.), interleukin-6 (IL-6), VEGF, acidic
fibroblast growth factor (aFGF), angiogenin, and other
macromolecules produced by the liver. Suitable assays include
mitogenesis assays using target cells responsive to the
macromolecule of interest, receptor-binding assays, competition
binding assays, immunological assays (e.g., ELISA), and other
formats known in the art. Metalloprotease secretion is measured
from treated primary human dermal fibroblasts, synoviocytes and
chondrocytes. The relative levels of collagenase, gelatinase and
stromalysin produced in response to culturing in the presence of a
Zcyto21 protein is measured using zymogram gels (Loita and
Stetler-Stevenson, Cancer Biology 1:96-106, 1990).
Procollagen/collagen synthesis by dermal fibroblasts and
chondrocytes in response to a test protein is measured using
.sup.3H-proline incorporation into nascent secreted collagen.
.sup.3H-labeled collagen is visualized by SDS-PAGE followed by
autoradiography (Unemori and Amento, J. Biol. Chem. 265:
10681-10685, 1990). Glycosaminoglycan (GAG) secretion from dermal
fibroblasts and chondrocytes is measured using a
1,9-dimethylmethylene blue dye binding assay (Famdale et al.,
Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays
are also carried out in the presence of IL-1.alpha. or TGF-.alpha.
to examine the ability of Zcyto21 protein to modify the established
responses to these cytokines.
[0100] Monocyte activation assays are carried out (1) to look for
the ability of Zcyto21 proteins to further stimulate monocyte
activation, and (2) to examine the ability of Zcyto21 proteins to
modulate attachment-induced or endotoxin-induced monocyte
activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987).
IL-1.alpha. and TNF.alpha. levels produced in response to
activation are measured by ELISA (Biosource, Inc. Camarillo,
Calif.). Monocyte/macrophage cells, by virtue of CD14 (LPS
receptor), are exquisitely sensitive to endotoxin, and proteins
with moderate levels of endotoxin-like activity will activate these
cells.
[0101] Hematopoietic activity of Zcyto21 proteins can be assayed on
various hematopoietic cells in culture. Preferred assays include
primary bone marrow colony assays and later stage
lineage-restricted colony assays, which are known in the art (e.g.,
Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on
a suitable semi-solid medium (e.g., 50% methylcellulose containing
15% fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN
antibiotic mix) are incubated in the presence of test polypeptide,
then examined microscopically for colony formation. Known
hematopoietic factors are used as controls. Mitogenic activity of
Zcyto21 polypeptides on hematopoietic cell lines can be measured as
disclosed above.
[0102] Cell migration is assayed essentially as disclosed by Kahler
et al. (Arteriosclerosis Thrombosis and Vascular Biology
17:932-939, 1997). A protein is considered to be chemotactic if it
induces migration of cells from an area of low protein
concentration to an area of high protein concentration. A typical
assay is performed using modified Boyden chambers with a
polystryrene membrane separating the two chambers (Transwell;
Corning Costar Corp.). The test sample, diluted in medium
containing 1% BSA, is added to the lower chamber of a 24-well plate
containing Transwells. Cells are then placed on the Transwell
insert that has been pretreated with 0.2% gelatin. Cell migration
is measured after 4 hours of incubation at 37.degree. C.
Non-migrating cells are wiped off the top of the Transwell
membrane, and cells attached to the lower face of the membrane are
fixed and stained with 0.1% crystal violet. Stained cells are then
extracted with 10% acetic acid and absorbance is measured at 600
nm. Migration is then calculated from a standard calibration curve.
Cell migration can also be measured using the matrigel method of
Grant et al. ("Angiogenesis as a component of
epithelial-mesenchymal interactions" in Goldberg and Rosen,
Epithelial-Mesenchymal Interaction in Cancer, Birkh{umlaut over
(auser)}Verlag, 1995, 235-248; Baatout, Anticancer Research
17:451-456, 1997).
[0103] Cell adhesion activity is assayed essentially as disclosed
by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,
microtiter plates are coated with the test protein, non-specific
sites are blocked with BSA, and cells (such as smooth muscle cells,
leukocytes, or endothelial cells) are plated at a density of
approximately 10.sup.4-10.sup.5 cells/well. The wells are incubated
at 37.degree. C. (typically for about 60 minutes), then
non-adherent cells are removed by gentle washing. Adhered cells are
quantitated by conventional methods (e.g., by staining with crystal
violet, lysing the cells, and determining the optical density of
the lysate). Control wells are coated with a known adhesive
protein, such as fibronectin or vitronectin.
[0104] The activity of Zcyto21 proteins can be measured with a
silicon-based biosensor microphysiometer that measures the
extracellular acidification rate or proton excretion associated
with receptor binding and subsequent physiologic cellular
responses. An exemplary such device is the Cytosensor.TM.
Microphysiometer manufactured by Molecular Devices, Sunnyvale,
Calif. A variety of cellular responses, such as cell proliferation,
ion transport, energy production, inflammatory response, regulatory
and receptor activation, and the like, can be measured by this
method. See, for example, McConnell et al., Science 257:1906-1912,
1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli
et al., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al.,
Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be
used for assaying adherent or non-adherent eukaryotic or
prokaryotic cells. By measuring extracellular acidification changes
in cell media over time, the microphysiometer directly measures
cellular responses to various stimuli, including Zcyto21 proteins,
their agonists, and antagonists. Preferably, the microphysiometer
is used to measure responses of a Zcyto21-responsive eukaryotic
cell, compared to a control eukaryotic cell that does not respond
to Zcyto21 polypeptide. Zcyto21-responsive eukaryotic cells
comprise cells into which a receptor for Zcyto21 has been
transfected, thereby creating a cell that is responsive to Zcyto21,
as well as cells naturally responsive to Zcyto21. Differences,
measured by a change, for example, an increase or diminution in
extracellular acidification, in the response of cells exposed to
Zcyto21 polypeptide, relative to a control not exposed to Zcyto21,
are a direct measurement of Zcyto21-modulated cellular responses.
Moreover, such Zcyto21-modulated responses can be assayed under a
variety of stimuli. The present invention thus provides methods of
identifying agonists and antagonists of Zcyto21 proteins,
comprising providing cells responsive to a Zcyto21 polypeptide,
culturing a first portion of the cells in the absence of a test
compound, culturing a second portion of the cells in the presence
of a test compound, and detecting a change, for example, an
increase or diminution, in a cellular response of the second
portion of the cells as compared to the first portion of the cells.
The change in cellular response is shown as a measurable change in
extracellular acidification rate. Culturing a third portion of the
cells in the presence of a Zcyto21 protein and the absence of a
test compound provides a positive control for the
Zcyto21-responsive cells and a control to compare the agonist
activity of a test compound with that of the Zcyto21 polypeptide.
Antagonists of Zcyto21 can be identified by exposing the cells to
Zcyto21 protein in the presence and absence of the test compound,
whereby a reduction in Zcyto21-stimulated activity is indicative of
antagonist activity in the test compound.
[0105] Expression of Zcyto21 polynucleotides in animals provides
models for further study of the biological effects of
overproduction or inhibition of protein activity in vivo.
Zcyto21-encoding polynucleotides and antisense polynucleotides can
be introduced into test animals, such as mice, using viral vectors
or naked DNA, or transgenic animals can be produced.
[0106] One in vivo approach for assaying proteins of the present
invention utilizes viral delivery systems. Exemplary viruses for
this purpose include adenovirus, herpesvirus, retroviruses,
vaccinia virus, and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acids. For
review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and
Douglas and Curiel, Science & Medicine 4:44-53, 1997. The
adenovirus system offers several advantages. Adenovirus can (i)
accommodate relatively large DNA inserts; (ii) be grown to
high-titer; (iii) infect a broad range of mammalian cell types; and
(iv) be used with many different promoters including ubiquitous,
tissue specific, and regulatable promoters. Because adenoviruses
are stable in the bloodstream, they can be administered by
intravenous injection.
[0107] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. In an
exemplary system, the essential E1 gene is deleted from the viral
vector, and the virus will not replicate unless the E1 gene is
provided by the host cell (e.g., the human 293 cell line). When
intravenously administered to intact animals, adenovirus primarily
targets the liver. If the adenoviral delivery system has an E1 gene
deletion, the virus cannot replicate in the host cells. However,
the host's tissue (e.g., liver) will express and process (and, if a
signal sequence is present, secrete) the heterologous protein.
Secreted proteins will enter the circulation in the highly
vascularized liver, and effects on the infected animal can be
determined.
[0108] An alternative method of gene delivery comprises removing
cells from the body and introducing a vector into the cells as a
naked DNA plasmid. The transformed cells are then re-implanted in
the body. Naked DNA vectors are introduced into host cells by
methods known in the art, including transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988;
Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang,
Meth. Cell Biol. 43:353-365, 1994.
[0109] Transgenic mice, engineered to express a Zcyto21 gene, and
mice that exhibit a complete absence of Zcyto21 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), can also be generated (Lowell et al., Nature 366:740-742,
1993). These mice can be employed to study the Zcyto21 gene and the
protein encoded thereby in an in vivo system. Transgenic mice are
particularly useful for investigating the role of Zcyto21 proteins
in early development in that they allow the identification of
developmental abnormalities or blocks resulting from the over- or
underexpression of a specific factor. See also, Maisonpierre et
al., Science 277:55-60, 1997 and Hanahan, Science 277:48-50, 1997.
Preferred promoters for transgenic expression include promoters
from metallothionein and albumin genes.
[0110] A loss of normal inhibitory control of muscle contraction
has been associated with damage or perturbation of selected
gamma-aminobutryric acid-secreting neurons. For example, Stiff Man
Syndrome exhibit remarkable stiffness of musculature, believed to
be mediated through interference of the functioning of their
gamma-aminobutryric acid (GABA) producing neurons. Other related
neuromuscular disorders include myotonia, metabolic myopathies,
Isaac's syndrome, dystonia, and tetanic spasms (Valldeoriola, J.
Neurol 246:423-431, 1999).
[0111] Similarly, direct measurement of Zcyto21 polypeptide, or its
loss of expression in a tissue can be determined in a tissue or
cells as they undergo tumor progression. Increases in invasiveness
and motility of cells, or the gain or loss of expression of Zcyto21
in a pre-cancerous or cancerous condition, in comparison to normal
tissue, can serve as a diagnostic for transformation, invasion and
metastasis in tumor progression. As such, knowledge of a tumor's
stage of progression or metastasis will aid the physician in
choosing the most proper therapy, or aggressiveness of treatment,
for a given individual cancer patient. Methods of measuring gain
and loss of expression (of either mRNA or protein) are well known
in the art and described herein and can be applied to Zcyto21
expression. For example, appearance or disappearance of
polypeptides that regulate cell motility can be used to aid
diagnosis and prognosis of prostate cancer (Banyard, J. and Zetter,
B. R., Cancer and Metast. Rev. 17:449-458, 1999). As an effector of
cell motility, or as a liver-specific marker, Zcyto21 gain or loss
of expression may serve as a diagnostic for brain and other
cancers. Moreover, analogous to the prostate specific antigen
(PSA), increased levels of Zcyto21 polypeptides, or anti-Zcyto21
antibodies in a patient, relative to a normal control can be
indicative of brain and other cancers (See, e.g., Mulders, TMT, et
al., Eur. J. Surgical Oncol. 16:37-41, 1990). Strong Zcyto21
expression in tissue not normally found to express Zcyto21 would
serve as a diagnostic of an abnormality in the cell or tissue type,
of invasion or metastasis of cancerous liver tissue into non-liver
tissue, and could aid a physician in directing further testing or
investigation, or aid in directing therapy.
[0112] In addition, Zcyto21 polynucleotide probes, anti-Zcyto21
antibodies, and detection the presence of Zcyto21 polypeptides in
tissue can be used to assess whether brain or other tissue found to
normally express Zcyto21 is present, for example, after surgery
involving the excision of a diseased or cancerous liver or neuronal
tissue. As such, the polynucleotides, polypeptides, and antibodies
of the present invention can be used as an aid to determine whether
all tissue is excised after surgery, for example, after surgery for
brain and other cancers. In such instances, it is especially
important to remove all potentially diseased tissue to maximize
recovery from the cancer, and to minimize recurrence. Preferred
embodiments include fluorescent, radiolabeled, or calorimetrically
labeled anti-Zcyto21 antibodies and Zcyto21 polypeptide binding
partners, that can be used histologically or in situ.
[0113] Moreover, the activity and effect of Zcyto21 on tumor
progression and metastasis can be measured in vivo. Several
syngeneic mouse models have been developed to study the influence
of polypeptides, compounds or other treatments on tumor
progression. In these models, tumor cells passaged in culture are
implanted into mice of the same strain as the tumor donor. The
cells will develop into tumors having similar characteristics in
the recipient mice, and metastasis will also occur in some of the
models. Appropriate tumor models for our studies include the Lewis
lung carcinoma (ATCC No. CRL-1642) and B16 melanoma (ATCC No.
CRL-6323), amongst others. These are both commonly used tumor
lines, syngeneic to the C57BL6 mouse, that are readily cultured and
manipulated in vitro. Tumors resulting from implantation of either
of these cell lines are capable of metastasis to the lung in C57BL6
mice. The Lewis lung carcinoma model has recently been used in mice
to identify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell
79: 315-328, 1994). C57BL6/J mice are treated with an experimental
agent either through daily injection of recombinant protein,
agonist or antagonist or a one-time injection of recombinant
adenovirus. Three days following this treatment, 10.sup.5 to
10.sup.6 cells are implanted under the dorsal skin. Alternatively,
the cells themselves may be infected with recombinant adenovirus,
such as one expressing Zcyto21, before implantation so that the
protein is synthesized at the tumor site or intracellularly, rather
than systemically. The mice normally develop visible tumors within
5 days. The tumors are allowed to grow for a period of up to 3
weeks, during which time they may reach a size of 1500-1800
mm.sup.3 in the control treated group. Tumor size and body weight
are carefully monitored throughout the experiment. At the time of
sacrifice, the tumor is removed and weighed along with the lungs
and the liver. The lung weight has been shown to correlate well
with metastatic tumor burden. As an additional measure, lung
surface metastases are counted. The resected tumor, lungs and liver
are prepared for histopathological examination,
immunohistochemistry, and in situ hybridization, using methods
known in the art and described herein. The influence of the
expressed polypeptide in question, e.g., Zcyto21, on the ability of
the tumor to recruit vasculature and undergo metastasis can thus be
assessed. In addition, aside from using adenovirus, the implanted
cells can be transiently transfected with Zcyto21. Use of stable
Zcyto21 transfectants as well as use of induceable promoters to
activate Zcyto21 expression in vivo are known in the art and can be
used in this system to assess Zcyto21 induction of metastasis.
Moreover, purified Zcyto21 or Zcyto21-conditioned media can be
directly injected in to this mouse model, and hence be used in this
system. For general reference see, O'Reilly M S, et al. Cell
79:315-328, 1994; and Rusciano D, et al. Murine Models of Liver
Metastasis. Invasion Metastasis 14:349-361, 1995.
[0114] Antisense methodology can be used to inhibit Zcyto21 gene
transcription to examine the effects of such inhibition in vivo.
Polynucleotides that are complementary to a segment of a
Zcyto21-encoding polynucleotide (e.g., a polynucleotide as set
forth in SEQ ID NO:1) are designed to bind to Zcyto21-encoding mRNA
and to inhibit translation of such mRNA. Such antisense
oligonucleotides can also be used to inhibit expression of Zcyto21
polypeptide-encoding genes in cell culture.
[0115] Most cytokines as well as other proteins produced by
activated lymphocytes play an important biological role in cell
differentiation, activation, recruitment and homeostasis of cells
throughout the body. Zcyto21 and inhibitors of Zcyto21 activity are
expected to have a variety of therapeutic applications. These
therapeutic applications include treatment of diseases which
require immune regulation, including autoimmune diseases such as
rheumatoid arthritis, multiple sclerosis, myasthenia gravis,
systemic lupus erythematosis, and diabetes. Zcyto21 may be
important in the regulation of inflammation, and therefore would be
useful in treating rheumatoid arthritis, asthma and sepsis. There
may be a role of Zcyto21 in mediating tumorgenesis, whereby a
Zcyto21 antagonist would be useful in the treatment of cancer.
Zcyto21 may be useful in modulating the immune system, whereby
Zcyto21 and Zcyto21 antagonists may be used for reducing graft
rejection, preventing graft-vs-host disease, boosting immunity to
infectious diseases, treating immunocompromised patients (e.g.,
HIV.sup.+ patients), or in improving vaccines.
[0116] As an interferon-like polypeptide in tissues of brain,
islet, prostate, testis, pituitary, placenta, ovarian tumor, lung
tumor, rectal tumor and ovarian tumor, as well as a CD3+ cell line,
and a virally infected prostate epithelial cell line, Zcyto21 is
useful to modulate viral infection, tumorigeneses and metastatis in
these and other tissues. In such cases, the interferon-like
molecule can be released by cell at the site of infection or
abnormal cell growth, or as a secreted molecule, it can migrate to
the site from a distant tissue.
[0117] The antiviral properties of Zcyto21 are particularly useful
in treating infection with papilloma viruses in vitro and in vivo.
For example, tumors caused by human papilloma viruses cause benign
tumors (i.e., genital warts) as well as malignant tumors such as
squamous-cell carcinomas. Treatment for these conditions commonly
is surgery or tissue destruction. Currently, however, some
antiviral/immunomodulatory drugs, including interferon alpha, have
been shown effective in reduce tumor size. See Baker, G. E et al.,
Dermatol. Clin. Apr. 15: 331-340, 1997. Further, as discussed by
Rockley, P. F. et al., (Pharmacol. Ther. 65(2): 265-287, 1995),
immunologic therapy with interferons can be directed against all
sites of infection, including clinical, subclinical, and latent
disease. In this example, IFN-alpha, IFN-beta and IFN-gamma have
been used successfully as monotherapy as well as in combination
with other therapies to treat anogenital condyloma acuminatum.
Zcyto21 will be a useful treatment similar to IFN-alpha, IFN-beta
and IFN-gamma in this treatment. Further more, there has been a
strong association between certain types of human papilloma virus
and cervical cancer. Zcyto21 can be used to detect, monitor and
treat cervical cancers.
[0118] As a small, secreted protein in islet cells Zcyto21 can
modulate the growth and differentiation of these cells.
Additionally, Zcyto21 may be useful in treating diabetes and
immunological conditions related to the growth and differentiation
of the cells.
[0119] The presence of Zcyto21 in brain and pituitary cells
indicates that it may also find use in growth and differentiation
of these cells. Further, the molecules of the present invention may
be responsible for nutritional homeostasis, including behavioral
disorders related to feeding and appetite suppression.
Additionally, Zcyto21 molecules may find use in treating
reproductive disorders in general.
[0120] Zcyto21 polypeptides can be administered alone or in
combination with other vasculogenic or angiogenic agents, including
VEGF. When using Zcyto21 in combination with an additional agent,
the two compounds can be administered simultaneously or
sequentially as appropriate for the specific condition being
treated.
[0121] Zcyto21 will be useful in treating tumorgenesis, and
therefore would be useful in the treatment of cancer. A Zcyto21
inhibition of anti-IgM stimulated normal B-cells and a similar
effect is observed in B-cell tumor lines suggest that there may be
therapeutic benefit in treating patients with the Zcyto21 in order
to induce the B cell tumor cells into a less proliferative state.
The ligand could be administered in combination with other agents
already in use including both conventional chemotherapeutic agents
as well as immune modulators such as interferon alpha. Alpha/beta
interferons have been shown to be effective in treating some
leukemias and animal disease models, and the growth inhibitory
effects of interferon-alpha and Zcyto21 may be additive for B-cell
tumor-derived cell lines.
[0122] The present invention provides a method of reducing
proliferation of a neoplastic B or T cells comprising administering
to a mammal with a B or T cell neoplasm an amount of a composition
of Zcyto21 sufficient to reduce proliferation of the neoplastic B
or T cells. Zcyto21 stimulation of lytic NK cells from marrow
progenitors and the proliferation of T-cells following activation
of the antigen receptors would enhance treatment for patients
receiving allogenic marrow transplants, and therefore, Zcyto21 will
enhance the generation of anti-tumor responses, with or without the
infusion of donor lymphocytes.
[0123] In another aspect, the present invention provides a method
of reducing proliferation of a neoplastic B or T cells comprising
administering to a mammal with a B or T cell neoplasm an amount of
a composition of Zcyto21 antagonist sufficient to reducing
proliferation of the neoplastic B or T cells. Furthermore, the
Zcyto21 antagonist can be a ligand/toxin fusion protein.
[0124] A Zcyto21-saporin fusion toxin may be employed against a
similar set of leukemias and lymphomas, extending the range of
leukemias that can be treated with Zcyto21. Fusion toxin mediated
activation of the Zcyto21 receptor provides two independent means
to inhibit the growth of the target cells, the first being
identical to the effects seen by the ligand alone, and the second
due to delivery of the toxin through receptor internalization.
[0125] Based on the teachings herein, the interferon-like Zcyto21
molecules of the present invention will be useful to detect,
monitor or treat such diverse conditions as hairy cell leukemia,
renal cell carcinoma, basal cell carcinoma, malignant melanoma,
AIDS-related Kaposi's sarcoma, multiple myeloma, chronic
myelogenous leukemia, non-Hodgkin's lymphoma, laryngeal
papillomatosis, mycosis fungoides, condyloma acuminata,
papillomavirus-induced epidermodysplasi verruciformis, chronic
hepatitis B, hepatitis C, chronic hepatitis D, and chronic non-A,
non-B/C hepatitis. The U.S. Food and Drug Administration has
approved the use of interferon-.beta. to treat multiple sclerosis,
a chronic disease of the nervous system. Interferon-.gamma. is used
to treat chronic granulomatous diseases, in which the interferon
enhances the patient's immune response to destroy infectious
bacterial, fungal, and protozoal pathogens. Clinical studies also
indicate that interferon-.gamma. may be useful in the treatment of
AIDS, leishmaniasis, and lepromatous leprosy
[0126] For pharmaceutical use, Zcyto21 proteins are formulated for
topical or parenteral, particularly intravenous or subcutaneous,
delivery according to conventional methods. In general,
pharmaceutical formulations will include a Zcyto21 polypeptide in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water, or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 19th ed., 1995. Zcyto21 will preferably be used
in a concentration of about 10 to 100 .mu.g/ml of total volume,
although concentrations in the range of 1 ng/ml to 1000 .mu.g/ml
may be used. For topical application, such as for the promotion of
wound healing, the protein will be applied in the range of 0.1-10
.mu.g/cm.sup.2 of wound area, with the exact dose determined by the
clinician according to accepted standards, taking into account the
nature and severity of the condition to be treated, patient traits,
etc. Determination of dose is within the level of ordinary skill in
the art. Dosing is daily or intermittently over the period of
treatment. Intravenous administration will be by bolus injection or
infusion over a typical period of one to several hours. Sustained
release formulations can also be employed. In general, a
therapeutically effective amount of Zcyto21 is an amount sufficient
to produce a clinically significant change in the treated
condition, such as a clinically significant change in hematopoietic
or immune function, a significant reduction in morbidity, or a
significantly increased histological score.
[0127] Zcyto21 proteins, agonists, and antagonists are useful for
modulating the expansion, proliferation, activation,
differentiation, migration, or metabolism of responsive cell types,
which include both primary cells and cultured cell lines. Of
particular interest in this regard are hematopoietic cells,
mesenchymal cells (including stem cells and mature myeloid and
lymphoid cells), endothelial cells, epithelial cells, smooth muscle
cells, fibroblasts, hepatocytes, neural cells and embryonic stem
cells. Zcyto21 polypeptides are added to tissue culture media for
these cell types at a concentration of about 10 pg/ml to about 100
ng/ml. Those skilled in the art will recognize that Zcyto21
proteins can be advantageously combined with other growth factors
in culture media.
[0128] Within the laboratory research field, Zcyto21 proteins can
also be used as molecular weight standards or as reagents in assays
for determining circulating levels of the protein, such as in the
diagnosis of disorders characterized by over- or under-production
of Zcyto21 protein or in the analysis of cell phenotype.
[0129] Zcyto21 proteins can also be used to identify inhibitors of
their activity. Test compounds are added to the assays disclosed
above to identify compounds that inhibit the activity of Zcyto21
protein. In addition to those assays disclosed above, samples can
be tested for inhibition of Zcyto21 activity within a variety of
assays designed to measure receptor binding or the
stimulation/inhibition of Zcyto21-dependent cellular responses. For
example, Zcyto21-responsive cell lines can be transfected with a
reporter gene construct that is responsive to a Zcyto21-stimulated
cellular pathway. Reporter gene constructs of this type are known
in the art, and will generally comprise a Zcyto21-activated serum
response element (SRE) operably linked to a gene encoding an
assayable protein, such as luciferase. Candidate compounds,
solutions, mixtures or extracts are tested for the ability to
inhibit the activity of Zcyto21 on the target cells as evidenced by
a decrease in Zcyto21 stimulation of reporter gene expression.
Assays of this type will detect compounds that directly block
Zcyto21 binding to cell-surface receptors, as well as compounds
that block processes in the cellular pathway subsequent to
receptor-ligand binding. In the alternative, compounds or other
samples can be tested for direct blocking of Zcyto21 binding to
receptor using Zcyto21 tagged with a detectable label (e.g.,
.sup.125I, biotin, horseradish peroxidase, FITC, and the like).
Within assays of this type, the ability of a test sample to inhibit
the binding of labeled Zcyto21 to the receptor is indicative of
inhibitory activity, which can be confirmed through secondary
assays. Receptors used within binding assays may be cellular
receptors or isolated, immobilized receptors.
[0130] As used herein, the term "antibodies" includes polyclonal
antibodies, monoclonal antibodies, antigen-binding fragments
thereof such as F(ab').sub.2 and Fab fragments, single chain
antibodies, and the like, including genetically engineered
antibodies. Non-human antibodies may be humanized by grafting
non-human CDRs onto human framework and constant regions, or by
incorporating the entire non-human variable domains (optionally
"cloaking" them with a human-like surface by replacement of exposed
residues, wherein the result is a "veneered" antibody). In some
instances, humanized antibodies may retain non-human residues
within the human variable region framework domains to enhance
proper binding characteristics. Through humanizing antibodies,
biological half-life may be increased, and the potential for
adverse immune reactions upon administration to humans is reduced.
One skilled in the art can generate humanized antibodies with
specific and different constant domains (i.e., different Ig
subclasses) to facilitate or inhibit various immune functions
associated with particular antibody constant domains. Antibodies
are defined to be specifically binding if they bind to a Zcyto21
polypeptide or protein with an affinity at least 10-fold greater
than the binding affinity to control (non-Zcyto21) polypeptide or
protein. The affinity of a monoclonal antibody can be readily
determined by one of ordinary skill in the art (see, for example,
Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).
[0131] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, Hurrell, J. G. R., Ed.,
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
Press, Inc., Boca Raton, Fla., 1982, which is incorporated herein
by reference). Of particular interest are generating antibodies to
hydrophilic antigenic sites which include, for example, residues
155 (Glu) to 160 (Glu); residues 51 (Lys) to 56 (Ala); residues 50
(Phe) to 55 (Asp); residues 140 (Pro) to 145 (Arg); and residues
154 (Gln) to 159 (Lys); as shown in SEQ ID NO: 2. As would be
evident to one of ordinary skill in the art, polyclonal antibodies
can be generated from a variety of warm-blooded animals such as
horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and
rats. The immunogenicity of a Zcyto21 polypeptide may be increased
through the use of an adjuvant such as alum (aluminum hydroxide) or
Freund's complete or incomplete adjuvant. Polypeptides useful for
immunization also include fusion polypeptides, such as fusions of a
Zcyto21 polypeptide or a portion thereof with an immunoglobulin
polypeptide or with maltose binding protein. The polypeptide
immunogen may be a full-length molecule or a portion thereof. If
the polypeptide portion is "hapten-like", such portion may be
advantageously joined or linked to a macromolecular carrier (such
as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or
tetanus toxoid) for immunization.
[0132] Alternative techniques for generating or selecting
antibodies include in vitro exposure of lymphocytes to Zcyto21
polypeptides, and selection of antibody display libraries in phage
or similar vectors (e.g., through the use of immobilized or labeled
Zcyto21 polypeptide). Human antibodies can be produced in
transgenic, non-human animals that have been engineered to contain
human immunoglobulin genes as disclosed in WIPO Publication WO
98/24893. It is preferred that the endogenous immunoglobulin genes
in these animals be inactivated or eliminated, such as by
homologous recombination.
[0133] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to Zcyto21
polypeptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radio-immunoassays, radio-immunoprecipitations, enzyme-linked
immunosorbent assays (ELISA), dot blot assays, Western blot assays,
inhibition or competition assays, and sandwich assays.
[0134] Antibodies to Zcyto21 may be used for affinity purification
of the protein, within diagnostic assays for determining
circulating levels of the protein; for detecting or quantitating
soluble Zcyto21 polypeptide as a marker of underlying pathology or
disease; for immunolocalization within whole animals or tissue
sections, including immunodiagnostic applications; for
immunohistochemistry; and as antagonists to block protein activity
in vitro and in vivo. Antibodies to Zcyto21 may also be used for
tagging cells that express Zcyto21; for affinity purification of
Zcyto21 polypeptides and proteins; in analytical methods employing
FACS; for screening expression libraries; and for generating
anti-idiotypic antibodies. Antibodies can be linked to other
compounds, including therapeutic and diagnostic agents, using known
methods to provide for targeting of those compounds to cells
expressing receptors for Zcyto21. For certain applications,
including in vitro and in vivo diagnostic uses, it is advantageous
to employ labeled antibodies. Suitable direct tags or labels
include radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like; indirect tags or labels may feature use of
biotin-avidin or other complement/anti-complement pairs as
intermediates. Antibodies of the present invention may also be
directly or indirectly conjugated to drugs, toxins, radionuclides
and the like, and these conjugates used for in vivo diagnostic or
therapeutic applications (e.g., inhibition of cell proliferation).
See, in general, Ramakrishnan et al., Cancer Res. 56:1324-1330,
1996.
[0135] Polypeptides and proteins of the present invention can be
used to identify and isolate receptors. Zcyto21 receptors may be
involved in growth regulation in the liver, blood vessel formation,
and other developmental processes. For example, Zcyto21 proteins
and polypeptides can be immobilized on a column, and membrane
preparations run over the column (as generally disclosed in
Immobilized Affinity Ligand Techniques, Hermanson et al., eds.,
Academic Press, San Diego, Calif., 1992, pp. 195-202). Proteins and
polypeptides can also be radiolabeled (Methods Enzymol., vol. 182,
"Guide to Protein Purification", M. Deutscher, ed., Academic Press,
San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al.,
Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem.
Pharmacol. 33:1167-1180, 1984) and used to tag specific
cell-surface proteins. In a similar manner, radiolabeled Zcyto21
proteins and polypeptides can be used to clone the cognate receptor
in binding assays using cells transfected with an expression cDNA
library.
[0136] Zcyto21 polypeptides can also be used to teach analytical
skills such as mass spectrometry, circular dichroism, to determine
conformation, especially of the four alpha helices, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing the Zcyto21 can be given to the student to analyze.
Since the amino acid sequence would be known by the instructor, the
protein can be given to the student as a test to determine the
skills or develop the skills of the student, the instructor would
then know whether or not the student has correctly analyzed the
polypeptide. Since every polypeptide is unique, the educational
utility of Zcyto21 would be unique unto itself.
[0137] The antibodies which bind specifically to Zcyto21 can be
used as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify Zcyto21, cloning and sequencing
the polynucleotide that encodes an antibody and thus as a practicum
for teaching a student how to design humanized antibodies. The
Zcyto21 gene, polypeptide, or antibody would then be packaged by
reagent companies and sold to educational institutions so that the
students gain skill in art of molecular biology. Because each gene
and protein is unique, each gene and protein creates unique
challenges and learning experiences for students in a lab
practicum. Such educational kits containing the Zcyto21 gene,
polypeptide, or antibody are considered within the scope of the
present invention. The present invention, thus generally described,
will be understood more readily by reference to the following
examples, which is provided by way of illustration and is not
intended to be limiting of the present invention.
EXAMPLES
Example 1
[0138] An expression plasmid containing all or part of a
polynucleotide encoding Zcyto21 is constructed via homologous
recombination. A fragment of Zcyto21 cDNA is isolated by PCR using
the polynucleotide sequence of SEQ ID NO: 1 with flanking regions
at the 5' and 3' ends corresponding to the vector sequences
flanking the Zcyto21 insertion point. The primers for PCR each
include from 5' to 3' end: 40 bp of flanking sequence from the
vector and 17 bp corresponding to the amino and carboxyl termini
from the open reading frame of Zcyto21.
[0139] Ten .mu.l of the 100 .mu.l PCR reaction mixture is run on a
0.8% low-melting-temperature agarose (SeaPlaque GTG.RTM.; FMC
BioProducts, Rockland, Me.) gel with 1.times.TBE buffer for
analysis. The remaining 90 .mu.l of the reaction misture is
precipitated with the addition of 5 .mu.l 1 M NaCl and 250 .mu.l of
absolute ethanol. The plasmid pZMP6, which has been cut with SmaI,
is used for recombination with the PCR fragment. Plamid pZMP6 is a
mammalian expression vector containing an expression cassette
having the cytomegalovirus immediate early promoter, multiple
restriction sites for insertion of coding sequences, a stop codon,
and a human growth hormone terminator; an E. coli origin of
replication; a mammalian selectable marker expression unit
comprising an SV40 promoter, enhancer and origin of replication, a
DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences
required for selection and replication in S. cerevisiae. It was
constructed from pZP9 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 98668) with the yeast genetic elements taken
from pRS316 (deposited at the American Type Culture Collection,
10801 University Boulevard, Manassas, Va. 20110-2209, under
Accession No. 77145), an internal ribosome entry site (IRES)
element from poliovirus, and the extracellular domain of CD8
truncated at the C-terminal end of the transmembrane domain.
[0140] One hundred microliters of competent yeast (S. cerevisiae)
cells are independently combined with 10 .mu.l of the various DNA
mixtures from above and transferred to a 0.2-cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed using power
supply (BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV
(5 kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600
.mu.l of 1.2 M sorbitol, and the yeast is plated in two 300-.mu.l
aliquots onto two URA-D plates and incubated at 30.degree. C. After
about 48 hours, the Ura.sup.+ yeast transformants from a single
plate are resuspended in 1 ml H.sub.2O and spun briefly to pellet
the yeast cells. The cell pellet is resuspended in 1 ml of lysis
buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1
mM EDTA). Five hundred microliters of the lysis mixture is added to
an Eppendorf tube containing 300 .mu.l acid-washed glass beads and
200 .mu.l phenol-chloroform, vortexed for 1 minute intervals two or
three times, and spun for 5 minutes in an Eppendorf centrifuge at
maximum speed. Three hundred microliters of the aqueous phase is
transferred to a fresh tube, and the DNA is precipitated with 600
.mu.l ethanol (EtOH), followed by centrifugation for 10 minutes at
4.degree. C. The DNA pellet is resuspended in 10 .mu.l
H.sub.2O.
[0141] Transformation of electrocompetent E. coli host cells
(Electromax DH10B.TM. cells; obtained from Life Technologies, Inc.,
Gaithersburg, Md.) is done with 0.5-2 ml yeast DNA prep and 40
.mu.l of cells. The cells are electropulsed at 1.7 kV, 25 pF, and
400 ohms. Following electroporation, 1 ml SOC (2% Bacto.TM.
Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM
glucose) is plated in 250-.mu.l aliquots on four LB AMP plates (LB
broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L
Ampicillin).
[0142] Individual clones harboring the correct expression construct
for Zcyto21 are identified by restriction digest to verify the
presence of the Zcyto21 insert and to confirm that the various DNA
sequences have been joined correctly to one another. The inserts of
positive clones are subjected to sequence analysis. Larger scale
plasmid DNA is isolated using a commercially available kit (QIAGEN
Plasmid Maxi Kit, Qiagen, Valencia, Calif.) according to
manufacturer's instructions. The correct construct is designated
pZMP6/Zcyto21.
Example 2
[0143] CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet.
12:555-666, 1986) are plated in 10-cm tissue culture dishes and
allowed to grow to approximately 50% to 70% confluency overnight at
37.degree. C., 5% CO.sub.2, in Ham's F12/FBS media (Ham's F12
medium (Life Technologies), 5% fetal bovine serum (Hyclone, Logan,
Utah), 1% L-glutamine (JRH Biosciences, Lenexa, Kans.), 1% sodium
pyruvate (Life Technologies)). The cells are then transfected with
the plasmid Zcyto21/pZMP6 by liposome-mediated transfection using a
3:1 (w/w) liposome formulation of the polycationic lipid
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanimini-
um-trifluoroacetate and the neutral lipid dioleoyl
phosphatidylethanolamine in membrane-filetered water
(Lipofectamine.TM. Reagent, Life Technologies), in serum free (SF)
media formulation (Ham's F12, 10 mg/ml transferrin, 5 mg/ml
insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).
Zcyto21/pZMP6 is diluted into 15-ml tubes to a total final volume
of 640 .mu.l with SF media. 35 .mu.l of Lipofectamine.TM. is mixed
with 605 .mu.l of SF medium. The resulting mixture is added to the
DNA mixture and allowed to incubate approximately 30 minutes at
room temperature. Five ml of SF media is added to the
DNA:Lipofectamine.TM. mixture. The cells are rinsed once with 5 ml
of SF media, aspirated, and the DNA:Lipofectamine.TM. mixture is
added. The cells are incubated at 37.degree. C. for five hours,
then 6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each
plate. The plates are incubated at 37.degree. C. overnight, and the
DNA:Lipofectamine.TM. mixture is replaced with fresh 5% FBS/Ham's
media the next day. On day 3 post-transfection, the cells are split
into T-175 flasks in growth medium. On day 7 postransfection, the
cells are stained with FITC-anti-CD8 monoclonal antibody
(Pharmingen, San Diego, Calif.) followed by anti-FITC-conjugated
magnetic beads (Miltenyi Biotec). The CD8-positive cells are
separated using commercially available columns (mini-MACS columns;
Miltenyi Biotec) according to the manufacturer's directions and put
into DMEM/Ham's F12/5% FBS without nucleosides but with 50 nM
methotrexate (selection medium).
[0144] Cells are plated for subcloning at a density of 0.5, 1 and 5
cells per well in 96-well dishes in selection medium and allowed to
grow out for approximately two weeks. The wells are checked for
evaporation of medium and brought back to 200 .mu.l per well as
necessary during this process. When a large percentage of the
colonies in the plate are near confluency, 100 .mu.l of medium is
collected from each well for analysis by dot blot, and the cells
are fed with fresh selection medium. The supernatant is applied to
a nitrocellulose filter in a dot blot apparatus, and the filter is
treated at 100.degree. C. in a vacuum oven to denature the protein.
The filter is incubated in 625 mM Tris-glycine, pH 9.1, 5 mM
.beta.-mercaptoethanol, at 65.degree. C., 10 minutes, then in 2.5%
non-fat dry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH
7.4, 150 mM NaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at
4.degree. C. on a rotating shaker. The filter is incubated with the
antibody-HRP conjugate in 2.5% non-fat dry milk Western A buffer
for 1 hour at room temperature on a rotating shaker. The filter is
then washed three times at room temperature in PBS plus 0.01% Tween
20, 15 minutes per wash. The filter is developed with
chemiluminescence reagents (ECL.TM. direct labelling kit; Amersham
Corp., Arlington Heights, Ill.) according to the manufacturer's
directions and exposed to film (Hyperfilm ECL, Amersham Corp.) for
approximately 5 minutes. Positive clones are trypsinized from the
96-well dish and transferred to 6-well dishes in selection medium
for scaleup and analysis by Western blot.
Example 3
[0145] Full-length Zcyto21 protein is produced in BHK cells
transfected with pZMP6/Zcyto21 (Example 1). BHK 570 cells (ATCC
CRL-10314) are plated in 10-cm tissue culture dishes and allowed to
grow to approximately 50 to 70% confluence overnight at 37.degree.
C., 5% CO.sub.2, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose;
Life Technologies), 5% fetal bovine serum (Hyclone, Logan, Utah), 1
mM L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium
pyruvate (Life Technologies). The cells are then transfected with
pZMP6/Zcyto21 by liposome-mediated transfection (using
Lipofectamine.TM.; Life Technologies), in serum free (SF) media
(DMEM supplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2
mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate). The plasmid
is diluted into 15-ml tubes to a total final volume of 640 .mu.l
with SF media. 35 .mu.l of the lipid mixture is mixed with 605
.mu.l of SF medium, and the resulting mixture is allowed to
incubate approximately 30 minutes at room temperature. Five
milliliters of SF media is then added to the DNA:lipid mixture. The
cells are rinsed once with 5 ml of SF media, aspirated, and the
DNA:lipid mixture is added. The cells are incubated at 37.degree.
C. for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media is
added to each plate. The plates are incubated at 37.degree. C.
overnight, and the DNA:lipid mixture is replaced with fresh 5%
FBS/DMEM media the next day. On day 5 post-transfection, the cells
are split into T-162 flasks in selection medium (DMEM+5% FBS, 1%
L-Gln, 1% NaPyr, 1 .mu.M methotrexate). Approximately 10 days
post-transfection, two 150-mm culture dishes of
methotrexate-resistant colonies from each transfection are
trypsinized, and the cells are pooled and plated into a T-162 flask
and transferred to large-scale culture.
Example 4
[0146] For construction of adenovirus vectors, the protein coding
region of human Zcyto21 is amplified by PCR using primers that add
PmeI and AscI restriction sites at the 5' and 3' termini
respectively. Amplification is performed with a full-length Zcyto21
cDNA template in a PCR reaction as follows: one cycle at 95.degree.
C. for 5 minutes; followed by 15 cycles at 95.degree. C. for 1
min., 61.degree. C. for 1 min., and 72.degree. C. for 1.5 min.;
followed by 72.degree. C. for 7 min.; followed by a 4.degree. C.
soak. The PCR reaction product is loaded onto a 1.2%
low-melting-temperature agarose gel in TAE buffer (0.04 M
Tris-acetate, 0.001 M EDTA). The Zcyto21 PCR product is excised
from the gel and purified using a commercially available kit
comprising a silica gel mambrane spin column (QIAquick.RTM. PCR
Purification Kit and gel cleanup kit; Qiagen, Inc.) as per kit
instructions. The PCR product is then digested with PmeI and AscI,
phenol/chloroform extracted, EtOH precipitated, and rehydrated in
20 ml TE (Tris/EDTA pH 8). The Zcyto21 fragment is then ligated
into the PmeI-AscI sites of the transgenic vector pTG12-8 and
transformed into E. coli DH10B.TM. competent cells by
electroporation. Vector pTG12-8 was derived from p2999B4 (Palmiter
et al., Mol Cell Biol 13:5266-5275, 1993) by insertion of a rat
insulin II intron (ca. 200 bp) and polylinker (Fse I/Pme I/Asc I)
into the Nru I site. The vector comprises a mouse metallothionein
(MT-1) promoter (ca. 750 bp) and human growth hormone (hGH)
untranslated region and polyadenylation signal (ca. 650 bp) flanked
by 10 kb of MT-1 5' flanking sequence and 7 kb of MT-1 3' flanking
sequence. The cDNA is inserted between the insulin II and hGH
sequences. Clones containing Zcyto21 are identified by plasmid DNA
miniprep followed by digestion with PmeI and AscI. A positive clone
is sequenced to insure that there were no deletions or other
anomalies in the construct.
[0147] DNA is prepared using a commercially available kit (Maxi
Kit, Qiagen, Inc.), and the Zcyto21 cDNA is released from the
pTG12-8 vector using PmeI and AscI enzymes. The cDNA is isolated on
a 1% low melting temperature agarose gel and excised from the gel.
The gel slice is melted at 70 .mu.C, and the DNA is extracted twice
with an equal volume of Tris-buffered phenol, precipitated with
EtOH, and resuspended in 10 .mu.l H.sub.2O.
[0148] The Zcyto21 cDNA is cloned into the EcoRV-AscI sites of a
modified pAdTrack-CMV (He, T-C. et al., Proc. Natl. Acad. Sci. USA
95:2509-2514, 1998). This construct contains the green fluorescent
protein (GFP) marker gene. The CMV promoter driving GFP expression
is replaced with the SV40 promoter, and the SV40 polyadenylation
signal is replaced with the human growth hormone polyadenylation
signal. In addition, the native polylinker is replaced with FseI,
EcoRV, and AscI sites. This modified form of pAdTrack-CMV is named
pZyTrack. Ligation is performed using a commercially available DNA
ligation and screening kit (Fast-Link.RTM. kit; Epicentre
Technologies, Madison, Wis.). Clones containing Zcyto21 are
identified by digestion of mini prep DNA with FseI and AscI. In
order to linearize the plasmid, approximately 5 .mu.g of the
resulting pZyTrack Zcyto21 plasmid is digested with PmeI.
Approximately 1 .mu.g of the linearized plasmid is cotransformed
with 200 ng of supercoiled pAdEasy (He et al., ibid.) into E. coli
BJ5183 cells (He et al., ibid.). The co-transformation is done
using a Bio-Rad Gene Pulser at 2.5 kV, 200 ohms and 25 .mu.Fa. The
entire co-transformation mixture is plated on 4 LB plates
containing 25 .mu.g/ml kanamycin. The smallest colonies are picked
and expanded in LB/kanamycin, and recombinant adenovirus DNA is
identified by standard DNA miniprep procedures. The recombinant
adenovirus miniprep DNA is transformed into E. coli DH10B.TM.
competent cells, and DNA is prepared using a Maxi Kit (Qiagen,
Inc.) according to kit instructions.
[0149] Approximately 5 .mu.g of recombinant adenoviral DNA is
digested with PacI enzyme (New England Biolabs) for 3 hours at
37.degree. C. in a reaction volume of 100 .mu.l containing 20-30 U
of PacI. The digested DNA is extracted twice with an equal volume
of phenol/chloroform and precipitated with ethanol. The DNA pellet
is resuspended in 10 .mu.l distilled water. A T25 flask of QBI-293A
cells (Quantum Biotechnologies, Inc. Montreal, Qc. Canada),
inoculated the day before and grown to 60-70% confluence, is
transfected with the PacI digested DNA. The PacI-digested DNA is
diluted up to a total volume of 50 .mu.l with sterile HBS (150 mM
NaCl, 20 mM HEPES). In a separate tube, 20 .mu.l of 1 mg/ml
N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts
(DOTAP) (Boehringer Mannheim, Indianapolis, Ind.) is diluted to a
total volume of 100 .mu.l with HBS. The DNA is added to the DOTAP,
mixed gently by pipeting up and down, and left at room temperature
for 15 minutes. The media is removed from the 293A cells and washed
with 5 ml serum-free minimum essential medium (MEM) alpha
containing 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino
acids, and 25 mM HEPES buffer (reagents obtained from Life
Technologies, Gaithersburg, Md.). 5 ml of serum-free MEM is added
to the 293A cells and held at 37.degree. C. The DNA/lipid mixture
is added drop-wise to the T25 flask of 293A cells, mixed gently,
and incubated at 37.degree. C. for 4 hours. After 4 hours the media
containing the DNA/lipid mixture is aspirated off and replaced with
5 ml complete MEM containing 5% fetal bovine serum. The transfected
cells are monitored for GFP expression and formation of foci (viral
plaques).
[0150] Seven days after transfection of 293A cells with the
recombinant adenoviral DNA, the cells express the GFP protein and
start to form foci (viral "plaques"). The crude viral lysate is
collected using a cell scraper to collect all of the 293A cells.
The lysate is transferred to a 50-ml conical tube. To release most
of the virus particles from the cells, three freeze/thaw cycles are
done in a dry ice/ethanol bath and a 37.degree. C. waterbath.
[0151] The crude lysate is amplified (Primary (1.degree.)
amplification) to obtain a working "stock" of Zcyto21 rAdV lysate.
Ten 10 cm plates of nearly confluent (80-90%) 293A cells are set up
20 hours previously, 200 ml of crude rAdV lysate is added to each
10-cm plate, and the cells are monitored for 48 to 72 hours for CPE
(cytopathic effect) under the white light microscope and expression
of GFP under the fluorescent microscope. When all of the 293A cells
show CPE, this stock lysate is collected and freeze/thaw cycles
performed as described above.
[0152] A secondary (2.degree.) amplification of Zcyto21 rAdV is
then performed. Twenty 15-cm tissue culture dishes of 293A cells
are prepared so that the cells are 80-90% confluent. All but 20 ml
of 5% MEM media is removed, and each dish is inoculated with
300-500 ml of the 1.degree. amplified rAdv lysate. After 48 hours
the 293A cells are lysed from virus production, the lysate is
collected into 250-ml polypropylene centrifuge bottles, and the
rAdV is purified.
[0153] NP-40 detergent is added to a final concentration of 0.5% to
the bottles of crude lysate in order to lyse all cells. Bottles are
placed on a rotating platform for 10 minutes agitating as fast as
possible without the bottles falling over. The debris is pelleted
by centrifugation at 20,000.times.G for 15 minutes. The supernatant
is transferred to 250-ml polycarbonate centrifuge bottles, and 0.5
volume of 20% PEG8000/2.5 M NaCl solution is added. The bottles are
shaken overnight on ice. The bottles are centrifuged at
20,000.times.G for 15 minutes, and the supernatant is discarded
into a bleach solution. Using a sterile cell scraper, the white,
virus/PEG precipitate from 2 bottles is resuspended in 2.5 ml PBS.
The resulting virus solution is placed in 2-ml microcentrifuge
tubes and centrifuged at 14,000.times.G in the microcentrifuge for
10 minutes to remove any additional cell debris. The supernatant
from the 2-ml microcentrifuge tubes is transferred into a 15-ml
polypropylene snapcap tube and adjusted to a density of 1.34 g/ml
with CsCl. The solution is transferred to 3.2-ml, polycarbonate,
thick-walled centrifuge tubes and spun at 348,000.times.G for 3-4
hours at 25 .mu.C. The virus forms a white band. Using wide-bore
pipette tips, the virus band is collected.
[0154] A commercially available ion-exchange columns (e.g., PD-10
columns prepacked with Sephadex.RTM. G-25M; Pharmacia Biotech,
Piscataway, N.J.) is used to desalt the virus preparation. The
column is equilibrated with 20 ml of PBS. The virus is loaded and
allowed to run into the column. 5 ml of PBS is added to the column,
and fractions of 8-10 drops are collected. The optical densities of
1:50 dilutions of each fraction are determined at 260 nm on a
spectrophotometer. Peak fractions are pooled, and the optical
density (OD) of a 1:25 dilution is determined. OD is converted to
virus concentration using the formula: (OD at 260
nm)(25)(1.1.times.10.sup.12)=virions/ml.
[0155] To store the virus, glycerol is added to the purified virus
to a final concentration of 15%, mixed gently but effectively, and
stored in aliquots at -80 .mu.C.
[0156] A protocol developed by Quantum Biotechnologies, Inc.
(Montreal, Canada) is followed to measure recombinant virus
infectivity. Briefly, two 96-well tissue culture plates are seeded
with 1.times.10.sup.4 293A cells per well in MEM containing 2%
fetal bovine serum for each recombinant virus to be assayed. After
24 hours 10-fold dilutions of each virus from 1.times.-2 to
1.times.10.sup.-14 are made in MEM containing 2% fetal bovine
serum. 100 .mu.l of each dilution is placed in each of 20 wells.
After 5 days at 37.degree. C., wells are read either positive or
negative for CPE, and a value for "Plaque Forming Units/ml" (PFU)
is calculated.
Example 5
[0157] Trangenic animals expressing Zcyto21 genes are producing
using adult, fertile males (studs) (B6C3fl, 2-8 months of age
(Taconic Farms, Germantown, N.Y.)), vasectomized males (duds) (CD1,
2-8 months, (Taconic Farms)), prepubescent fertile females (donors)
(B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females
(recipients) (CD1, 2-4 months, (Taconic Farms)).
[0158] The donors are acclimated for 1 week and then injected with
approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin
(Sigma, St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse of
human Chorionic Gonadotropin (hCG (Sigma)) I.P. to induce
superovulation. Donors are mated with studs subsequent to hormone
injections. Ovulation generally occurs within 13 hours of hCG
injection. Copulation is confirmed by the presence of a vaginal
plug the morning following mating.
[0159] Fertilized eggs are collected under a surgical scope (Leica
MZ12 Stereo Microscope, Leica, Wetzlar, Del.). The oviducts are
collected and eggs are released into urinanalysis slides containing
hyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and
twice in Whitten's W640 medium (Table 4) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are then stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
[0160] 10-20 micrograms of plasmid DNA containing a cDNA of the
Zcyto21 gene is linearized, gel-purified, and resuspended in 10 mM
Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a final concentration of 5-10
nanograms per microliter for microinjection.
[0161] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0162] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pregassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0163] The following day, 12-17 healthy 2-cell embryos from the
previous day's injection are transferred into the recipient. The
swollen ampulla is located and holding the oviduct between the
ampulla and the bursa, a nick in the oviduct is made with a 28 g
needle close to the bursa, making sure not to tear the ampulla or
the bursa. The embryos are implanted through this nick, and by
holding onto the peritoneal wall, the reproductive organs are
guided back into the abdominal cavity.
[0164] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0165] Genomic DNA is prepared from the tail snips using a Qiagen
Dneasy kit following the manufacturer's instructions. Genomic DNA
is analyzed by PCR using primers designed to the human growth
hormone (hGH) 3' UTR portion of the transgenic vector. A region
unique to the human sequence was identified from an alignment of
the human and mouse growth hormone 3' UTR DNA sequences, ensuring
that the PCR reaction does not amplify the mouse sequence. Primers
which amplify a 368 base pair fragment of hGH and primers which
hybridize to vector sequences and amplify the cDNA insert, are
often used along with the hGH primers. In these experiments, DNA
from animals positive for the transgene will generate two bands, a
368 base pair band corresponding to the hGH 3' UTR fragment and a
band of variable size corresponding to the cDNA insert.
[0166] Once animals are confirmed to be transgenic (TG), they may
be back-crossed into an inbred strain by placing a TG female with a
wild-type male, or a TG male with one or two wild-type female(s).
As pups are born and weaned, the sexes are separated, and their
tails snipped for genotyping.
[0167] Analysis of the mRNA expression level of each transgene is
done using an RNA solution hybridization assay or real-time PCR on
an ABI Prism 7700 (PE Applied Biosystems, Inc., Foster City,
Calif.) following manufacturer's instructions. TABLE-US-00005 TABLE
5 WHITTEN'S 640 MEDIA mgs/200 m mgs/500/ml NaCl 1280 3200 KCl 72
180 KH.sub.2PO.sub.4 32 80 MgSO.sub.4.cndot.7H.sub.2O 60 150
Glucose 200 500 Ca.sup.2+ Lactate 106 265 K Penn 15 37.5
Streptomycin SO.sub.4 10 25 NaHCO.sub.3 380 950 Na Pyruvate 5 12.5
H.sub.20 200 500 EDTA 100 .mu.l 250 .mu.l 5% Phenol Red 200 .mu.l
500 .mu.l BSA 600 1500 All reagents are available from Sigma.
Example 6
1. Stimulation of Expression from an Interferon-Responsive
Promoter
[0168] In one series of experiments, conditioned medium (CM)
containing Zcyto21 protein is generated by infecting 293A cells
with recombinant adenovirus containing the cDNA for Zcyto21
(AdZy-Zcyto21) at a multiplicity of infection of 400 particles per
cell. CM is harvested at time points between 40 hours post
infection and stored at -20.degree. C. CM is also generated from an
infection with a recombinant adenovirus lacking a cDNA
(AdZy-parental). Prior to use, a portion of the CM is concentrated
14 fold in a Millipore Ultrafree-15 (5,000 nominal molecular weight
limit) centrifugal filter, and then, filtered through a Millipore
Ultrafree-15 (100,000 nominal molecular weight limit) centrifugal
filter to reduce the amount of viral particles present in the
media, and finally filtered through a Millipore 0.2 .mu.m syringe
filter to sterilize the CM. Concentrated CM samples are diluted 1:2
in binding buffer and incubated with cells from a murine cell line
for 5 hours at 37.degree. C.
2. Anti-Viral Activity of Zcyto21
[0169] Another series of experiments examines the anti-viral
activity of Zcyto21. In these studies, the anti-viral assay is
performed by plating L929 cells (ATCC No. CCL-1) in growth media
RPMI medium 1640 containing 10% fetal bovine serum, penicillin,
streptomycin, and L-glutamine in 96-well format at 50,000 cells per
well. Adenovirus CM from 293A cells infected with either
AdZy-Zcyto21m or AdZy-parental, as described above, are incubated
with cells overnight. A positive control in the assay is provided
by murine interferon-.alpha. serially diluted 1:10, starting at 100
ng/ml. L929 cells with growth media alone provided the negative
control. Treated cells are incubated for 24 hours. The media are
discarded, fresh medium are added, and encephalomyocarditis virus
(ATCC No. vr129b) is introduced at a multiplicity of infection of
0.1 (i.e., one virus particle for every ten L929 cells). The cells
are incubated in the presence of the virus for 24 hours, and then,
the wells are scored for percent cytopathic effect (CPE
3. Antiproliferation Assay Using a BAF3 Cell Line
[0170] BaF3 is used to determine if Zcyto21 has anti-proliferative
properties. Baby hamster kidney (BHK) cells are stably transfected
with an expression vector containing the CMV promoter plus intron A
upstream of the Zcyto21 cDNA or an unrelated cDNA, called
Z.alpha.30, using BRL lipofectamine. Stably transfected cells are
seeded in a cell factory in serum free media and allowed to grow
for three days before conditioned media is harvested and
concentrated in a 5 K filter to 10.times.. Concentrated conditioned
medium samples are stored at 4.degree. C.
[0171] The following assay is used to test for anti-proliferation
of BaF3. In a 96 well plate, eight 1:2 serial dilutions are made of
growth media alone (RPMI 1640, 10% fetal bovine serum, 1 mM sodium
pyruvate, 2 mM L-glutamine), or murine IL-3 (starting at 50 pg/ml
in growth medium) with final volume of 100 .mu.l. Fifty microliters
of the following are added to both growth media alone or mIL-3
diluted lanes: human interferon-.alpha. (100 ng/ml, 10 ng/ml, or 1
ng/ml diluted in growth medium), human interferon-.beta. (100
ng/ml, 10 ng/ml, or 1 ng/ml diluted in growth medium), murine
interferon-.alpha. (100 ng/ml, 10 ng/ml, or 1 ng/ml diluted in
growth medium), murine interferon-.beta. (100 ng/ml, 10 ng/ml, or 1
ng/ml diluted in growth medium), Zcyto21 (at 2.5.times.,
0.5.times., or 0.1.times.), and murine Z.alpha.30 (at 2.5.times.,
0.5.times., or 0.1.times.).
[0172] The BaF3cell line is washed three times in growth medium,
pellets are resuspended in growth medium, cells are counted and
diluted in growth medium to 5,000 cells/50 .mu.l. Fifty microliters
of diluted cells are then added to each dilution of samples. Assay
plates are incubated in a 37.degree. C. incubator for three to four
days. Twenty microliters of Alomar blue are then added to each well
and the plate are incubated overnight at 37.degree. C. The plates
are read on the fluorescent plate reader at excitation wavelength
of 544 and emission wavelength 590.
Example 7
[0173] Tissue Distribution of Human Zcyto21 in Tissue Panels Using
PCR
[0174] A panel of cDNA samples from human tissues was screened for
Zcyto21 expression using PCR. The panel was made in house and
contained 77 marathon cDNA and cDNA samples from various norman and
cancerous human tissues and cell lines as shown in Table 6, below.
The cDNA samples came from in-house libraries or marathon cDNA
preparations of RNA that were prepared in-house, or from a
commercial supplier such as Clontech (Palo Alto, Calif.) or
Invitrogen (Carlsbad, Calif.). The marathon cDNAs were made using
the Marathon cDNA Amplification Kit (Clontech). To assure quality
of the panel samples, three tests for quality control (QC) were
run: (1) To assess the RNA quality used for the libraries, the
in-house cDNAs were tested for average insert size by PCR with
vector oligos that were specific for the vector sequences for an
individual cDNA library; (2) Standardization of the concentration
of the cDNA in panel samples was achieved using standard PCR
methods to amplify full length alpha tubulin or G3PDH cDNA; and (3)
a sample was sent to sequencing to check for possible ribosomal or
mitochondrial DNA contamination. The panel was set up in a 96-well
format that included a human genomic DNA (Clontech) positive
control sample. Each well contained approximately 0.2-100 pg/.mu.l
of cDNA. The first PCR reactions were set up using oligos ZC39,270
(SEQ ID NO:14) and ZC39,272 (SEQ ID NO:15), Advantage 2 DNA
Polymerase Mix (Clontech) and Rediload dye (Research Genetics,
Inc., Huntsville, Ala.). The amplification was carried out as
follows: 1 cycle at 94.degree. for 1 minute then 35 cycles of
94.degree., 10 seconds; 67.degree., 45 seconds and ended with a 3
minute final extension at 72.degree.. The correct DNA fragment size
was observed in brain, islet, prostate, testis, pituitary,
placenta, ovarian tumor, lung tumor, CD3+ and HPVS. Another PCR
reaction was set up using oligos ZC39,270 (SEQ ID NO:14) and
ZC39,271 (SEQ ID NO:16), Advangtage 2 DNA Polymerase Mix (Clontech)
and Rediload dye (Research Genetics). The amplification was carried
out as follows: 1 cycle at 94.degree., 1 minute then 35 cycles of
94.degree., 10 seconds; 65.degree., 30 seconds, 72.degree., 30
seconds and ended with a 3 minute extension at 72.degree.. The
correct DNA fragment size was observed in pituitary, rectal tumor
and ovarian tumor. TABLE-US-00006 TABLE 6 #samples #samples Tissue
tested Tissue tested adrenal gland 1 bladder 1 bone marrow 3 brain
2 cervix 1 colon 1 fetal brain 3 fetal heart 2 fetal kidney 1 fetal
liver 2 fetal lung 1 fetal skin 1 heart 2 fetal muscle 1 kidney 2
liver 1 lung 1 lymph node 1 mammary gland 1 melanoma 1 ovary 1
pancreas 1 pituitary 2 placenta 3 prostate 3 rectum 1 salivary
gland 2 skeletal muscle 1 small intestine 1 spinal cord 2 spleen 1
uterus 1 stomach 1 adipocyte library 1 testis 5 islet 1 thymus 1
prostate SMC 1 thyroid 2 RPMI 1788 (ATCC # CCL-156) 1 trachea 1
WI38 (ATCC # CCL-75) 1 esophageal tumor 1 lung tumor 1 liver tumor
1 ovarian tumor 1 rectal tumor 1 stomach tumor 1 uterine tumor 2
CD3+ library selected PBMC's 1 (stimulated) HaCAT library 1 HPV
library(ATCC # CRL-2221) 1 HPVS library (ATCC # CRL- 1 MG63 library
1 2221) - selected K562 (ATCC#CCL-243) 1
[0175] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
16 1 603 DNA Homo sapiens CDS (1)...(603) 1 atg gct gca gct tgg acc
gtg gtg ctg gtg act ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr
Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 gcc gtg gca ggc
cct gtc ccc act tcc aag ccc acc aca act ggg aag 96 Ala Val Ala Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 ggc tgc
cac att ggc agg ttc aaa tct ctg tca cca cag gag cta gcg 144 Gly Cys
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45
agc ttc aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa 192
Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50
55 60 aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg
agg 240 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg 65 70 75 80 ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct
gag ctg gcc 288 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala 85 90 95 ctg acg ctg aag gtc ctg gag gcc gct gct ggc
cca gcc ctg gag gac 336 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu Asp 100 105 110 gtc cta gac cag ccc ctt cac acc ctg
cac cac atc ctc tcc cag ctc 384 Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln Leu 115 120 125 cag gcc tgt atc cag cct cag
ccc aca gca ggg ccc agg ccc cgg ggc 432 Gln Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140 cgc ctc cac cac tgg
ctg cac cgg ctc cag gag gcc ccc aaa aag gag 480 Arg Leu His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 tcc gct
ggc tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc 528 Ser Ala
Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175
ctc acg cga gac ctc aaa tat gtg gcc gat ggg gac ctg tgt ctg aga 576
Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg 180
185 190 acg tca acc cac cct gag tcc acc tga 603 Thr Ser Thr His Pro
Glu Ser Thr * 195 200 2 200 PRT Homo sapiens 2 Met Ala Ala Ala Trp
Thr Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala
Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly
Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40
45 Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys
50 55 60 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp
Leu Arg 65 70 75 80 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu
Ala Glu Leu Ala 85 90 95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala
Gly Pro Ala Leu Glu Asp 100 105 110 Val Leu Asp Gln Pro Leu His Thr
Leu His His Ile Leu Ser Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro
Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140 Arg Leu His His
Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 Ser
Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170
175 Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asp Leu Cys Leu Arg
180 185 190 Thr Ser Thr His Pro Glu Ser Thr 195 200 3 600 DNA
Artificial Sequence degenerate sequence misc_feature (1)...(600) n
= A,T,C or G 3 atggcngcng cntggacngt ngtnytngtn acnytngtny
tnggnytngc ngtngcnggn 60 ccngtnccna cnwsnaarcc nacnacnacn
ggnaarggnt gycayathgg nmgnttyaar 120 wsnytnwsnc cncargaryt
ngcnwsntty aaraargcnm gngaygcnyt ngargarwsn 180 ytnaarytna
araaytggws ntgywsnwsn ccngtnttyc cnggnaaytg ggayytnmgn 240
ytnytncarg tnmgngarmg nccngtngcn ytngargcng arytngcnyt nacnytnaar
300 gtnytngarg cngcngcngg nccngcnytn gargaygtny tngaycarcc
nytncayacn 360 ytncaycaya thytnwsnca rytncargcn tgyathcarc
cncarccnac ngcnggnccn 420 mgnccnmgng gnmgnytnca ycaytggytn
caymgnytnc argargcncc naaraargar 480 wsngcnggnt gyytngargc
nwsngtnacn ttyaayytnt tymgnytnyt nacnmgngay 540 ytnaartayg
tngcngaygg ngayytntgy ytnmgnacnw snacncaycc ngarwsnacn 600 4 603
DNA Homo sapiens CDS (1)...(603) 4 atg gct gca gct tgg acc gtg gtg
ctg gtg act ttg gtg cta ggc ttg 48 Met Ala Ala Ala Trp Thr Val Val
Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 gcc gtg gca ggc cct gtc
ccc act tcc aag ccc acc aca act ggg aag 96 Ala Val Ala Gly Pro Val
Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 ggc tgc cac att
ggc agg ttc aaa tct ctg tca cca cag gag cta gcg 144 Gly Cys His Ile
Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 agc ttc
aag aag gcc agg gac gcc ttg gaa gag tca ctc aag ctg aaa 192 Ser Phe
Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60
aac tgg agt tgc agc tct cct gtc ttc ccc ggg aat tgg gac ctg agg 240
Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65
70 75 80 ctt ctc cag gtg agg gag cgc cct gtg gcc ttg gag gct gag
ctg gcc 288 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu
Leu Ala 85 90 95 ctg acg ctg aag gtc ctg gag gcc gct gct ggc cca
gcc ctg gag gac 336 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro
Ala Leu Glu Asp 100 105 110 gtc cta gac cag ccc ctt cac acc ctg cac
cac atc ctc tcc cag ctc 384 Val Leu Asp Gln Pro Leu His Thr Leu His
His Ile Leu Ser Gln Leu 115 120 125 cag gcc tgt atc cag cct cag ccc
aca gca ggg ccc agg ccc cgg ggc 432 Gln Ala Cys Ile Gln Pro Gln Pro
Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140 cgc ctc cac cac tgg ctg
cac cgg ctc cag gag gcc ccc aaa aag gag 480 Arg Leu His His Trp Leu
His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 tcc gct ggc
tgc ctg gag gca tct gtc acc ttc aac ctc ttc cgc ctc 528 Ser Ala Gly
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175 ctc
acg cga gac ctc aaa tat gtg gcc gat ggg aac ctg tgt ctg aga 576 Leu
Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg 180 185
190 acg tca acc cac cct gag tcc acc tga 603 Thr Ser Thr His Pro Glu
Ser Thr * 195 200 5 200 PRT Homo sapiens 5 Met Ala Ala Ala Trp Thr
Val Val Leu Val Thr Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala Gly
Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly Cys
His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45
Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50
55 60 Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu
Arg 65 70 75 80 Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala
Glu Leu Ala 85 90 95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly
Pro Ala Leu Glu Asp 100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu
His His Ile Leu Ser Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro Gln
Pro Thr Ala Gly Pro Arg Pro Arg Gly 130 135 140 Arg Leu His His Trp
Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu 145 150 155 160 Ser Ala
Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175
Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg 180
185 190 Thr Ser Thr His Pro Glu Ser Thr 195 200 6 856 DNA Homo
sapiens CDS (98)...(700) 6 aattaccttt tcactttaca cacatcatct
tggattgccc attttgcgtg gctaaaaagc 60 agagccatgc cgctggggaa
gcagttgcga tttagcc atg gct gca gct tgg acc 115 Met Ala Ala Ala Trp
Thr 1 5 gtg gtg ctg gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct
gtc 163 Val Val Leu Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro
Val 10 15 20 ccc act tcc aag ccc acc aca act ggg aag ggc tgc cac
att ggc agg 211 Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His
Ile Gly Arg 25 30 35 ttc aaa tct ctg tca cca cag gag cta gcg agc
ttc aag aag gcc agg 259 Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser
Phe Lys Lys Ala Arg 40 45 50 gac gcc ttg gaa gag tca ctc aag ctg
aaa aac tgg agt tgc agc tct 307 Asp Ala Leu Glu Glu Ser Leu Lys Leu
Lys Asn Trp Ser Cys Ser Ser 55 60 65 70 cct gtc ttc ccc ggg aat tgg
gac ctg agg ctt ctc cag gtg agg gag 355 Pro Val Phe Pro Gly Asn Trp
Asp Leu Arg Leu Leu Gln Val Arg Glu 75 80 85 cgc cct gtg gcc ttg
gag gct gag ctg gcc ctg acg ctg aag gtc ctg 403 Arg Pro Val Ala Leu
Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu 90 95 100 gag gcc gct
gct ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt 451 Glu Ala Ala
Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu 105 110 115 cac
acc ctg cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct 499 His
Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro 120 125
130 cag ccc aca gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg
547 Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu
135 140 145 150 cac cgg ctc cag gag gcc ccc aaa aag gag tcc gct ggc
tgc ctg gag 595 His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
Cys Leu Glu 155 160 165 gca tct gtc acc ttc aac ctc ttc cgc ctc ctc
acg cga gac ctc aaa 643 Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg Asp Leu Lys 170 175 180 tat gtg gcc gat ggg aac ctg tgt ctg
aga acg tca acc cac cct gag 691 Tyr Val Ala Asp Gly Asn Leu Cys Leu
Arg Thr Ser Thr His Pro Glu 185 190 195 tcc acc tga caccccacac
cttatttatg cgctgagccc tactccttcc 740 Ser Thr * 200 ttaatttatt
tcctctcacc ctttatttat gaagctgcag ccctgactga gacatagggc 800
tgagtttatt gttttacttt tatacattat gcacaaataa acaacaagga attgga 856 7
200 PRT Homo sapiens 7 Met Ala Ala Ala Trp Thr Val Val Leu Val Thr
Leu Val Leu Gly Leu 1 5 10 15 Ala Val Ala Gly Pro Val Pro Thr Ser
Lys Pro Thr Thr Thr Gly Lys 20 25 30 Gly Cys His Ile Gly Arg Phe
Lys Ser Leu Ser Pro Gln Glu Leu Ala 35 40 45 Ser Phe Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys 50 55 60 Asn Trp Ser
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg 65 70 75 80 Leu
Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala 85 90
95 Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
100 105 110 Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser
Gln Leu 115 120 125 Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg Pro Arg Gly 130 135 140 Arg Leu His His Trp Leu His Arg Leu Gln
Glu Ala Pro Lys Lys Glu 145 150 155 160 Ser Ala Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu Phe Arg Leu 165 170 175 Leu Thr Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg 180 185 190 Thr Ser Thr
His Pro Glu Ser Thr 195 200 8 676 DNA Homo sapiens CDS (17)...(676)
8 gccctcggcc aattgg atg gtg ccc acc aca ttg gca gag cca tgc cgc tgg
52 Met Val Pro Thr Thr Leu Ala Glu Pro Cys Arg Trp 1 5 10 gga agc
agt tgc gat tta gcc atg gct gca gct tgg acc gtg gtg ctg 100 Gly Ser
Ser Cys Asp Leu Ala Met Ala Ala Ala Trp Thr Val Val Leu 15 20 25
gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct gtc ccc act tcc 148
Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro Val Pro Thr Ser 30
35 40 aag ccc acc aca act ggg aag ggc tgc cac att ggc agg ttc aaa
tct 196 Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
Ser 45 50 55 60 ctg tca cca cag gag cta gcg agc ttc aag aag gcc agg
gac gcc ttg 244 Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg
Asp Ala Leu 65 70 75 gaa gag tca ctc aag ctg aaa aac tgg agt tgc
agc tct cct gtc ttc 292 Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys
Ser Ser Pro Val Phe 80 85 90 ccc ggg aat tgg gac ctg agg ctt ctc
cag gtg agg gag cgc cct gtg 340 Pro Gly Asn Trp Asp Leu Arg Leu Leu
Gln Val Arg Glu Arg Pro Val 95 100 105 gcc ttg gag gct gag ctg gcc
ctg acg ctg aag gtc ctg gag gcc gct 388 Ala Leu Glu Ala Glu Leu Ala
Leu Thr Leu Lys Val Leu Glu Ala Ala 110 115 120 gct ggc cca gcc ctg
gag gac gtc cta gac cag ccc ctt cac acc ctg 436 Ala Gly Pro Ala Leu
Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu 125 130 135 140 cac cac
atc ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc aca 484 His His
Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr 145 150 155
gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg ctg cac cgg ctc 532
Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu 160
165 170 cag gag gcc ccc aaa aag gag tcc gct ggc tgc ctg gag gca tct
gtc 580 Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
Val 175 180 185 acc ttc aac ctc ttc cgc ctc ctc acg cga gac ctc aaa
tat gtg gcc 628 Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys
Tyr Val Ala 190 195 200 gat ggg gac ctg tgt ctg aga acg tca acc cac
cct gag tcc acc tga 676 Asp Gly Asp Leu Cys Leu Arg Thr Ser Thr His
Pro Glu Ser Thr * 205 210 215 9 219 PRT Homo sapiens 9 Met Val Pro
Thr Thr Leu Ala Glu Pro Cys Arg Trp Gly Ser Ser Cys 1 5 10 15 Asp
Leu Ala Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val 20 25
30 Leu Gly Leu Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr
35 40 45 Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser
Pro Gln 50 55 60 Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu
Glu Glu Ser Leu 65 70 75 80 Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro
Val Phe Pro Gly Asn Trp 85 90 95 Asp Leu Arg Leu Leu Gln Val Arg
Glu Arg Pro Val Ala Leu Glu Ala 100 105 110 Glu Leu Ala Leu Thr Leu
Lys Val Leu Glu Ala Ala Ala Gly Pro Ala 115 120 125 Leu Glu Asp Val
Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu 130 135 140 Ser Gln
Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg 145 150 155
160 Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro
165 170 175 Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe
Asn Leu 180 185 190 Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala
Asp Gly Asp Leu 195 200 205 Cys Leu Arg Thr Ser Thr His Pro Glu Ser
Thr 210 215 10 660 DNA Artificial Sequence degenerate sequence
misc_feature (1)...(660) n = A,T,C or G 10 atggtnccna cnacnytngc
ngarccntgy mgntggggnw snwsntgyga yytngcnatg 60 gcngcngcnt
ggacngtngt nytngtnacn ytngtnytng gnytngcngt ngcnggnccn 120
gtnccnacnw snaarccnac nacnacnggn aarggntgyc ayathggnmg nttyaarwsn
180 ytnwsnccnc argarytngc nwsnttyaar aargcnmgng aygcnytnga
rgarwsnytn 240 aarytnaara aytggwsntg ywsnwsnccn gtnttyccng
gnaaytggga yytnmgnytn 300 ytncargtnm gngarmgncc ngtngcnytn
gargcngary tngcnytnac nytnaargtn 360 ytngargcng cngcnggncc
ngcnytngar gaygtnytng aycarccnyt ncayacnytn 420 caycayathy
tnwsncaryt ncargcntgy athcarccnc arccnacngc
nggnccnmgn 480 ccnmgnggnm gnytncayca ytggytncay mgnytncarg
argcnccnaa raargarwsn 540 gcnggntgyy tngargcnws ngtnacntty
aayytnttym gnytnytnac nmgngayytn 600 aartaygtng cngayggnga
yytntgyytn mgnacnwsna cncayccnga rwsnacntrr 660 11 628 DNA Homo
sapiens CDS (17)...(628) 11 gccctcggcc aattgg atg gtg ccc acc aca
ttg gct tgg acc gtg gtg ctg 52 Met Val Pro Thr Thr Leu Ala Trp Thr
Val Val Leu 1 5 10 gtg act ttg gtg cta ggc ttg gcc gtg gca ggc cct
gtc ccc act tcc 100 Val Thr Leu Val Leu Gly Leu Ala Val Ala Gly Pro
Val Pro Thr Ser 15 20 25 aag ccc acc aca act ggg aag ggc tgc cac
att ggc agg ttc aaa tct 148 Lys Pro Thr Thr Thr Gly Lys Gly Cys His
Ile Gly Arg Phe Lys Ser 30 35 40 ctg tca cca cag gag cta gcg agc
ttc aag aag gcc agg gac gcc ttg 196 Leu Ser Pro Gln Glu Leu Ala Ser
Phe Lys Lys Ala Arg Asp Ala Leu 45 50 55 60 gaa gag tca ctc aag ctg
aaa aac tgg agt tgc agc tct cct gtc ttc 244 Glu Glu Ser Leu Lys Leu
Lys Asn Trp Ser Cys Ser Ser Pro Val Phe 65 70 75 ccc ggg aat tgg
gac ctg agg ctt ctc cag gtg agg gag cgc cct gtg 292 Pro Gly Asn Trp
Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val 80 85 90 gcc ttg
gag gct gag ctg gcc ctg acg ctg aag gtc ctg gag gcc gct 340 Ala Leu
Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala 95 100 105
gct ggc cca gcc ctg gag gac gtc cta gac cag ccc ctt cac acc ctg 388
Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu 110
115 120 cac cac atc ctc tcc cag ctc cag gcc tgt atc cag cct cag ccc
aca 436 His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
Thr 125 130 135 140 gca ggg ccc agg ccc cgg ggc cgc ctc cac cac tgg
ctg cac cgg ctc 484 Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp
Leu His Arg Leu 145 150 155 cag gag gcc ccc aaa aag gag tcc gct ggc
tgc ctg gag gca tct gtc 532 Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
Cys Leu Glu Ala Ser Val 160 165 170 acc ttc aac ctc ttc cgc ctc ctc
acg cga gac ctc aaa tat gtg gcc 580 Thr Phe Asn Leu Phe Arg Leu Leu
Thr Arg Asp Leu Lys Tyr Val Ala 175 180 185 gat ggg gac ctg tgt ctg
aga acg tca acc cac cct gag tcc acc tga 628 Asp Gly Asp Leu Cys Leu
Arg Thr Ser Thr His Pro Glu Ser Thr * 190 195 200 12 203 PRT Homo
sapiens 12 Met Val Pro Thr Thr Leu Ala Trp Thr Val Val Leu Val Thr
Leu Val 1 5 10 15 Leu Gly Leu Ala Val Ala Gly Pro Val Pro Thr Ser
Lys Pro Thr Thr 20 25 30 Thr Gly Lys Gly Cys His Ile Gly Arg Phe
Lys Ser Leu Ser Pro Gln 35 40 45 Glu Leu Ala Ser Phe Lys Lys Ala
Arg Asp Ala Leu Glu Glu Ser Leu 50 55 60 Lys Leu Lys Asn Trp Ser
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp 65 70 75 80 Asp Leu Arg Leu
Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala 85 90 95 Glu Leu
Ala Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala 100 105 110
Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu 115
120 125 Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro
Arg 130 135 140 Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu Gln
Glu Ala Pro 145 150 155 160 Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala
Ser Val Thr Phe Asn Leu 165 170 175 Phe Arg Leu Leu Thr Arg Asp Leu
Lys Tyr Val Ala Asp Gly Asp Leu 180 185 190 Cys Leu Arg Thr Ser Thr
His Pro Glu Ser Thr 195 200 13 612 DNA Artificial Sequence
degenerate sequence misc_feature (1)...(612) n = A,T,C or G 13
atggtnccna cnacnytngc ntggacngtn gtnytngtna cnytngtnyt nggnytngcn
60 gtngcnggnc cngtnccnac nwsnaarccn acnacnacng gnaarggntg
ycayathggn 120 mgnttyaarw snytnwsncc ncargarytn gcnwsnttya
araargcnmg ngaygcnytn 180 gargarwsny tnaarytnaa raaytggwsn
tgywsnwsnc cngtnttycc nggnaaytgg 240 gayytnmgny tnytncargt
nmgngarmgn ccngtngcny tngargcnga rytngcnytn 300 acnytnaarg
tnytngargc ngcngcnggn ccngcnytng argaygtnyt ngaycarccn 360
ytncayacny tncaycayat hytnwsncar ytncargcnt gyathcarcc ncarccnacn
420 gcnggnccnm gnccnmgngg nmgnytncay caytggytnc aymgnytnca
rgargcnccn 480 aaraargarw sngcnggntg yytngargcn wsngtnacnt
tyaayytntt ymgnytnytn 540 acnmgngayy tnaartaygt ngcngayggn
gayytntgyy tnmgnacnws nacncayccn 600 garwsnacnt rr 612 14 23 DNA
Artificial Sequence oligonucleotide ZC39270 14 aggcggaaga
ggttgaaggt gac 23 15 22 DNA Artificial Sequence oligonucleotide
ZC39272 15 cgtcctagac cagccccttc ac 22 16 22 DNA Artificial
Sequence oligonucleotide ZC39271 16 gaccgtggtg ctggtgactt tg 22
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