U.S. patent application number 11/549772 was filed with the patent office on 2007-05-03 for cytokine zalpha11 ligand.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Stacey R. Dillon, Donald C. Foster, Jane A. Gross, Angela K. Hammond, Richard D. Holly, Janet V. Johnston, Andrew J. Nelson, JULIA E. NOVAK, Scott R. Presnell, Cindy A. Sprecher.
Application Number | 20070099269 11/549772 |
Document ID | / |
Family ID | 27494488 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070099269 |
Kind Code |
A1 |
NOVAK; JULIA E. ; et
al. |
May 3, 2007 |
CYTOKINE ZALPHA11 LIGAND
Abstract
Antibodies that bind to polypeptides and peptides comprising the
sequence of zalpha11 Ligand as shown in SEQ ID NO: 2 are described.
The antibodies may bind the full length sequence of 162 amino acid
residues or a fragment thereof, including a mature polypeptide of
131 amino acid residues and smaller polypeptide and peptide
sequences. The antibodies may include antibodies that are
polyclonal, monoclonal, murine, humanized or neutralizing. Methods
for producing the antibodies are also described.
Inventors: |
NOVAK; JULIA E.; (Bainbridge
Island, WA) ; Presnell; Scott R.; (Tacoma, WA)
; Sprecher; Cindy A.; (Seattle, WA) ; Foster;
Donald C.; (Lake Forest Park, WA) ; Holly; Richard
D.; (Seattle, WA) ; Gross; Jane A.; (Seattle,
WA) ; Johnston; Janet V.; (Seattle, WA) ;
Nelson; Andrew J.; (Shoreline, WA) ; Dillon; Stacey
R.; (Seattle, WA) ; Hammond; Angela K.; (Maple
Valley, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
27494488 |
Appl. No.: |
11/549772 |
Filed: |
October 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11346580 |
Feb 2, 2006 |
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11549772 |
Oct 16, 2006 |
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10295723 |
Nov 15, 2002 |
6686178 |
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11346580 |
Feb 2, 2006 |
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09923246 |
Aug 3, 2001 |
6605272 |
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10295723 |
Nov 15, 2002 |
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09522217 |
Mar 9, 2000 |
6307024 |
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09923246 |
Aug 3, 2001 |
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60123547 |
Mar 9, 1999 |
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60123904 |
Mar 11, 1999 |
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60142013 |
Jul 1, 1999 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/488; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/5406 20130101;
C07K 14/52 20130101; C07K 14/001 20130101; A01K 2217/05 20130101;
A61K 38/00 20130101; C07K 16/2866 20130101; C07K 16/24 20130101;
G01N 2333/52 20130101; A61P 7/00 20180101; C07K 14/54 20130101;
C12N 15/62 20130101; A61P 37/02 20180101; C07K 2319/55 20130101;
C12N 2799/026 20130101; A61P 35/02 20180101; A01K 2267/03 20130101;
A61K 38/18 20130101; C07K 14/715 20130101; C07K 2317/73 20130101;
C07K 2319/24 20130101; C07K 2319/30 20130101; A01K 67/0275
20130101; A01K 2227/105 20130101; C07K 2319/00 20130101; C12N
15/8509 20130101; C07K 14/535 20130101; C07K 2319/21 20130101; A61P
35/00 20180101; A61K 38/18 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/252.3; 435/488; 530/350; 536/023.5 |
International
Class: |
C07K 14/705 20070101
C07K014/705; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 15/74 20060101 C12N015/74; C12N 1/21 20070101
C12N001/21 |
Claims
1. An isolated polynucleotide molecule comprising a sequence of
nucleotides that encodes a polypeptide comprising residues 41 to
148 of SEQ ID NO:2, wherein the polypeptide binds a zalpha11
receptor as shown in SEQ ID NO: 115.
2. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment that
encodes a polypeptide comprising residues 41 to 148 of SEQ ID NO:2,
wherein the polypeptide binds a zalpha11 receptor as shown in SEQ
ID NO:115; and (c) a transcription terminator.
3. A cultured cell comprising the expression vector of claim 2.
4. The cultured cell of claim 3, wherein the cultured cell is a
prokaryotic cell.
5. The prokaryotic cell of claim 4, wherein the prokaryotic cell is
an E. coli cell.
6. A process comprising culturing the cell of claim 3 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
7. A process comprising culturing the cell of claim 4 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
8. A process comprising culturing the cell of claim 5 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
9. An isolated polynucleotide molecule comprising a sequence of
nucleotides that encodes a polypeptide comprising a sequence of
amino acids that is at least 90% identical to residues 32 to 162 of
SEQ ID NO:2, wherein the polypeptide binds a zalpha11 receptor as
shown in SEQ ID NO: 115.
10. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment that
encodes a polypeptide comprising a sequence of amino acids that is
at least 90% identical to residues 32 to 162 of SEQ ID NO:2,
wherein the polypeptide binds a zalpha11 receptor as shown in SEQ
ID NO:115; and (c) a transcription terminator.
11. A cultured cell comprising the expression vector of claim
10.
12. The cultured cell of claim 11, wherein the cultured cell is a
prokaryotic cell.
13. The prokaryotic cell of claim 12, wherein the prokaryotic cell
is an E. coli cell.
14. A process comprising culturing the cell of claim 11 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
15. A process comprising culturing the cell of claim 12 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
16. A process comprising culturing the cell of claim 13 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
17. An isolated polynucleotide molecule comprising a sequence of
nucleotides that encodes a polypeptide comprising residues 41 to
148 of SEQ ID NO:2, wherein the polypeptide stimulates
proliferation of NK cells or NK cell progenitors, stimulates
activation of NK cells, stimulates proliferation of T cells,
stimulates proliferation of B cells stimulated with anti-CD40
antibodies, or reduces proliferation of B cells stimulated with
anti-IgM antibodies.
18. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment that
encodes a polypeptide comprising residues 41 to 148 of SEQ ID NO:2,
wherein the polypeptide stimulates proliferation of NK cells or NK
cell progenitors, stimulates activation of NK cells, stimulates
proliferation of T cells, stimulates proliferation of B cells
stimulated with anti-CD40 antibodies, or reduces proliferation of B
cells stimulated with anti-IgM antibodies; and (c) a transcription
terminator.
19. A cultured cell comprising the expression vector of claim
18.
20. The cultured cell of claim 19, wherein the cultured cell is a
prokaryotic cell.
21. The prokaryotic cell of claim 20, wherein the prokaryotic cell
is an E. coli cell.
22. A process comprising culturing the cell of claim 19 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
23. A process comprising culturing the cell of claim 20 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
24. A process comprising culturing the cell of claim 21 under
conditions whereby the DNA segment is expressed and further
comprising recovering the polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/346,580, which is a Reissue Application of U.S. Pat. No.
6,686,178, which was issued from U.S. Ser. No. 10/295,723, filed on
Nov. 15, 2002, which are incorporated herein by reference, which
was a divisional of U.S. Ser. No. 09/923,246, filed on Aug. 3,
2001, issued as U.S. Pat. No. 6,605,272 which was a divisional of
U.S. Ser. No. 09/522,217, filed on Mar. 9, 2000, which was issued
as U.S. Pat. No. 6,307,024, and which claims benefit to Provisional
Application 60/123,547, filed on Mar. 9, 1999; 60/123,904, filed on
Mar. 11, 1999; and 60/142,013, filed on Jul. 1, 1999.
BACKGROUND OF THE INVENTION
[0002] Proliferation and differentiation of cells of multicellular
organisms are controlled by hormones and polypeptide growth
factors. These diffusable molecules allow cells to communicate with
each other and act in concert to form cells, tissues and organs,
and to repair damaged tissue. Examples of hormones and growth
factors include the steroid hormones (e.g. estrogen, testosterone),
parathyroid hormone, follicle stimulating hormone, the
interleukins, platelet derived growth factor (PDGF), epidermal
growth factor (EGF), granulocyte-macrophage colony stimulating
factor (GM-CSF), erythropoietin (EPO) and calcitonin.
[0003] Hormones and growth factors influence cellular metabolism by
binding to receptors. Receptors may be integral membrane proteins
that are linked to signaling pathways within the cell, such as
second messenger systems. Other classes of receptors are soluble
molecules, such as the transcription factors.
[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.
[0005] 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.
[0006] Mature T cells may be activated, i.e., by an antigen or
other stimulus, to produce, for example, cytokines, biochemical
signaling molecules, or receptors that further influence the fate
of the T cell population.
[0007] B cells can be activated via receptors on their cell surface
including B cell receptor and other accessory molecules to perform
accessory cell functions, such as production of cytokines.
[0008] Natural killer (NK) cells have a common progenitor cell with
T cells and B cells, and play a role in immune surveillance. NK
cells, which comprise up to 15% of blood lymphocytes, do not
express antigen receptors, and therefore do not use MHC recognition
as requirement for binding to a target cell. NK cells are involved
in the recognition and killing of certain tumor cells and virally
infected cells. In vivo, NK cells are believed to require
activation, however, in vitro, NK cells have been shown to kill
some types of tumor cells without activation.
[0009] 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.
[0010] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is an illustration of a multiple alignment of human
IL-2, human IL-15, zalpha11 Ligand (SEQ ID NO: 2), human IL-4,
mouse IL-4, human GM-CSF and mouse GM-CSF.
[0012] FIG. 2 is a Hopp/Woods hydrophilicity plot of human zalpha11
Ligand.
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention provides an antibody
which binds to a polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO: 2 from residue 1 to residue 162.
Additional embodiments of the invention include antibodies that are
polyclonal, monoclonal, murine, humaninzed murine antibodies and
antibodies which are neutralizing.
[0014] In another aspect, the antibody binds a polypeptide
comprising residues 32 to 162 as shown in SEQ ID NO: 2. Additional
embodiments of the invention include antibodies that are
polyclonal, monoclonal, murine, humaninzed murine antibodies and
antibodies which are neutralizing.
[0015] The present invention also provides an antibody fragment
which binds to a polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO: 2 from residue 1 to residue
162.
[0016] Other aspects of the present invention provide antibodies
that bind to peptides comprising a sequence of amino acid residues
as shown in SEQ ID NO: 2 from residues 114 to 119, 101 to 105, 126
to 131, 113 to 118 or 158 to 162. In certain embodiments, these
antibodies are polyclonal, monoclonal, murine, humaninzed murine
antibodies and antibodies which are neutralizing.
[0017] Another aspect of the present invention provides a method of
producing an antibody to a polypeptide comprising: inoculating an
animal with a polypeptide selected from the group consisting of:
(a) a polypeptide consisting of 9 to 131 amino acids, wherein the
polypeptide is identical to a contiguous sequence of amino acid
residues in SEQ ID NO:2 from amino acid number 32 (Gln) to amino
acid number 162 (Ser); (b) a polypeptide according to claim 1; (c)
a polypeptide comprising the amino acid sequence of SEQ ID NO:2
from amino acid number 41 (Gln) to amino acid number 148 (Ile); (d)
a polypeptide comprising the amino acid sequence of SEQ ID NO:2
from amino acid number 41 (Gln) to amino acid number 56 (Val); (e)
a polypeptide comprising the amino acid sequence of SEQ ID NO:2
from amino acid number 69 (Thr) to amino acid number 84 (Leu); (f)
a polypeptide comprising the amino acid sequence of SEQ ID NO:2
from amino acid number 92 (Asn) to amino acid number 105 (Arg); (g)
a polypeptide comprising the amino acid sequence of SEQ ID NO:2
from amino acid number 135 (Glu) to amino acid number 148 (Ile);
(h) a polypeptide comprising the amino acid sequence of SEQ ID
NO:72; (i) a polypeptide comprising the amino acid sequence of SEQ
ID NO:73; (j) a polypeptide comprising the amino acid sequence of
SEQ ID NO:2 from amino acid number 32 (Gln) to amino acid number
162 (Ser); (k) a polypeptide comprising the amino acid sequence of
SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 162
(Ser); (l) a polypeptide comprising the amino acid sequence of SEQ
ID NO:2 from amino acid number 114 to amino acid number 119; (m) a
polypeptide comprising the amino acid sequence of SEQ ID NO:2 from
amino acid number 101 to amino acid number 105; (n) a polypeptide
comprising the amino acid sequence of SEQ ID NO:2 from amino acid
number 126 to amino acid number 131; (o) a polypeptide comprising
the amino acid sequence of SEQ ID NO:2 from amino acid number 113
to amino acid number 118; (p) a polypeptide comprising the amino
acid sequence of SEQ ID NO:2 from amino acid number 158 to amino
acid number 162; and wherein the polypeptide elicits an immune
response in the animal to produce the antibody; and isolating the
antibody from the animal. The present invention also includes
antibodies produced by the methods described above, wherein the
antibody binds to a zalpha11 Ligand polypeptide.
DESCRIPTION OF THE INVENTION
[0018] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0019] 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* 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.).
[0020] 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.
[0021] 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.
[0022] 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 <109 M-1.
[0023] 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'.
[0024] 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).
[0025] 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.
[0026] 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).
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, *-globin, *-globin, and myoglobin are
paralogs of each other.
[0032] 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.
[0033] 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".
[0034] 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.
[0035] 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.
[0036] 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).
[0037] 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.
[0038] 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.
[0039] 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%.
[0040] All references cited herein are incorporated by reference in
their entirety.
[0041] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a protein having the structure of
a four-helical-bundle cytokine. Through processes of cloning,
proliferation assays and binding studies described in detail
herein, a polynucleotide sequence encoding a novel ligand
polypeptide has been identified that is a ligand with high
specificity for the previously orphan receptor zalpha11. This
polypeptide ligand, designated zalpha11 Ligand, was isolated from a
cDNA library generated from activated human peripheral blood cells
(hPBCs), which were selected for CD3. CD3 is a cell surface marker
unique to cells of lymphoid origin, particularly T cells.
[0042] In the examples which follow, a cell line that is dependent
on the zalpha11 orphan receptor-linked pathway for survival and
growth in the absence of other growth factors was used to screen
for a source of the cDNA encoding the zalpha11 Ligand. The
preferred growth factor-dependent cell line that was used for
transfection and expression of zalpha11 receptor was BaF3 (Palacios
and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et al., Mol.
Cell. Biol. 6: 4133-4135, 1986). However, other growth
factor-dependent cell lines, such as FDC-P1 (Hapel et al., Blood
64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7: 235-240,
1993) are suitable for this purpose.
[0043] The amino acid sequence for the zalpha11 receptor indicated
that the encoded receptor belonged to the Class I cytokine receptor
subfamily that includes, but is not limited to, the receptors for
IL-2, IL-4, IL-7, IL-15, EPO, TPO, GM-CSF and G-CSF (for a review
see, Cosman, "The Hematopoietin Receptor Superfamily" in Cytokine
5(2): 95-106, 1993). The zalpha11 receptor is fully described in
commonly-owned PCT Patent Application No. US99/22149. Analysis of
the tissue distribution of the mRNA of the zalpha11 receptor
revealed expression in lymph node, peripheral blood leukocytes
(PBLs), spleen, bone marrow, and thymus. Moreover, the mRNA was
abundant in the Raji cell line (ATCC No. CCL-86) derived from a
Burkitt's lymphoma. The tissue distribution of the receptor
suggests that a target for the predicted zalpha11 Ligand is
hematopoietic lineage cells, in particular lymphoid progenitor
cells and lymphoid cells. Other known four-helical-bundle cytokines
that act on lymphoid cells include IL-2, IL-4, IL-7, and IL-15. For
a review of four-helical-bundle cytokines, see, Nicola et al.,
Advances in Protein Chemistry 52:1-65, 1999 and Kelso, A., Immunol.
Cell Biol. 76:300-317, 1998.
[0044] Conditioned media (CM) from CD3+ selected,
PMA/Ionomycin-stimulated human peripheral blood cells supported the
growth of BaF3 cells that expressed the zalpha11 receptor and were
otherwise dependent on IL-3. Conditioned medias from cells that
were not: 1) PMA/Ionomycin-stimulated; or were not: 2) CD3 selected
(with or without PMA/Ionomycin stimulation) did not support the
growth of BaF3/zalpha11 receptor cells. Control experiments
demonstrated that this proliferative activity was not attributable
to other known growth factors, and that the ability of such
conditioned media to stimulate proliferation of zalpha11
receptor-expressing cells could be neutralized by a soluble form of
the receptor.
[0045] Proliferation of zalpha11 receptor-expressing BaF3 cells
exposed to CM from CD3+ selected, PMA/Ionomycin-stimulated human
peripheral blood cells were identified by visual inspection of the
cultures and/or by proliferation assay. Many suitable proliferation
assays are known in the art, and include assays for reduction of a
dye such as alamarBlue.TM. (AccuMed International, Inc. Westlake,
Ohio), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (Mosman, J. Immunol. Meth. 65: 55-63, 1983); 3, (4,5
dimethyl thiazol-2yl)-5-3-carboxymethoxyphenyl-2H-tetrazolium;
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tet-
razolium hydroxide; and cyanoditolyl-tetrazolium chloride (which
are commercially available from Polysciences, Inc., Warrington,
Pa.); mitogenesis assays, such as measurement of incorporation of
.sup.3H-thymidine; dye exclusion assays using, for example,
naphthalene black or trypan blue; dye uptake using diacetyl
fluorescein; and chromium release. See, in general, Freshney,
Culture of Animal Cells: A Manual of Basic Technique, 3rd ed.,
Wiley-Liss, 1994, which is incorporated herein by reference.
[0046] A cDNA library was prepared from CD3+ selected, PMA- and
Ionomycin-stimulated primary human peripheral blood cells. The CD3+
selected, PMA- and Ionomycin-stimulated human peripheral blood
cells cDNA library was divided into pools containing multiple cDNA
molecules and was transfected into a host cell line, for example,
BHK 570 cells (ATCC accession no. 10314). The transfected host
cells were cultured in a medium that did not contain exogenous
growth factors and conditioned medium was collected. The
conditioned media were assayed for the ability to stimulate
proliferation of BaF3 cells transfected with the zalpha11 receptor.
cDNA pools producing conditioned medium that stimulated
BaF3/zalpha11 receptor cells were identified. This pooled plasmid
cDNA was electroporated into E. coli. cDNA was isolated from single
colonies and transfected individually into BHK 570 cells. Positive
clones were identified by a positive result in the BaF3/zalpha11
receptor proliferation assay, and specificity was tested by
neutralization of proliferation using the soluble zalpha11
receptor.
[0047] A positive clone was isolated, and sequence analysis
revealed that the polynucleotide sequence contained within the
plasmid DNA was novel. The secretory signal sequence is comprised
of amino acid residues 1 (Met) to 31 (Gly), and the mature
polypeptide is comprised of amino acid residues 32 (Gln) to 162
(Ser) (as shown in SEQ ID NO: 2).
[0048] 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. Referring to the human zalpha11 Ligand amino
acid sequence shown in SEQ ID NO:2, alignment of human zalpha11
Ligand, human IL-15, human IL-4, and human GM-CSF amino acid
sequences it is predicted that zalpha11 Ligand helix A is defined
by amino acid residues 41-56; helix B by amino acid residues 69-84;
helix C by amino acid residues 92-105; and helix D by amino acid
residues 135-148; as shown in SEQ ID NO: 2. Structural analysis
suggests that the A/B loop is long, the B/C loop is short and the
C/D loop is parallel long. This loop structure results in an
up-up-down-down helical organization. The cysteine residues are
absolutely conserved between zalpha11 Ligand and IL-15, as shown in
FIG. 1. The cysteine residues that are conserved between IL-15 and
zalpha11 Ligand correspond to amino acid residues 71, 78, 122 and
125 of SEQ ID NO: 2. Conservation of some of the cysteine residues
is also found in IL-2, IL-4, GM-CSF and zalpha11 Ligand
corresponding to amino acid residues 78 and 125 of SEQ ID NO: 2, as
shown in FIG. 1. Consistent cysteine placement is further
confirmation of the four-helical-bundle structure. Also highly
conserved in the family comprising IL-15, IL-2, IL-4, GM-CSF and
zalpha11 Ligand is the Glu-Phe-Leu sequence as shown in SEQ ID NO:
2 at residues 136-138, as in FIG. 1.
[0049] Further analysis of zalpha11 Ligand based on multiple
alignments (as shown in FIG. 1) predicts that amino acid residues
44, 47 and 135 (as shown in SEQ ID NO: 2) play an important role in
zalpha11 Ligand binding to its cognate receptor. Moreover, the
predicted amino acid sequence of murine zalpha11 Ligand shows 57%
identity to the predicted human protein. Based on comparison
between sequences of human and murine zalpha11 Ligand
well-conserved residues were found in the regions predicted to
encode alpha helices A and D. The corresponding polynucleotides
encoding the zalpha11 Ligand polypeptide regions, domains, motifs,
residues and sequences described herein are as shown in SEQ ID
NO:1.
[0050] Detailed mutational analysis has been performed for IL-4 and
IL-2, both of which are highly related to zalpha11 ligand. 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 Asp34, Asn99, and Asn 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,
Gln141 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
Tyr124 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).
[0051] While helix A is relatively well-conserved between human and
murine zalpha11 Ligand, helix C is more divergent. While both
species have predominant acidic amino acids in this region, the
differences may account for species specificity in interaction
between zalpha11 Ligand and its "beta" type receptor, zalpha11.
Loop A/B and helix B of zalpha11 Ligand are well-conserved between
species; although no receptor subunit corresponding to IL-2R* has
yet been identified, conservation through this region suggests that
it is functionally significant. The D helices of human and murine
zalpha11 Ligand are also highly conserved. Zalpha11 receptor
antagonists may be designed through mutations within zalpha11
Ligand helix D. These may include truncation of the protein from
residue Gln145 (SEQ ID NO: 2), or mutations of Gln145 or Ile148 (of
SEQ ID NO: 2; corresponding to Tyr124 in human IL-4) to residues
such as Ala or Asp. Any mutation which disrupts the zalpha11 Ligand
helical structure may abolish binding with its receptor and thus
inhibit signaling.
[0052] 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. Zalpha11 Ligand is believed to be a new member of the
short-helix form cytokine group. 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 zalpha11 Ligand will be useful
for preparing chimeric fusion molecules, particularly with other
short-helix form cytokines to determine and modulate receptor
binding specificity. Of particular interest are fusion proteins
engineered with helix A and/or helix D, and fusion proteins that
combine helical and loop domains from other short-form cytokines
such as IL-2, IL-4, IL-15 and GM-CSF. The amino acid residues
comprising helices A, B, C, and D, and loops A/B, B/C and C/D for
zalpha11 Ligand, IL-2, IL-4, IL-15 and GM-CSF are shown in Table 1.
TABLE-US-00001 TABLE 1 Helix A A/B Loop Helix B B/C Loop Helix C
C/D Loop Helix D zalpha11 Ligand 41-56 57-68 69-84 85-91 92-105
106-134 135-148 SEQ ID NO: 2 residues IL-2 residues 36-46 47-52
53-75 76-86 87-99 100-102 103-121 SEQ ID NO: 111 IL-4 residues
29-43 44-64 65-83 84-94 95-118 119-133 134-151 SEQ ID NO: 112 IL-15
residues 45-68 69-83 84-101 102-106 107-119 120-133 134-160 SEQ ID
NO: 113 GM-CSF residues 30-44 45-71 72-81 82-90 91-102 103-119
120-131 SEQ ID NO: 114
[0053] The present invention provides polynucleotide molecules,
including DNA and RNA molecules, that encode the zalpha11 Ligand
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 NO:3 is a degenerate DNA sequence
that encompasses all DNAs that encode the zalpha11 Ligand
polypeptide of SEQ ID NO:2. Those skilled in the art will recognize
that the degenerate sequence of SEQ ID NO:3 also provides all RNA
sequences encoding SEQ ID NO:2 by substituting U for T. Thus,
zalpha11 Ligand polypeptide-encoding polynucleotides comprising
nucleotide 1 or 97 to nucleotide 486 of SEQ ID NO:3 and their RNA
equivalents are contemplated by the present invention. Table 2 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-00002
TABLE 2 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
[0054] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table 3.
TABLE-US-00003 TABLE 3 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 B
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
[0055] 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.
[0056] 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.
[0057] 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 zalpha11 Ligand
RNA. 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)+ RNA using known
methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding zalpha11 Ligand polypeptides are then
identified and isolated by, for example, hybridization or PCR.
[0058] A full-length clone encoding zalpha11 Ligand 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 zalpha11 receptor fragments, or other specific
binding partners.
[0059] Zalpha11 Ligand polynucleotide sequences disclosed herein
can also be used as probes or primers to clone 5' non-coding
regions of a zalpha11 Ligand gene. In view of the tissue-specific
expression observed for zalpha11 Ligand this gene region is
expected to provide for hematopoietic- and lymphoid-specific
expression. Promoter elements from a zalpha11 Ligand gene could
thus be used to direct the tissue-specific expression of
heterologous genes in, for example, transgenic animals or patients
treated with gene therapy. Cloning of 5' flanking sequences also
facilitates production of zalpha11 Ligand proteins by "gene
activation" as disclosed in U.S. Pat. No. 5,641,670. Briefly,
expression of an endogenous zalpha11 Ligand gene in a cell is
altered by introducing into the zalpha11 Ligand locus a DNA
construct comprising at least a targeting sequence, a regulatory
sequence, an exon, and an unpaired splice donor site. The targeting
sequence is a zalpha11 Ligand 5' non-coding sequence that permits
homologous recombination of the construct with the endogenous
zalpha11 Ligand locus, whereby the sequences within the construct
become operably linked with the endogenous zalpha11 Ligand coding
sequence. In this way, an endogenous zalpha11 Ligand promoter can
be replaced or supplemented with other regulatory sequences to
provide enhanced, tissue-specific, or otherwise regulated
expression.
[0060] 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 zalpha11 Ligand
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zalpha11 Ligand 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 zalpha11 Ligand 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 zalpha11 Ligand-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
zalpha11 Ligand 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 zalpha11 Ligand polypeptide, binding studies or
activity assays. Similar techniques can also be applied to the
isolation of genomic clones.
[0061] The polynucleotide sequence for the mouse ortholog of
zalpha11 Ligand has been identified and is shown in SEQ ID NO: 55
and the corresponding amino acid sequence shown in SEQ ID NO: 56.
There is 62% identity between the mouse and human sequences over a
124 amino acid region that corresponds to residues 30 to 153 in SEQ
ID NO: 2 and residues 23 to 146 of SEQ ID NO: 56 of zalpha11
Ligand. Mature sequence for the mouse zalpha11 Ligand putatively
begins at His18 (as shown in SEQ ID NO: 56), which corresponds to
His25 (as shown in SEQ ID NO: 2) in the human sequence. Because a
truncated form of the human polypeptide is active, it is likely
that an equivalent polypeptide of the mouse zalpha11 Ligand (i.e.
without residues His18 to Pro22 of SEQ ID NO: 56) is active as
well. Tissue analysis revealed that expression of mouse zalpha11
Ligand is found in testis, spleen and thymus.
[0062] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
zalpha11 Ligand and that allelic variation and alternative splicing
are expected to occur. Allelic variants of this sequence can be
cloned by probing cDNA or genomic libraries from different
individuals according to standard procedures. Allelic variants of
the 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 zalpha11 Ligand 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.
[0063] The zalpha11 Ligand gene has been mapped to the IL-2
framework marker SHGC-12342, positioning zalpha11 Ligand
approximately 180 kb from the IL-2 marker. The use of surrounding
markers positions the zalpha11 Ligand gene in the 4q27 region on
the integrated LDB chromosome 4 map (The Genetic Location Database,
University of Southhampton,). The present invention also provides
reagents which will find use in diagnostic applications. For
example, the zalpha11 Ligand gene, a probe comprising zalpha11
Ligand DNA or RNA or a subsequence thereof can be used to determine
if the zalpha11 Ligand gene is present on a human chromosome, such
as chromosome 4, or if a gene mutation has occurred. Based on
annotation of a fragment of human genomic DNA containing a part of
zalpha11 Ligand genomic DNA (Genbank Accession No. AC007458),
zalpha11 Ligand is located at the 4q27 region of chromosome 4.
Detectable chromosomal aberrations at the zalpha11 Ligand 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).
[0064] 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.
[0065] As stated previously, human zalpha11 Ligand gene resides
near the IL-2 gene, which is in a region of chromosome 4q that has
been shown to have linkage with susceptibility to inflammatory
bowel disease (IBD) (including Crohn's disease (CD) and ulcerative
colitis) in some families (Hampe et al. Am. J. Hum. Genet.
64:808-816, 1999; Cho et al. Proc. Natl. Acad. Sci. 95:7502-7507,
1998). In addition, the zalpha11 receptor gene maps to 16 p11,
another genomic region which is associated with susceptibility to
CD (Hugot et al., Nature 379:821-823, 1996; Ohmen et al., Hum. Mol.
Genet. 5:1679-1683, 1996). CD is a chronic inflammation of the gut
with frequent systemic involvement; while the exact etiology is
unknown, immunoregulatory dysfunction involving failure of
tolerance to ordinary gut antigens is a major component (for
reviews, see (Braegger et al., Annals Allergy 72:135-141, 1994;
Sartor, Am. J. Gastroenterol. 92:5S-11S, 1997)). Several studies
have found abnormal NK activity in CD patients (see, for example,
(Egawa et. al., J. Clin. Lab. Immunol. 20:187-192, 1986;
Aparicio-Pages et al. J. Clin. Lab. Immunol. 29:119-124, 1989; van
Tol et al., Scand. J. Gastroenterol. 27:999-1005, 1992)), and
defective memory B cell formation has also been documented (Brogan
et al., J. Clin. Lab. Immunol. 24:69-74, 1987). Since zalpha11
Ligand plays a role in immune regulation, and since the genes for
both receptor and ligand lie within CD susceptibility regions, both
receptor and ligand are candidate genes for genetic predisposition
to Crohn's disease.
[0066] Determination of the involvement of zalpha11 receptor and/or
zalpha11 Ligand in the pathology of IBD can be accomplished by
several methods. Sequencing of exons from genomic DNA can reveal
coding mutations (including missense, nonsense, and frameshift
mutations), as can sequencing of cDNAs. An additional advantage of
sequencing from genomic DNA is that splice junctions are also
contained within the sequenced fragments and may reveal splicing
abnormalities, which might not appear in cDNA samples if, for
example, misspliced RNAs were rapidly degraded. The genomic
structure of zalpha11 Ligand has been determined. Other methods for
analysis of zalpha11 Ligand and receptor in IBD patients include:
(1) assessment of ligand production from activated T cells from
patients vs. normal controls (i.e. by bioassay); (2) in situ
hybridization of zalpha11 receptor or zalpha11 Ligand RNA to
sections of inflamed intestine from IBD patients, compared to
similar sections from normal controls; (3) immunohistochemistry on
sections from IBD patients vs. normal controls; and (4) assessment
of the responsiveness of patients' peripheral B cells to zalpha11
Ligand, as measured by mitogenesis assays.
[0067] A diagnostic could assist physicians in determining the type
of disease and appropriate associated therapy, or could assist in
genetic counseling. As such, the inventive anti-zalpha11 Ligand
antibodies, polynucleotides, and polypeptides can be used for the
detection of zalpha11 Ligand polypeptide, mRNA or anti-zalpha11
Ligand antibodies, thus serving as markers and be directly used for
detecting or genetic diseases or cancers, as described herein,
using methods known in the art and described herein. Further,
zalpha11 Ligand polynucleotide probes can be used to detect
abnormalities involving chromosome 4q27 as described herein. These
abnormalities may be associated with human diseases, or
tumorigenesis, spontaneous abortion or other genetic disorders.
Thus, zalpha11 Ligand polynucleotide probes can be used to detect
abnormalities or genotypes associated with these defects.
[0068] As discussed above, defects in the zalpha11 Ligand gene
itself may result in a heritable human disease state. Molecules of
the present invention, such as the polypeptides, antagonists,
agonists, polynucleotides and antibodies of the present invention
would aid in the detection, diagnosis prevention, and treatment of
diseases associated with a zalpha11 Ligand genetic defect. In
addition, zalpha11 Ligand polynucleotide probes can be used to
detect allelic differences between diseased or non-diseased
individuals at the zalpha11 Ligand chromosomal locus. As such, the
zalpha11 Ligand sequences can be used as diagnostics in forensic
DNA profiling.
[0069] 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 (i) obtaining a genetic sample from a potentially diseased
patient, diseased patient or potential non-diseased carrier of a
recessive disease allele; (ii) producing a first reaction product
by incubating the genetic sample with a zalpha11 Ligand
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 zalpha11 Ligand 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 a normal or control individual. 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 zalpha11 Ligand genetic locus, and the like, are
indicative of a genetic abnormality, genetic aberration, or allelic
difference in comparison to the normal 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 from 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, 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)).
[0070] Mutations associated with the zalpha11 Ligand 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 zalpha11 Ligand 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)).
[0071] Positions of introns in the zalpha11 Ligand gene were
determined by identification of genomic clones, followed by
sequencing the intron/exon junctions. The first intron lies between
amino acid residue 56 (Leu) and residue 57 (Val) in Seq. ID. No. 2,
and is 115 base pairs in length. The second intron is the largest
at 4.4 kilobases, and lies between amino acid residue 68 (Glu) and
residue 69 (Thr) in Seq. ID. No. 2. The third intron is 2.6
kilobases, and lies between amino acid residue 120 (Leu) and
residue 121 (Thr) in Seq. ID. No. 2. The final intron, 89 base
pairs, lies between amino acid residue 146 (Lys) and residue 147
(Met) in Seq. ID. No. 2. The complete gene spans about 8 kb.
[0072] The structure of the zalpha11 Ligand gene is similar to that
of the IL-2 gene (Fujita et al. Proc. Natl. Acad. Sci.
80:7437-7441, 1983), though the zalpha11 Ligand gene contains one
additional intron (intron 4). The pattern of a short first intron
and long second and third introns is conserved between the two
genes, though the IL-2 gene is slightly smaller overall (about 6
kb). The IL-15 gene, on the other hand, consists of 8 exons and
spans at least 34 kb (Anderson et al. Genomics 25:701-706, 1995).
Thus the zalpha11 Ligand gene is more similar in structure to the
IL-2 gene than to the IL-15 gene.
[0073] Within embodiments of the invention, isolated zalpha11
Ligand-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 47 to 532 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 (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe.
[0074] 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 Tm 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.
[0075] It is well within the abilities of one skilled in the art to
adapt these conditions for use with a particular polynucleotide
hybrid. The Tm 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 Tm 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 Tm 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 Tm 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 Tm. For
smaller probes, <50 base pairs, hybridization is typically
carried out at the Tm or 5-10.degree. C. below the calculated Tm.
This allows for the maximum rate of hybridization for DNA-DNA and
DNA-RNA hybrids.
[0076] 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
zalpha11 Ligand 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.
[0077] 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 zalpha11 Ligand 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.
[0078] The present invention also provides isolated zalpha11 Ligand
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% 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% sequence identity to the sequence of amino
acid residues 1 to 162 or 32 to 162 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.
[0079] The present invention also contemplates variant zalpha11
Ligand 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 zalpha11 Ligand
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% sequence identity to the amino acid sequence of
SEQ ID NO:2. Alternatively, zalpha11 Ligand 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.
[0080] 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 4 (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. .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. .times. the .times. .times. two .times. .times.
sequences ] .times. 100 ##EQU1## TABLE-US-00004 TABLE 4 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
[0081] 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 zalpha11 Ligand. 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).
[0082] 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).
[0083] 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.
[0084] Variant zalpha11 Ligand 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 5) 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 108 to 216 amino acid
residues that comprise a sequence that is at least 70%, preferably
at least 90%, and more preferably 95% 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 zalpha11 Ligand polypeptide and the affinity tag.
Preferred such sites include thrombin cleavage sites and factor Xa
cleavage sites. TABLE-US-00005 TABLE 5 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
[0085] 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.
[0086] Amino acid sequence changes are made in zalpha11 Ligand
polypeptides so as to minimize disruption of higher order structure
essential to biological activity. For example, where the zalpha11
Ligand 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, e.g., A and D helices, residues 44, 47 and
135 of SEQ ID NO: 2. 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).
[0087] A Hopp/Woods hydrophilicity profile of the zalpha11 Ligand
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 zalpha11 Ligand, hydrophilic
regions include amino acid residues 114-119 of SEQ ID NO: 2, amino
acid residues 101-105 of SEQ ID NO: 2, amino acid residues 126-131
of SEQ ID NO: 2, amino acid residues 113-118 of SEQ ID NO: 2, and
amino acid residues 158-162 of SEQ ID NO: 2.
[0088] 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 zalpha11 Ligand
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.
For example, residues tolerant of substitution could include
residues 100 and 103 as shown in SEQ ID NO: 2. Cysteine residues at
positions 71, 78, 122 and 125 of SEQ ID NO: 2, will be relatively
intolerant of substitution.
[0089] The identities of essential amino acids can also be inferred
from analysis of sequence similarity between IL-15, IL-2, IL-4 and
GM-CSF with zalpha11 Ligand. 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 zalpha11 Ligand polynucleotide on the basis of structure is
to determine whether a nucleic acid molecule encoding a potential
variant zalpha11 Ligand gene can hybridize to a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1, as
discussed above.
[0090] 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).
[0091] The present invention also includes functional fragments of
zalpha11 Ligand polypeptides and nucleic acid molecules encoding
such functional fragments. A "functional" zalpha11 Ligand 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-zalpha11 Ligand antibody or zalpha11
receptor (either soluble or immobilized). As previously described
herein, zalpha11 Ligand is characterized by a four-helical-bundle
structure comprising helix A (amino acid residues 41-56), helix B
(amino acid residues 69-84), helix C (amino acid residues 92-105)
and helix D (amino acid residues 135-148), 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
four-helical-bundle cytokine, such as IL-15, IL-2, IL-4 and GM-CSF,
or by a non-native and/or an unrelated secretory signal peptide
that facilitates secretion of the fusion protein.
[0092] Thus the present invention provides fusion proteins
comprising at least four polypeptides, wherein the order of
polypeptides from N-terminus to C-terminus are: a first polypeptide
comprises amino acids selected from a group consisting of: (a) IL-2
helix A amino acid residues 36-46 of SEQ ID NO: 111; (b) IL-15
helix A amino acid residues 29-43 of SEQ ID NO: 112; (c) IL-4 helix
A amino acid residues 45-68 of SEQ ID NO: 113; (d) GMCSF helix A
amino acid residues 30-44 of SEQ ID NO: 114; and (e) amino acids
residues 41 to 56 of SEQ ID NO: 2; a first spacer of 6-27 amino
acids; and a second polypeptide that comprises amino acid residues
selected from the group consisting of: (a) IL-2 helix B amino acid
residues 53-75 of SEQ ID NO: 111; (b) IL-4 helix B amino acid
residues 65-83 of SEQ ID NO: 112; (c) IL-15 helix B amino acid
residues 84-101 of SEQ ID NO: 113; (d) GMCSF helix B amino acid
residues 72-81 of SEQ ID NO: 114; and (e) amino acid residues 69-84
of SEQ ID NO: 2; a second spacer of 5-11 amino acid residues; a
third polypeptide that comprises a sequence of amino acid residues
selected from the group consisting of: (a) IL-2 helix C residues
87-99 of SEQ ID NO: 111; (b) IL-4 helix C residues 95-118 of SEQ ID
NO: 112; (c) IL-15 helix C residues 107-119 of SEQ ID NO: 113; (d)
GMCSF helix C residues 91-102 of SEQ ID NO: 114; and (e) amino acid
residues 92-105 of SEQ ID NO: 2; a third spacer of 3-29 amino acid
residues; and a fourth polypeptide that comprises amino acid
residues selected from the group consisting of: (a) IL-2 helix D
amino acid residues 103-121 of SEQ ID NO: 111; (b) IL-15 helix D
amino acid residues 134-157 of SEQ ID NO: 112; (c) IL-4 helix D
amino acid residues 134-160 of SEQ ID NO: 113; (d) GMCSF helix D
amino acid residues 120-131 of SEQ ID NO: 114; and (e) amino acid
residues 135-148 of SEQ ID NO: 2, wherein at least one of the four
polypeptides is from zalpha11 Ligand. In other embodiments that the
spacer peptides will be selected from the A/B, B/C and C/D loops of
zalpha11 Ligand, IL-2, IL-4, IL-15 or GM-CSF, as shown in Table
1.
[0093] Routine deletion analyses of nucleic acid molecules can be
performed to obtain functional fragments of a nucleic acid molecule
that encodes a zalpha11 Ligand polypeptide. As an illustration, DNA
molecules having the nucleotide sequence of SEQ ID NO:1 or
fragments thereof, can be digested with Bal31 nuclease to obtain a
series of nested deletions. These DNA fragments are then inserted
into expression vectors in proper reading frame, and the expressed
polypeptides are isolated and tested for zalpha11 Ligand activity,
or for the ability to bind anti-zalpha11 Ligand antibodies or
zalpha11 receptor. One alternative to exonuclease digestion is to
use oligonucleotide-directed mutagenesis to introduce deletions or
stop codons to specify production of a desired zalpha11 Ligand
fragment. Alternatively, particular fragments of a zalpha11 Ligand
gene can be synthesized using the polymerase chain reaction.
[0094] Standard methods for identifying functional domains are
well-known to those of skill in the art. For example, studies on
the truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993); Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2 5A synthetase induced by human
interferon," in Biological Interferon Systems Proceedings of ISIR
TNO Meeting on Interferon Systems, Cantell (ed.), pages 65 72
(Nijhoff 1987); Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985); Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995); and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0095] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204), and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)).
[0096] Variants of the disclosed zalpha11 Ligand nucleotide and
polypeptide sequences can also be generated through DNA shuffling
as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc.
Natl. Acad. Sci. USA 91:10747 (1994), and international publication
No. WO 97/20078. Briefly, variant DNA molecules are generated by in
vitro homologous recombination by random fragmentation of a parent
DNA followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent DNA molecules, such as allelic variants or DNA
molecules from different species, to introduce additional
variability into the process. Selection or screening for the
desired activity, followed by additional iterations of mutagenesis
and assay provides for rapid "evolution" of sequences by selecting
for desirable mutations while simultaneously selecting against
detrimental changes.
[0097] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-zalpha11 Ligand antibodies or
soluble zalpha11 receptor, can be recovered from the host cells and
rapidly sequenced using modern equipment. These methods allow the
rapid determination of the importance of individual amino acid
residues in a polypeptide of interest, and can be applied to
polypeptides of unknown structure.
[0098] In addition, the proteins of the present invention (or
polypeptide fragments thereof) can be joined to other bioactive
molecules, particularly other cytokines, to provide
multi-functional molecules. For example, one or more helices from
zalpha11 Ligand can be joined to other cytokines to enhance their
biological properties or efficiency of production.
[0099] The present invention thus provides a series of novel,
hybrid molecules in which a segment comprising one or more of the
helices of zalpha11 Ligand is fused to another polypeptide. Fusion
is preferably done by splicing at the DNA level to allow expression
of chimeric molecules in recombinant production systems. The
resultant molecules are then assayed for such properties as
improved solubility, improved stability, prolonged clearance
half-life, improved expression and secretion levels, and
pharmacodynamics. Such hybrid molecules may further comprise
additional amino acid residues (e.g. a polypeptide linker) between
the component proteins or polypeptides.
[0100] Non-naturally occurring amino acids include, without
limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,
allo-threonine, methylthreonine, hydroxyethylcysteine,
hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic
acid, thiazolidine carboxylic acid, dehydroproline, 3- and
4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and
4-fluorophenylalanine. Several methods are known in the art for
incorporating non-naturally occurring amino acid residues into
proteins. For example, an in vitro system can be employed wherein
nonsense mutations are suppressed using chemically aminoacylated
suppressor tRNAs. Methods for synthesizing amino acids and
aminoacylating tRNA are known in the art. Transcription and
translation of plasmids containing nonsense mutations is typically
carried out in a cell-free system comprising an E. coli S30 extract
and commercially available enzymes and other reagents. Proteins are
purified by chromatography. See, for example, Robertson et al., J.
Am. Chem. Soc. 113:2722 (1991), Ellman et al., Methods Enzymol.
202:301 (1991), Chung et al., Science 259:806 (1993), and Chung et
al., Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0101] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993). It may
be advantageous to stabilize zalpha11 Ligand to extend the
half-life of the molecule, particularly for extending metabolic
persistence in an active state. To achieve extended half-life,
zalpha11 Ligand molecules can be chemically modified using methods
described herein. PEGylation is one method commonly used that has
been demonstrated to increase plasma half-life, increased
solubility, and decreased antigenicity and immunogenicity (Nucci et
al., Advanced Drug Delivery Reviews 6:133-155, 1991 and Lu et al.,
Int. J. Peptide Protein Res. 43:127-138, 1994).
[0102] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for zalpha11 Ligand amino acid residues.
[0103] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a zalpha11 Ligand
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0104] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein. Hopp/Woods hydrophilicity
profiles can be used to determine regions that have the most
antigenic potential (Hopp et al., 1981, ibid. and Hopp, 1986,
ibid.). In zalpha11 Ligand these regions include: amino acid
residues 114-119, 101-105, 126-131, 113-118, and 158-162 of SEQ ID
NO: 2.
[0105] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fourteen amino acids, or about fourteen to about thirty amino
acids of SEQ ID NO:2 or SEQ ID NO:56. Such epitope-bearing peptides
and polypeptides can be produced by fragmenting a zalpha11 Ligand
polypeptide, or by chemical peptide synthesis, as described herein.
Moreover, epitopes can be selected by phage display of random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin.
Immunol. 5:268 (1993); and Cortese et al., Curr. Opin. Biotechnol.
7:616 (1996)). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an epitope
are described, for example, by Mole, "Epitope Mapping," in Methods
in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992); Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production Engineering and Clinical Application, Ritter and Ladyman
(eds.), pages 60-84 (Cambridge University Press 1995), and Coligan
et al. (eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5
and pages 9.4.1-9.4.11 (John Wiley & Sons 1997).
[0106] Regardless of the particular nucleotide sequence of a
variant zalpha11 Ligand polynucleotide, the polynucleotide encodes
a polypeptide that is characterized by its proliferative or
differentiating activity, its ability to induce or inhibit
specialized cell functions, or by the ability to bind specifically
to an anti-zalpha11 Ligand antibody or zalpha11 receptor. More
specifically, variant zalpha11 Ligand polynucleotides will encode
polypeptides which exhibit at least 50% and preferably, greater
than 70%, 80% or 90%, of the activity of the polypeptide as shown
in SEQ ID NO: 2.
[0107] For any zalpha11 Ligand polypeptide, including variants and
fusion proteins, one of ordinary skill in the art can readily
generate a fully degenerate polynucleotide sequence encoding that
variant using the information set forth in Tables 1 and 2
above.
[0108] The present invention further provides a variety of other
polypeptide fusions (and related multimeric proteins comprising one
or more polypeptide fusions). For example, a zalpha11 Ligand
polypeptide can be prepared as a fusion to a dimerizing protein as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant
region domains. Immunoglobulin-zalpha11 Ligand polypeptide fusions
can be expressed in genetically engineered cells (to produce a
variety of multimeric zalpha11 Ligand analogs). Auxiliary domains
can be fused to zalpha11 Ligand polypeptides to target them to
specific cells, tissues, or macromolecules. For example, a zalpha11
Ligand polypeptide or protein could be targeted to a predetermined
cell type by fusing a zalpha11 Ligand polypeptide to a ligand that
specifically binds to a receptor on the surface of that target
cell. In this way, polypeptides and proteins can be targeted for
therapeutic or diagnostic purposes. A zalpha11 Ligand polypeptide
can be fused to two or more moieties, such as an affinity tag for
purification and a targeting domain. Polypeptide fusions can also
comprise one or more cleavage sites, particularly between domains.
See, Tuan et al., Connective Tissue Research 34:1-9, 1996.
[0109] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptides that
have substantially similar sequence identity to residues 1-162 or
32-162 of SEQ ID NO: 2, or functional fragments and fusions
thereof, wherein such polypeptides or fragments or fusions retain
the properties of the wild-type protein such as the ability to
stimulate proliferation, differentiation, induce specialized cell
function or bind the zalpha11 receptor or zalpha11 Ligand
antibodies.
[0110] The zalpha11 Ligand polypeptides of the present invention,
including full-length polypeptides, functional fragments, and
fusion polypeptides, can be produced in genetically engineered host
cells according to conventional techniques. 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. Eukaryotic cells,
particularly cultured cells of multicellular organisms, are
preferred. 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.
[0111] In general, a DNA sequence encoding a zalpha11 Ligand
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.
[0112] To direct a zalpha11 Ligand 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
zalpha11 Ligand, or may be derived from another secreted protein
(e.g., t-PA) or synthesized de novo. The secretory signal sequence
is operably linked to the zalpha11 Ligand 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 secretory 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).
[0113] Alternatively, the secretory signal sequence contained in
the polypeptides of the present invention is used to direct other
polypeptides into the secretory pathway. The present invention
provides for such fusion polypeptides. A signal fusion polypeptide
can be made wherein a secretory signal sequence derived from amino
acid residue 1-31 of SEQ ID NO:2 is be operably linked to a DNA
sequence encoding another polypeptide using methods known in the
art and disclosed herein. The secretory signal sequence contained
in the fusion polypeptides of the present invention is preferably
fused amino-terminally to an additional peptide to direct the
additional peptide into the secretory pathway. Such constructs have
numerous applications known in the art. For example, these novel
secretory signal sequence fusion constructs can direct the
secretion of an active component of a normally non-secreted
protein. Such fusions may be used in vivo or in vitro to direct
peptides through the secretory pathway.
[0114] Cultured mammalian cells are suitable 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.
1:841-5, 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, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996). 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) 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.
[0115] 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.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins such as CD4,
CD8, Class I MHC, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0116] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect 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. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols, Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. The second method of making
recombinant baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system is sold in the Bac-to-Bac kit (Life
Technologies, Rockville, Md.). This system utilizes a transfer
vector, pFastBac1.TM. (Life Technologies) containing a Tn7
transposon to move the DNA encoding the zalpha11 Ligand polypeptide
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." The pFastBac1.TM. transfer vector utilizes the
AcNPV polyhedrin promoter to drive the expression of the gene of
interest, in this case zalpha11 Ligand. However, pFastBac1.TM. can
be modified to a considerable degree. The polyhedrin promoter can
be removed and substituted with the baculovirus basic protein
promoter (also known as Pcor, p6.9 or MP promoter) which is
expressed earlier in the baculovirus infection, and has been shown
to be advantageous for expressing secreted proteins. See,
Hill-Perkins, M. S. and Possee, R. D., J. Gen. Virol. 71:971-6,
1990; Bonning, B. C. et al., J. Gen. Virol. 75:1551-6, 1994; and,
Chazenbalk, G. D., and Rapoport, B., J. Biol. Chem. 270:1543-9,
1995. In such transfer vector constructs, a short or long version
of the basic protein promoter can be used. Moreover, transfer
vectors can be constructed which replace the native zalpha11 Ligand
secretory signal sequences with secretory signal sequences derived
from insect proteins. For example, a secretory signal sequence from
Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin
(Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen,
San Diego, Calif.) can be used in constructs to replace the native
zalpha11 Ligand secretory signal sequence. In addition, transfer
vectors can include an in-frame fusion with DNA encoding an epitope
tag at the C- or N-terminus of the expressed zalpha11 Ligand
polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T.
et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using techniques
known in the art, a transfer vector containing zalpha11 Ligand is
transformed into E. coli, and screened for bacmids which contain an
interrupted lacZ gene indicative of recombinant baculovirus. The
bacmid DNA containing the recombinant baculovirus genome is
isolated, using common techniques, and used to transfect Spodoptera
frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses
zalpha11 Ligand is subsequently produced. Recombinant viral stocks
are made by methods commonly used the art.
[0117] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435).
[0118] 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.
132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0119] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (.OMEGA.) of from
1 to 40 milliseconds, most preferably about 20 milliseconds.
[0120] 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 zalpha11 Ligand 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.
[0121] 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. P. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0122] It is preferred to purify the polypeptides 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
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0123] Expressed recombinant zalpha11 Ligand polypeptides (or
chimeric zalpha11 Ligand polypeptides) can be purified using
fractionation and/or conventional purification methods and media.
Ammonium sulfate precipitation and acid or chaotrope extraction may
be used for fractionation of samples. Exemplary purification steps
may include hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable chromatographic
media include derivatized dextrans, agarose, cellulose,
polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and
Q derivatives are preferred. Exemplary chromatographic media
include those media derivatized with phenyl, butyl, or octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl
650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso
Haas) and the like. Suitable solid supports include glass beads,
silica-based resins, cellulosic resins, agarose beads, cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide
resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties. Examples of coupling chemistries include
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, hydrazide activation,
and carboxyl and amino derivatives for carbodiimide coupling
chemistries. These and other solid media are well known and widely
used in the art, and are available from commercial suppliers.
Methods for binding receptor polypeptides to support media are well
known in the art. Selection of a particular method is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods, Pharmacia LKB Biotechnology, Uppsala,
Sweden, 1988.
[0124] The polypeptides of the present invention can be isolated by
exploitation of their physical or biochemical properties. For
example, immobilized metal ion adsorption (IMAC) chromatography can
be used to purify histidine-rich proteins, including those
comprising polyhistidine tags. Briefly, a gel is first charged with
divalent metal ions to form a chelate (Sulkowski, Trends in
Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to
this matrix with differing affinities, depending upon the metal ion
used, and will be eluted by competitive elution, lowering the pH,
or use of strong chelating agents. Other methods of purification
include purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp. 529-39) and use of the
soluble zalpha11 receptor. Within additional embodiments of the
invention, a fusion of the polypeptide of interest and an affinity
tag (e.g., maltose-binding protein, an immunoglobulin domain) may
be constructed to facilitate purification.
[0125] Moreover, using methods described in the art, polypeptide
fusions, or hybrid zalpha11 Ligand proteins, are constructed using
regions or domains of the inventive zalpha11 Ligand in combination
with those of other human cytokine family proteins (e.g.
interleukins or GM-CSF), or heterologous proteins (Sambrook et al.,
ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5,
1994, and references therein). These methods allow the
determination of the biological importance of larger domains or
regions in a polypeptide of interest. Such hybrids may alter
reaction kinetics, binding, constrict or expand the substrate
specificity, or alter tissue and cellular localization of a
polypeptide, and can be applied to polypeptides of unknown
structure.
[0126] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a helix conferring a biological function may be swapped
between zalpha11 Ligand of the present invention with the
functionally equivalent helices from another family member, such as
IL-15, IL-2, IL-4 or GM-CSF. Such components include, but are not
limited to, the secretory signal sequence; helices A, B, C, D;
loops A/B, B/C, C/D; of four-helical-bundle cytokines. Such fusion
proteins would be expected to have a biological functional profile
that is the same or similar to polypeptides of the present
invention or other known four-helical-bundle cytokine family
proteins, depending on the fusion constructed. Moreover, such
fusion proteins may exhibit other properties as disclosed
herein.
[0127] Standard molecular biological and cloning techniques can be
used to swap the equivalent domains between the zalpha11 Ligand
polypeptide and those polypeptides to which they are fused.
Generally, a DNA segment that encodes a domain of interest, e.g.,
zalpha11 Ligand helices A through D, or other domain described
herein, is operably linked in frame to at least one other DNA
segment encoding an additional polypeptide (for instance a domain
or region from another cytokine, such as the IL-2, or the like),
and inserted into an appropriate expression vector, as described
herein. Generally DNA constructs are made such that the several DNA
segments that encode the corresponding regions of a polypeptide are
operably linked in frame to make a single construct that encodes
the entire fusion protein, or a functional portion thereof. For
example, a DNA construct would encode from N-terminus to C-terminus
a fusion protein comprising a signal polypeptide followed by a
mature four helical bundle cytokine fusion protein containing helix
A, followed by helix B, followed by helix C, followed by helix D.
Such fusion proteins can be expressed, isolated, and assayed for
activity as described herein.
[0128] Zalpha11 Ligand polypeptides or fragments thereof may also
be prepared through chemical synthesis. zalpha11 Ligand
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; and may or may not
include an initial methionine amino acid residue. For example, the
polypeptides can be prepared by solid phase peptide synthesis, for
example as described by Merrifield, J. Am. Chem. Soc. 85:2149,
1963.
[0129] The activity of molecules of the present invention can be
measured using a variety of assays that measure proliferation of
and/or binding to cells expressing the zalpha11 receptor. Of
particular interest are changes in zalpha11 Ligand-dependent cells.
Suitable cell lines to be engineered to be zalpha11
Ligand-dependent include the IL-3-dependent BaF3 cell line
(Palacios and Steinmetz, Cell 41: 727-734, 1985; Mathey-Prevot et
al., Mol. Cell. Biol. 6: 4133-4135, 1986), FDC-P1 (Hapel et al.,
Blood 64: 786-790, 1984), and MO7e (Kiss et al., Leukemia 7:
235-240, 1993). Growth factor-dependent cell lines can be
established according to published methods (e.g. Greenberger et
al., Leukemia Res. 8: 363-375, 1984; Dexter et al., in Baum et al.
Eds., Experimental Hematology Today, 8th Ann. Mtg. Int. Soc. Exp.
Hematol. 1979, 145-156, 1980).
[0130] Proteins of the present invention are useful for stimulating
proliferation, activation, differentiation and/or induction or
inhibition of specialized cell function of cells of the involved
homeostasis of the hematopoiesis and immune function. In
particular, zalpha11 Ligand polypeptides are useful for stimulating
proliferation, activation, differentiation, induction or inhibition
of specialized cell functions of cells of the hematopoietic
lineages, including, but not limited to, T cells, B cells, NK
cells, dendritic cells, monocytes, and macrophages, as well as
epithelial cells. Proliferation and/or differentiation of
hematopoietic cells can be measured in vitro using cultured cells
or in vivo by administering molecules of the claimed invention to
the 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, incorporated herein by reference), incorporation
of radiolabelled nucleotides (Cook et al., Analytical Biochem.
179:1-7, 1989, incorporated herein by reference), incorporation of
5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells
(Porstmann et al., J. Immunol. Methods 82:169-179, 1985,
incorporated herein by reference), 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; all
incorporated herein by reference). 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).
[0131] The molecules of the present invention can be assayed in
vivo using 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 acid (for
review, see T. C. Becker et al., Meth. Cell Biol. 43:161-89, 1994;
and J. T. Douglas and D. T. Curiel, Science & Medicine 4:44-53,
1997).
[0132] As a ligand, the activity of zalpha11 Ligand polypeptide can
be measured by a silicon-based biosensor microphysiometer which
measures the extracellular acidification rate or proton excretion
associated with receptor binding and subsequent physiologic
cellular responses. An exemplary 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, H. M. et al., Science
257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol.
228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59,
1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95,
1998.
[0133] Moreover, zalpha11 Ligand can be used to identify cells,
tissues, or cell lines which respond to a zalpha11
Ligand-stimulated pathway. The microphysiometer, described above,
can be used to rapidly identify ligand-responsive cells, such as
cells responsive to zalpha11 Ligand of the present invention. Cells
can be cultured in the presence or absence of zalpha11 Ligand
polypeptide. Those cells which elicit a measurable change in
extracellular acidification in the presence of zalpha11 Ligand are
responsive to zalpha11 Ligand. Such cells or cell lines, can be
used to identify antagonists and agonists of zalpha11 Ligand
polypeptide as described above.
[0134] In view of the tissue distribution observed for zalpha11
receptor agonists (including the natural zalpha11 Ligand
substrate/cofactor/etc.) and/or antagonists have enormous potential
in both in vitro and in vivo applications. Compounds identified as
zalpha11 Ligand agonists are useful for expansion, proliferation,
activation, differentiation, and/or induction or inhibition of
specialized cell functions of cells involved in homeostasis of
hematopoiesis and immune function. For example, zalpha11 Ligand and
agonist compounds are useful as components of defined cell culture
media, and may be used alone or in combination with other cytokines
and hormones to replace serum that is commonly used in cell
culture. Agonists are thus useful in specifically promoting the
growth and/or development of T-cells, B-cells, NK cells, cytotoxic
lymphocytes, and other cells of the lymphoid and myeloid lineages
in culture.
[0135] Antagonists are also useful as research reagents for
characterizing sites of ligand-receptor interaction. Antagonists
are useful to inhibit expansion, proliferation, activation, and/or
differentiation of cells involved in regulating hematopoiesis.
Inhibitors of zalpha11 Ligand activity (zalpha11 Ligand
antagonists) include anti-zalpha11 Ligand antibodies and soluble
zalpha11 Ligand receptors, as well as other peptidic and
non-peptidic agents (including ribozymes).
[0136] zalpha11 Ligand can also be used to identify inhibitors
(antagonists) of its activity. Test compounds are added to the
assays disclosed herein to identify compounds that inhibit the
activity of zalpha11 Ligand. In addition to those assays disclosed
herein, samples can be tested for inhibition of zalpha11 Ligand
activity within a variety of assays designed to measure receptor
binding, the stimulation/inhibition of zalpha11 Ligand-dependent
cellular responses or proliferation of zalpha11 receptor-expressing
cells.
[0137] A zalpha11 Ligand polypeptide can be expressed as a fusion
with an immunoglobulin heavy chain constant region, typically an Fc
fragment, which contains two constant region domains and lacks the
variable region. Methods for preparing such fusions are disclosed
in U.S. Pat. Nos. 5,155,027 and 5,567,584. Such fusions are
typically secreted as multimeric molecules wherein the Fc portions
are disulfide bonded to each other and two non-Ig polypeptides are
arrayed in closed proximity to each other. Fusions of this type can
be used for example, for dimerization, increasing stability and in
vivo half-life, to affinity purify ligand, as in vitro assay tool
or antagonist. For use in assays, the chimeras are bound to a
support via the Fe region and used in an ELISA format.
[0138] A zalpha11 Ligand-binding polypeptide can also be used for
purification of ligand. The polypeptide is immobilized on a solid
support, such as beads of agarose, cross-linked agarose, glass,
cellulosic resins, silica-based resins, polystyrene, cross-linked
polyacrylamide, or like materials that are stable under the
conditions of use. Methods for linking polypeptides to solid
supports are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide
activation. The resulting medium will generally be configured in
the form of a column, and fluids containing ligand are passed
through the column one or more times to allow ligand to bind to the
receptor polypeptide. The ligand is then eluted using changes in
salt concentration, chaotropic agents (guanidine HCl), or pH to
disrupt ligand-receptor binding.
[0139] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available biosensor
instrument (BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may be
advantageously employed. Such receptor, antibody, member of a
complement/anti-complement pair or fragment is immobilized onto the
surface of a receptor chip. Use of this instrument is disclosed by
Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and
Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member
or fragment is covalently attached, using amine or sulfhydryl
chemistry, to dextran fibers that are attached to gold film within
the flow cell. A test sample is passed through the cell. If a
ligand, epitope, or opposite member of the
complement/anti-complement pair is present in the sample, it will
bind to the immobilized receptor, antibody or member, respectively,
causing a change in the refractive index of the medium, which is
detected as a change in surface plasmon resonance of the gold film.
This system allows the determination of on- and off-rates, from
which binding affinity can be calculated, and assessment of
stoichiometry of binding. Alternatively, ligand/receptor binding
can be analyzed using SELDI(.TM.) technology (Ciphergen, Inc., Palo
Alto, Calif.).
[0140] Ligand-binding receptor polypeptides can also be used within
other assay systems known in the art. Such systems include
Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949) and calorimetric
assays (Cunningham et al., Science 253:545-48, 1991; Cunningham et
al., Science 245:821-25, 1991).
[0141] Zalpha11 Ligand polypeptides can also be used to prepare
antibodies that bind to zalpha11 Ligand epitopes, peptides or
polypeptides. The zalpha11 Ligand polypeptide or a fragment thereof
serves as an antigen (immunogen) to inoculate an animal and elicit
an immune response. One of skill in the art would recognize that
antigenic, epitope-bearing polypeptides contain a sequence of at
least 6, preferably at least 9, and more preferably at least 15 to
about 30 contiguous amino acid residues of a zalpha11 Ligand
polypeptide (e.g., SEQ ID NO:2). Polypeptides comprising a larger
portion of a zalpha11 Ligand polypeptide, i.e., from 30 to 100
residues up to the entire length of the amino acid sequence are
included. Antigens or immunogenic epitopes can also include
attached tags, adjuvants and carriers, as described herein.
Suitable antigens include the zalpha11 Ligand polypeptide encoded
by SEQ ID NO:2 from amino acid number 32 to amino acid number 162,
or a contiguous 9 to 131 amino acid fragment thereof. Other
suitable antigens include, the full length and the mature zalpha11
Ligand, helices A-D, and individual or multiple helices A, B, C,
and D, of the zalpha11 Ligand four-helical-bundle structure, as
described herein. Preferred peptides to use as antigens are
hydrophilic peptides such as those predicted by one of skill in the
art from a hydrophobicity plot, as described herein, for example,
amino acid residues 114-119, 101-105, 126-131, 113-118, and 158-162
of SEQ ID NO:2.
[0142] Antibodies from an immune response generated by inoculation
of an animal with these antigens can be isolated and purified as
described herein. Methods for preparing and isolating polyclonal
and monoclonal antibodies are well known in the art. See, for
example, Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc., 1995;
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R.,
Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications,
CRC Press, Inc., Boca Raton, Fla., 1982.
[0143] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated from inoculating a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, and rats with a zalpha11 Ligand
polypeptide or a fragment thereof. The immunogenicity of a zalpha11
Ligand 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 zalpha11 Ligand 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.
[0144] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab')2 and Fab
proteolytic fragments. Genetically engineered intact antibodies or
fragments, such as chimeric antibodies, Fv fragments, single chain
antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. 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. Moreover, 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.
[0145] Antibodies are considered to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and 2) they do
not significantly cross-react with related polypeptide molecules. A
threshold level of binding is determined if anti-zalpha11 Ligand
antibodies herein bind to a zalpha11 Ligand polypeptide, peptide or
epitope with an affinity at least 10-fold greater than the binding
affinity to control (non-zalpha11 Ligand) polypeptide. It is
preferred that the antibodies exhibit a binding affinity (Ka) of
10.sup.6 M.sup.-1 or greater, preferably 107 M.sup.-1 or greater,
more preferably 108 M.sup.-1 or greater, and most preferably 109
M.sup.-1 or greater. The binding affinity of an antibody can be
readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, G., Ann. NY Acad. Sci.
51: 660-672, 1949).
[0146] Whether anti-zalpha11 Ligand antibodies do not significantly
cross-react with related polypeptide molecules is shown, for
example, by the antibody detecting zalpha11 Ligand polypeptide but
not known related polypeptides using a standard Western blot
analysis (Ausubel et al., ibid.). Examples of known related
polypeptides are those disclosed in the prior art, such as known
orthologs, and paralogs, and similar known members of a protein
family. Screening can also be done using non-human zalpha11 Ligand,
and zalpha11 Ligand mutant polypeptides. Moreover, antibodies can
be "screened against" known related polypeptides, to isolate a
population that specifically binds to the zalpha11 Ligand
polypeptides. For example, antibodies raised to zalpha11 Ligand are
adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to zalpha11 Ligand will flow through the matrix
under the proper buffer conditions. Screening allows isolation of
polyclonal and monoclonal antibodies non-crossreactive to known
closely related polypeptides (Antibodies: A Laboratory Manual,
Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current Protocols in Immunology, Cooligan, et al. (eds.), National
Institutes of Health, John Wiley and Sons, Inc., 1995). Screening
and isolation of specific antibodies is well known in the art. See,
Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et
al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd.,
1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.
Specifically binding anti-zalpha11 Ligand antibodies can be
detected by a number of methods in the art, and disclosed
below.
[0147] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which bind to zalpha11 Ligand
proteins or 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,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
zalpha11 Ligand protein or polypeptide.
[0148] Antibodies to zalpha11 Ligand may be used for tagging cells
that express zalpha11 Ligand; for isolating zalpha11 Ligand by
affinity purification; for diagnostic assays for determining
circulating levels of zalpha11 Ligand polypeptides; for detecting
or quantitating soluble zalpha11 Ligand as a marker of underlying
pathology or disease; in analytical methods employing FACS; for
screening expression libraries; for generating anti-idiotypic
antibodies; and as neutralizing antibodies or as antagonists to
block zalpha11 Ligand activity in vitro and in vivo. 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
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to zalpha11 Ligand or fragments thereof may be used in
vitro to detect denatured zalpha11 Ligand or fragments thereof in
assays, for example, Western Blots or other assays known in the
art.
[0149] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria, toxin, saporin, Pseudomonas exotoxin, ricin,
abrin and the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/anticomplementary
pair.
[0150] Binding polypeptides can also act as zalpha11 Ligand
"antagonists" to block zalpha11 Ligand binding and signal
transduction in vitro and in vivo. These anti-zalpha11 Ligand
binding polypeptides would be useful for inhibiting zalpha11 Ligand
activity or protein-binding.
[0151] Polypeptide-toxin fusion proteins or antibody-toxin fusion
proteins can be used for targeted cell or tissue inhibition or
ablation (for instance, to treat cancer cells or tissues).
Alternatively, if the polypeptide has multiple functional domains
(i.e., an activation domain or a receptor binding domain, plus a
targeting domain), a fusion protein including only the targeting
domain may be suitable for directing a detectable molecule, a
cytotoxic molecule or a complementary molecule to a cell or tissue
type of interest. In instances where the domain only fusion protein
includes a complementary molecule, the anti-complementary molecule
can be conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0152] Zalpha11 Ligand cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for enhancing in vivo
killing of target tissues (for example, blood and bone marrow
cancers), if the zalpha11 Ligand polypeptide or anti-zalpha11
Ligand antibody targets the hyperproliferative blood or bone marrow
cell (See, generally, Hornick et al., Blood 89:4437-47, 1997). The
described fusion proteins enable targeting of a cytokine to a
desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable zalpha11 Ligand polypeptides or
anti-zalpha11 Ligand antibodies target an undesirable cell or
tissue (i.e., a tumor or a leukemia), and the fused cytokine
mediated improved target cell lysis by effector cells. Suitable
cytokines for this purpose include interleukin 2 and
granulocyte-macrophage colony-stimulating factor (GM-CSF), for
instance.
[0153] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products, and receptors. The stage of a cell population's
differentiation is monitored by identification of markers present
in the cell population.
[0154] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation affect the entire cell population originating
from a common precursor or stem cell. Thus, the present invention
includes stimulating or inhibiting the proliferation of lymphoid
cells, hematopoietic cells and epithelial cells.
[0155] Zalpha11 Ligand was isolated from tissue known to have
important immunological function and which contain cells that play
a role in the immune system. Zalpha11 Ligand is expressed in CD3+
selected, activated peripheral blood cells, and it has been shown
that zalpha11 Ligand expression increases after T cell activation.
Moreover, results of experiments described in the Examples section
herein demonstrate that polypeptides of the present invention have
an effect on the growth/expansion and/or differentiated state of NK
cells or NK progenitors. Additional evidence demonstrates that
zalpha11 Ligand affects proliferation and/or differentiation of T
cells and B cells in vivo. Factors that both stimulate
proliferation of hematopoietic progenitors and activate mature
cells are generally known. NK cells are responsive to IL-2 alone,
but proliferation and activation generally require additional
growth factors. For example, it has been shown that IL-7 and Steel
Factor (c-kit ligand) were required for colony formation of NK
progenitors. IL-15+IL-2 in combination with IL-7 and Steel Factor
was more effective (Mrozek et al., Blood 87:2632-2640, 1996).
However, unidentified cytokines may be necessary for proliferation
of specific subsets of NK cells and/or NK progenitors (Robertson
et. al., Blood 76:2451-2438, 1990). A composition comprising
zalpha11 Ligand and IL-15 stimulates NK progenitors and NK cells,
with evidence that this composition is more potent than previously
described factors and combinations of factors.
[0156] Assays measuring differentiation include, for example,
measuring cell 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).
Alternatively, zalpha11 Ligand polypeptide itself can serve as an
additional cell-surface or secreted marker associated with
stage-specific expression of a tissue. As such, direct measurement
of zalpha11 Ligand polypeptide, or its loss of expression in a
tissue as it differentiates, can serve as a marker for
differentiation of tissues.
[0157] Similarly, direct measurement of zalpha11 Ligand
polypeptide, or its loss of expression in a tissue can be
determined in a tissue or in cells as they undergo tumor
progression. Increases in invasiveness and motility of cells, or
the gain or loss of expression of zalpha11 Ligand 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 zalpha11
Ligand 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, zalpha11 Ligand gain or loss of expression may serve
as a diagnostic for lymphoid, B-cell, epithelial, hematopoietic and
other cancers.
[0158] Moreover, the activity and effect of zalpha11 Ligand 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/J 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/J 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 zalpha11 Ligand,
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 mm3 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.,
zalpha11 Ligand, 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 zalpha11 Ligand. Use of stable zalpha11 Ligand
transfectants as well as use of induceable promoters to activate
zalpha11 Ligand expression in vivo are known in the art and can be
used in this system to assess zalpha11 Ligand induction of
metastasis. Moreover, purified zalpha11 Ligand or zalpha11 Ligand
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.
[0159] Zalpha11 Ligand will be useful in treating tumorgenesis, and
therefore would be useful in the treatment of cancer. Zalpha11
Ligand inhibits IL-4 stimulated proliferation of anti-IgM
stimulated normal B-cells and a similar effect is observed in
B-cell tumor lines suggesting that there may be therapeutic benefit
in treating patients with the zalpha11 Ligand 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 zalpha11 Ligand are additive for at least one
B-cell tumor-derived cell line.
[0160] 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 zalpha11 Ligand sufficient to reduce proliferation of the
neoplastic B or T cells. In other embodiments, the composition can
comprise at least one other cytokine selected from the group
consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand or stem cell
factor.
[0161] 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 zalpha11 Ligand antagonist sufficient to reducing
proliferation of the neoplastic B or T cells. In other embodiments,
the composition can comprise at least one other cytokine selected
from the group consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand
or stem cell factor. Furthermore, the zalpha11 Ligand antagonist
can be a ligand/toxin fusion protein.
[0162] A zalpha11 Ligand-saporin fusion toxin may be employed
against a similar set of leukemias and lymphomas, extending the
range of leukemias that can be treated with zalpha11 Ligand. Fusion
toxin mediated activation of the zalpha11 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. The lymphoid restricted expression pattern of the
zalpha11 receptor suggests that the ligand-saporin conjugate can be
tolerated by patients.
[0163] When treatment for malignancies includes allogeneic bone
marrow or stem cell transplantion, zalpha11 Ligand may be valuable
in enhancement of the graft-vs-tumor effect. zalpha11 Ligand
stimulates the generation of lytic NK cells from marrow progenitors
and stimulates the proliferation of T-cells following activation of
the antigen receptors. Therefore, when patients receive allogenic
marrow transplants, zalpha11 Ligand will enhance the generation of
anti-tumor responses, with or without the infusion of donor
lymphocytes.
[0164] The tissue distribution of a receptor for a given cytokine
offers a strong indication of the potential sites of action of that
cytokine. Northern analysis of zalpha11 receptor revealed
transcripts in human spleen, thymus, lymph node, bone marrow, and
peripheral blood leukocytes. Specific cell types were identified as
expressing zalpha11 receptors, and strong signals were seen in a
mixed lymphocyte reaction (MLR) and in the Burkitt's lymphoma Raji.
The two monocytic cell lines, THP-1 (Tsuchiya et al., Int. J.
Cancer 26:171-176, 1980) and U937 (Sundstrom et al., Int. J. Cancer
17:565-577, 1976), were negative.
[0165] Zalpha11 receptor is expressed at relatively high levels in
the MLR, in which peripheral blood mononuclear cells (PBMNC) from
two individuals are mixed, resulting in mutual activation.
Detection of high levels of transcript in the MLR but not in
resting T or B cell populations suggests that zalpha11 receptor
expression may be induced in one or more cell types during
activation. Activation of isolated populations of T and B cells can
be artificially achieved by stimulating cells with PMA and
ionomycin. When sorted cells were subjected to these activation
conditions, levels of zalpha11 receptor transcript increased in
both cell types, supporting a role for this receptor and zalpha11
Ligand in immune responses, especially in autocrine and paracrine T
and B cell expansions during activation. Zalpha11 Ligand may also
play a role in the expansion of more primitive progenitors involved
in lymphopoiesis.
[0166] zalpha11 receptor was found to be present at low levels in
resting T and B cells, and was upregulated during activation in
both cell types. Interestingly, the B cells also down-regulate the
message more quickly than do T cells, suggesting that amplitude of
signal and timing of quenching of signal are important for the
appropriate regulation of B cell responses.
[0167] In addition, a large proportion of intestinal lamina propria
cells show positive hybridization signals with zalpha11 receptor.
This tissue consists of a mixed population of lymphoid cells,
including activated CD4+ T cells and activated B cells. Immune
dysfunction, in particular chronic activation of the mucosal immune
response, plays an important role in the etiology of Crohn's
disease; abnormal response to and/or production of proinflammatory
cytokines is also a suspected factor (Braegger et al., Annals
Allergy 72:135-141, 1994; Sartor R B Am. J. Gastroenterol.
92:5S-11S, 1997. zalpha11 Ligand in concert with IL-15 expands NK
cells from bone marrow progenitors and augments NK cell effector
function. zalpha11 Ligand also co-stimulates mature B cells
stimulated with anti-CD40 antibodies, but inhibits B cell
proliferation to signals through IgM. zalpha11 Ligand enhances T
cell proliferation in concert with a signal through the T cell
receptor, and overexpression in transgenic mice leads to
lymphopenia and an expansion of monocytes and granulocytes. These
pleiotropic effects of zalpha11 Ligand suggest that it can provide
therapeutic utility for a wide range of diseases arising from
defects in the immune system, including (but not limited to)
systemic lupus erythematosus (SLE), rheumatoid arthritis (RA),
multiple sclerosis (MS), myasthenia gravis, and diabetes. It is
important to note that these diseases are the result of a complex
network of immune dysfunction (SLE, for example, is the
manifestation of defects in both T and B cells), and that immune
cells are dependent upon interaction with one another to elicit a
potent immune response. Therefore, zalpha11 Ligand (or an
antagonist of the Ligand) that can be used to manipulate more than
one type of immune cell is an attractive therapeutic candidate for
intervention at multiple stages of disease.
[0168] The polypeptides and proteins of the present invention can
also be used ex vivo, such as in autologous marrow culture.
Briefly, bone marrow is removed from a patient prior to
chemotherapy or organ transplant and treated with zalpha11 Ligand,
optionally in combination with one or more other cytokines. The
treated marrow is then returned to the patient after chemotherapy
to speed the recovery of the marrow or after transplant to suppress
graft vs. Host disease. In addition, the proteins of the present
invention can also be used for the ex vivo expansion of marrow or
peripheral blood progenitor (PBPC) cells. Prior to treatment,
marrow can be stimulated with stem cell factor (SCF) to release
early progenitor cells into peripheral circulation. These
progenitors can be collected and concentrated from peripheral blood
and then treated in culture with zalpha11 Ligand, optionally in
combination with one or more other cytokines, including but not
limited to SCF, IL-2, IL-4, IL-7 or IL-15, to differentiate and
proliferate into high-density lymphoid cultures, which can then be
returned to the patient following chemotherapy or
transplantation.
[0169] The present invention provides a method for expansion of
hematopoietic cells and hematopoietic cell progenitors comprising
culturing bone marrow or peripheral blood cells with a composition
comprising an amount of zalpha11 Ligand sufficient to produce an
increase in the number of lymphoid cells in the bone marrow or
peripheral blood cells as compared to bone marrow or peripheral
blood cells cultured in the absence of zalpha11 Ligand. In other
embodiments, the hematopoietic cells and hematopoietic progenitor
cells are lymphoid cells. In another embodiment, the lymphoid cells
are NK cells or cytotoxic T cells. Furthermore, the composition can
also comprise at least one other cytokine selected from the group
consisting of IL-2, IL-15, IL-4, GM-CSF, Flt3 ligand and stem cell
factor.
[0170] Alternatively, zalpha11 Ligand may activate the immune
system which would be important in boosting immunity to infectious
diseases, treating immunocompromised patients, such as HIV+
patients, or in improving vaccines. In particular, zalpha11 Ligand
stimulation or expansion of NK cells, or their progenitors, would
provide therapeutic value in treatment of viral infection, and as
an anti-neoplastic factor. NK cells are thought to play a major
role in elimination of metastatic tumor cells and patients with
both metastases and solid tumors have decreased levels of NK cell
activity (Whiteside et. al., Curr. Top. Microbiol. Immunol.
230:221-244, 1998). Similarly, zalpha11 Ligand stimulation of the
immune response against viral and non-viral pathogenic agents
(including bacteria, protozoa, and fungi) would provide therapeutic
value in treatment of such infections by inhibiting the growth of
such infections agents. Determining directly or indirectly the
levels of a pathogen or antigen, such as a tumor cell, present in
the body can be achieved by a number of methods known in the art
and described herein.
[0171] The present invention include a method of stimulating an
immune response in a mammal exposed to an antigen or pathogen
comprising the steps of: (1) determining directly or indirectly the
level of antigen or pathogen present in said mammal; (2)
administering a composition comprising zalpha11 Ligand polypeptide
in an acceptable pharmaceutical vehicle; (3) determining directly
or indirectly the level of antigen or pathogen in said mammal; and
(4) comparing the level of the antigen or pathogen in step 1 to the
antigen or pathogen level in step 3, wherein a change in the level
is indicative of stimulating an immune response. In another
embodiment the zalpha11 Ligand composition is re-administered. In
other embodiments, the antigen is a B cell tumor; a virus; a
parasite or a bacterium.
[0172] In another aspect, the present invention provides a method
of stimulating an immune response in a mammal exposed to an antigen
or pathogen comprising: (1) determining a level of an antigen- or
pathogen-specific antibody; (2) administering a composition
comprising zalpha11 Ligand polypeptide in an acceptable
pharmaceutical vehicle; (3) determining a post administration level
of antigen- or pathogen-specific antibody; (4) comparing the level
of antibody in step (1) to the level of antibody in step (3),
wherein an increase in antibody level is indicative of stimulating
an immune response.
[0173] Polynucleotides encoding zalpha11 Ligand polypeptides are
useful within gene therapy applications where it is desired to
increase or inhibit zalpha11 Ligand activity. If a mammal has a
mutated or absent zalpha11 Ligand gene, the zalpha11 Ligand gene
can be introduced into the cells of the mammal. In one embodiment,
a gene encoding a zalpha11 Ligand polypeptide is introduced in vivo
in a viral vector. Such vectors include an attenuated or defective
DNA virus, such as, but not limited to, herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus (EBV), adenovirus,
adeno-associated virus (AAV), and the like. Defective viruses,
which entirely or almost entirely lack viral genes, are preferred.
A defective virus is not infective after introduction into a cell.
Use of defective viral vectors allows for administration to cells
in a specific, localized area, without concern that the vector can
infect other cells. Examples of particular vectors include, but are
not limited to, a defective herpes simplex virus 1 (HSV1) vector
(Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an
attenuated adenovirus vector, such as the vector described by
Stratford-Perricaudet et al., J. Clin. Invest. 90:626-30, 1992; and
a defective adeno-associated virus vector (Samulski et al., J.
Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8,
1989).
[0174] A zalpha11 Ligand gene can be introduced in a retroviral
vector, e.g., as described in Anderson et al., U.S. Pat. No.
5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S. Pat.
No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et
al., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat. No.
5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845, 1993. Alternatively, the vector can be introduced by
lipofection in vivo using liposomes. Synthetic cationic lipids can
be used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA
85:8027-31, 1988). The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. More particularly, directing
transfection to particular cells represents one area of benefit.
For instance, directing transfection to particular cell types would
be particularly advantageous in a tissue with cellular
heterogeneity, such as the immune system, pancreas, liver, kidney,
and brain. Lipids may be chemically coupled to other molecules for
the purpose of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide
molecules can be coupled to liposomes chemically.
[0175] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0176] Antisense methodology can be used to inhibit zalpha11 Ligand
gene transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a zalpha11
Ligand-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to zalpha11 Ligand-encoding
mRNA and to inhibit translation of such mRNA. Such antisense
polynucleotides are used to inhibit expression of zalpha11 Ligand
polypeptide-encoding genes in cell culture or in a subject.
[0177] Mice engineered to express the zalpha11 Ligand gene,
referred to as "transgenic mice," and mice that exhibit a complete
absence of zalpha11 Ligand gene function, referred to as "knockout
mice," may also be generated (Snouwaert et al., Science 257:1083,
1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M. R.,
Science 244: 1288-1292, 1989; Palmiter, R. D. et al. Annu Rev
Genet. 20: 465-499, 1986). For example, transgenic mice that
over-express zalpha11 Ligand, either ubiquitously or under a
tissue-specific or tissue-restricted promoter can be used to ask
whether over-expression causes a phenotype. For example,
over-expression of a wild-type zalpha11 Ligand polypeptide,
polypeptide fragment or a mutant thereof may alter normal cellular
processes, resulting in a phenotype that identifies a tissue in
which zalpha11 Ligand expression is functionally relevant and may
indicate a therapeutic target for the zalpha11 Ligand, its agonists
or antagonists. For example, a preferred transgenic mouse to
engineer is one that over-expresses the zalpha11 Ligand (amino acid
residues 32-162 of SEQ ID NO:2). Moreover, such over-expression may
result in a phenotype that shows similarity with human diseases.
Similarly, knockout zalpha11 Ligand mice can be used to determine
where zalpha11 Ligand is absolutely required in vivo. The phenotype
of knockout mice is predictive of the in vivo effects of that a
zalpha11 Ligand antagonist, such as those described herein, may
have. The human or mouse zalpha11 Ligand cDNA can be used to
generate knockout mice. These mice may be employed to study the
zalpha11 Ligand gene and the protein encoded thereby in an in vivo
system, and can be used as in vivo models for corresponding human
diseases. Moreover, transgenic mice expression of zalpha11 Ligand
antisense polynucleotides or ribozymes directed against zalpha11
Ligand, described herein, can be used analogously to transgenic
mice described above. Studies may be carried out by administration
of purified zalpha11 Ligand protein, as well.
[0178] For pharmaceutical use, the proteins of the present
invention are formulated for parenteral, particularly intravenous
or subcutaneous, delivery according to conventional methods. The
bioactive polypeptide or antibody conjugates described herein can
be delivered intravenously, intraarterially or intraductally, or
may be introduced locally at the intended site of action.
Intravenous administration will be by bolus injection or infusion
over a typical period of one to several hours. In general,
pharmaceutical formulations will include a zalpha11 Ligand protein
in combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. 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. Therapeutic doses will generally
be in the range of 0.1 to 100 .mu.g/kg of patient weight per day,
preferably 0.5-20 .mu.g/kg per day, with the exact dose determined
by the clinician according to accepted standards, taking into
account the nature and severity of the condition to be treated,
patient traits, etc. Determination of dose is within the level of
ordinary skill in the art. The proteins may be administered for
acute treatment, over one week or less, often over a period of one
to three days or may be used in chronic treatment, over several
months or years. In general, a therapeutically effective amount of
zalpha11 Ligand is an amount sufficient to produce a clinically
significant change in hematopoietic or immune function.
[0179] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0180] Construction of MPL-zalpha11 Polypeptide Chimera: MPL
Extracellular and .TM. Domain Fused to the zalpha11 Intracellular
Signaling Domain
[0181] The extracellular and transmembrane domains of the murine
MPL receptor were isolated from a plasmid containing the murine MPL
receptor (PHZ1/MPL plasmid) using PCR with primers ZC17,212 (SEQ ID
NO:5) and ZC19,914 (SEQ ID NO:6). The reaction conditions were as
follows: 95.degree. C. for 1 min.; 35 cycles at 95.degree. C. for 1
min., 45.degree. C. for 1 min., 72.degree. C. for 2 min.; followed
by 72.degree. C. at 10 min.; then a 10.degree. C. soak. The PCR
product was run on a 1% low melting point agarose (Boerhinger
Mannheim, Indianapolis, Ind.) and the approximately 1.5 kb MPL
receptor fragment isolated using Qiaquick.TM. gel extraction kit
(Qiagen) as per manufacturer's instructions.
[0182] The intracellular domains of human zalpha11 were isolated
from a plasmid containing zalpha11 receptor cDNA using PCR with
primers ZC19,913 (SEQ ID NO:8) and ZC20,097 (SEQ ID NO:9). The
polynucleotide sequence corresponding to the zalpha11 receptor
coding sequence is shown in SEQ ID NO:7, and the corresponding
amino acid sequence shown in SEQ ID NO: 115. The reaction
conditions were as per above. The PCR product was run on a 1% low
melting point agarose (Boerhinger Mannheim) and the approximately
900 bp zalpha11 fragment isolated using Qiaquick gel extraction kit
as per manufacturer's instructions.
[0183] Each of the isolated fragments described above were mixed at
a 1:1 volumetric ratio and used in a PCR reaction using ZC17,212
(SEQ ID NO:5) and ZC20,097 (SEQ ID NO:9) to create the MPL-zalpha11
chimera. The reaction conditions were as follows: 95.degree. C. for
1 min.; 35 cycles at 95.degree. C. for 1 min., 55.degree. C. for 1
min., 72.degree. C. for 2 min.; followed by 72.degree. C. at 10
min.; then a 10.degree. C. soak. The entire PCR product was run on
a 1% low melting point agarose (Boehringer Mannheim) and the
approximately 2.4 kb MPL-zalpha11 chimera fragment isolated using
Qiaquick gel extraction kit (Qiagen) as per manufacturer's
instructions. The MPL-zalpha11 chimera fragment was digested with
EcoRI (BRL) and XbaI (Boerhinger Mannheim) as per manufacturer's
instructions. The entire digest was run on a 1% low melting point
agarose (Boehringer Mannheim) and the cleaved MPL-zalpha11 chimera
isolated using Qiaquick.TM. gel extraction kit (Qiagen) as per
manufacturer's instructions. The resultant cleaved MPL-zalpha11
chimera was inserted into an expression vector as described
below.
[0184] Recipient expression vector pZP-5N was digested with EcoRI
(BRL) and HindIII (BRL) as per manufacturer's instructions, and gel
purified as described above. This vector fragment was combined with
the EcoRI and XbaI cleaved MPL-zalpha11 chimera isolated above and
a XbaI/HindIII linker fragment in a ligation reaction. The ligation
was run using T4 Ligase (BRL), at 15.degree. C. overnight. A sample
of the ligation was electroporated in to DH10B ElectroMAX.TM.
electrocompetent E. coli cells (25 .mu.F, 200.OMEGA., 2.3V).
Transformants were plated on LB+Ampicillin plates and single
colonies screened by PCR to check for the MPL-zalpha11 chimera
using ZC17,212 (SEQ ID NO:5) and ZC20,097 (SEQ ID NO:9) using the
PCR conditions as described above.
[0185] Confirmation of the MPL-zalpha11 chimera sequence was made
by sequence analyses using the following primers: ZC12,700 (SEQ ID
NO:10), ZC5,020 (SEQ ID NO:11), ZC6,675 (SEQ ID NO:12), ZC7,727
(SEQ ID NO:13), ZC8,290 (SEQ ID NO:14), ZC19,572 (SEQ ID NO:15),
ZC6,622 (SEQ ID NO:16), ZC7,736 (SEQ ID NO:17), and ZC9,273 (SEQ ID
NO:18). The insert was approximately 2.4 bp, and was
full-length.
Example 2
[0186] MPL-zalpha11 Chimera Based Proliferation in BAF3 Assay Using
Alamar Blue
[0187] A. Construction of BaF3 Cells Expressing MPL-zalpha11
Chimera
[0188] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, pZP-5N/MPL-zalpha11 plasmid DNA (Example
1) was prepared and purified using a Qiagen Maxi Prep kit (Qiagen)
as per manufacturer's instructions. BaF3 cells for electroporation
were washed once in RPMI media and then resuspended in RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the pZP-5N/MPL-zalpha11 plasmid
DNA and transferred to separate disposable electroporation chambers
(GIBCO BRL). Following a 15 minute incubation at room temperature
the cells were given two serial shocks (800 lFad/300 V.; 1180
lFad/300 V.) delivered by an electroporation apparatus
(CELL-PORATOR.TM.; GIBCO BRL). After a 5 minute recovery time, the
electroporated cells were transferred to 50 ml of complete media
and placed in an incubator for 15-24 hours (37.degree. C., 5% CO2).
The cells were then spun down and resuspended in 50 ml of complete
media containing Geneticin.TM. (Gibco) selection (500 .mu.g/ml
G418) in a T-162 flask to isolate the G418-resistant pool. Pools of
the transfected BaF3 cells, hereinafter called BaF3/MPL-zalpha11
cells, were assayed for signaling capability as described
below.
[0189] B. Testing the Signaling Capability of the BaF3/MPL-zalpha11
Cells Using an Alamar Blue Proliferation Assay
[0190] BaF3 MPL-zalpha11 cells were spun down and washed in the
complete media, described above, but without mIL-3 (hereinafter
referred to as "mIL-3 free media"). The cells were spun and washed
3 times to ensure the removal of the mIL-3. Cells were then counted
in a hemacytometer. Cells were plated in a 96-well format at 5000
cells per well in a volume of 100 .mu.l per well using the mIL-3
free media.
[0191] Proliferation of the BaF3/MPL-zalpha11 cells was assessed
using murine mthrombopoietin (mTPO) diluted with mIL-3 free media
to 500 ng/ml, 250 ng/ml, 125 ng/ml, 62 ng/ml, 30 ng/ml, 15 ng/ml,
7.5 ng/ml, 3.75 ng/ml, 1.8 ng/ml, 0.9 ng/ml, 0.5 ng/ml and 0.25
ng/ml concentrations. 100 .mu.l of the diluted mTPO was added to
the BaF3/MPL-zalpha11 cells. The total assay volume is 200 .mu.l.
Negative controls were run in parallel using mIL-3 free media only,
without the addition of mTPO. The assay plates were incubated at
37.degree. C., 5% CO2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Alamar Blue
gives a fluourometric readout based on the metabolic activity of
cells, and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emmission).
[0192] Results confirmed the signaling capability of the
intracellular portion of the zalpha11 receptor, as the
thrombopoietin induced proliferation at approximately 10 fold over
back ground at mTPO concentrations of 62 ng/ml and greater.
Example 3
[0193] Construction of Expression Vector Expressing Full-Length
zalpha11
[0194] The entire zalpha11 receptor was isolated from a plasmid
containing zalpha11 receptor cDNA (SEQ ID NO:7) using PCR with
primers ZC19,905 (SEQ ID NO:19) and ZC19,906 (SEQ ID NO:20). The
reaction conditions were as follows: 95.degree. C. for 1 min; 35
cycles at 95.degree. C. for 1 min, 55.degree. C. for 1 min,
72.degree. C. for 2 min; followed by 72.degree. C. at 10 min; then
a 10.degree. C. soak. The PCR product was run on a 1% low melting
point agarose (Boerhinger Mannheim) gel and the approximately 1.5
kb zalpha11 cDNA isolated using Qiaquick.TM. gel extraction kit
(Qiagen) as per manufacturer's instructions.
[0195] The purified zalpha11 cDNA was digested with BamHI
(Boerhinger Mannheim) and EcoRI (BRL) as per manufacturer's
instructions. The entire digest was run on a 1% low melting point
agarose (Boerhinger Mannheim) gel and the cleaved zalpha11 fragment
was purified the using Qiaquick.TM. gel extraction kit as per
manufacturer's instructions. The resultant cleaved zalpha11
fragment was inserted into an expression vector as described
below.
[0196] Recipient expression vector pZP-5N was digested with BamHI
(Boerhinger Mannheim) and EcoRI (BRL) as per manufacturer's
instructions, and gel purified as described above. This vector
fragment was combined with the BamHI and EcoRI cleaved zalpha11
fragment isolated above in a ligation reaction using T4 Ligase
(BRL). The ligation was incubated at 15.degree. C. overnight. A
sample of the ligation was electroporated in to DH10B
electroMAX.TM. electrocompetent E. coli cells (25 .mu.F,
200.OMEGA., 2.3V). Transformants were plated on LB+Ampicillin
plates and single colonies screened by PCR to check for the
zalpha11 sequence using ZC19,905 (SEQ ID NO: 19) and ZC19,906 (SEQ
ID NO:20) using the PCR conditions as described above.
[0197] Confirmation of the zalpha11 sequence was made by sequence
analyses using the following primers: ZC12,700 (SEQ ID NO:10),
ZC5,020 (SEQ ID NO:11), ZC20,114 (SEQ ID NO:21), ZC19,459 (SEQ ID
NO:22), ZC19,954 (SEQ ID NO:23), and ZC20,116 (SEQ ID NO:24). The
insert was approximately 1.6 kb, and was full-length.
Example 4
[0198] Zalpha11 Based Proliferation in BAF3 Assay Using Alamar
Blue
[0199] A. Construction of BaF3 Cells Expressing zalpha11
Receptor
[0200] BaF3 cells expressing the full-length zalpha11 receptor was
constructed as per Example 2A above, using 30 .mu.g of the zalpha11
expression vector, described in Example 3 above. The BaF3 cells
expressing the zalpha11 receptor mRNA were designated as
BaF3/zalpha11. These cells were used to screen for zalpha11 Ligand
as described below in Examples 5 and 6.
Example 5
[0201] Screening for zalpha11 Ligand Using BaF3/Zalpha11 Cells
Using an Alamar Blue Proliferation Assay
[0202] A. Activation of Primary Monkey Splenocytes to Test for
Presence of zalpha11 Ligand
[0203] Monkey splenocytes were stimulated in vitro to produce
conditioned media to test for the presence of zalpha11 Ligand
activity as described below. Monkey spleens were obtained from 8
year old female M. nesestrian monkeys. The spleens were teased part
to produce a single cell suspension. The mononuclear cells were
isolated by Ficoll-Paque.RTM. PLUS (Pharmacia Biotech, Uppsala,
Sweden) density gradient. The mononuclear cells were seeded at
2.times.10.sup.6 cells/ml in RPMI-1640 media supplemented with 10%
FBS and activated with 5 ng/ml Phorbol-12-myristate-13-acetate
(PMA) (Calbiochem, San Diego, Calif.), and 0.5 mg/ml Ionomycin.TM.
(Calbiochem) for 48 hrs. The supernatant from the stimulated monkey
spleen cells was used to assay proliferation of the BaF3/zalpha11
cells as described below.
[0204] B. Screening for zalpha11 Ligand Using BaF3/Zalpha11 Cells
Using an Alamar Blue Proliferation Assay
[0205] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0206] Proliferation of the BaF3/Zalpha11 cells was assessed using
conditioned media from activated monkey spleen (see Example 5A).
Conditioned media was diluted with mIL-3 free media to 50%, 25%,
12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100
.mu.l of the diluted conditioned media was added to the
BaF3/Zalpha11 cells. The total assay volume is 200 .mu.l. The assay
plates were incubated at 37.degree. C., 5% CO.sub.2 for 3 days at
which time Alamar Blue (Accumed, Chicago, Ill.) was added at 20
.mu.l/well. Plates were again incubated at 37.degree. C., 5%
CO.sub.2 for 24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular devices) as described above (Example 2).
[0207] Results confirmed the proliferative response of the
BaF3/Zalpha11 cells to a factor present in the activated monkey
spleen conditioned media. The response, as measured, was
approximately 4-fold over background at the 50% concentration. The
untransfected BaF3 cells did not proliferate in response to this
factor, showing that this factor is specific for the Zalpha11
receptor.
[0208] C. Human Primary Source Used to Isolate zalpha11 Ligand
[0209] 100 ml blood draws were taken from each of six donors. The
blood was drawn using 10.times. 10 ml vacutainer tubes containing
heparin. Blood was pooled from six donors (600 ml), diluted 1:1 in
PBS, and separated using a Ficoll-Paque.RTM. PLUS (Pharmacia
Biotech). The isolated primary human cell yield after separation on
the ficoll gradient was 1.2.times.10.sup.9 cells.
[0210] Cells were suspended in 9.6 ml MACS buffer (PBS, 0.5% EDTA,
2 mM EDTA). 1.6 ml of cell suspension was removed and 0.4 ml CD3
microbeads (Miltenyi Biotec, Auburn, Calif.) added. The mixture was
incubated for 15 min. at 4.degree. C. These cells labeled with CD3
beads were washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0211] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS.TM. magnetic field (Miltenyi). The column was
equilibrated with 5 ml MACS buffer. The isolated primary human
cells were then applied to the column. The CD3 negative cells were
allowed to pass through. The column was rinsed with 9 ml (3.times.3
ml) MACS buffer. The column was then removed from the magnet and
placed over a 15 ml falcon tube. CD3+ cells were eluted by adding 5
ml MACS buffer to the column and bound cells flushed out using the
plunger provided by the manufacturer. The incubation of the cells
with the CD3 magnetic beads, washes, and VS+ column steps
(incubation through elution) above were repeated five more times.
The resulting CD3+ fractions from the six column separations were
pooled. The yield of CD3+ selected human cells were
3.times.10.sup.8 total cells.
[0212] A sample of the pooled CD3+ selected human cells was removed
for staining and sorting on a fluorescent antibody cell sorter
(FACS) to assess their purity. The human CD3+ selected cells were
91% CD3+ cells.
[0213] The human CD3+ selected cells were activated by incubating
in RPMI+5% FBS+PMA 10 ng/ml and Ionomycin 0.5 .mu.g/ml (Calbiochem)
for 13 hours 37.degree. C. The supernatant from these activated
CD3+ selected human cells was tested for zalpha11 Ligand activity
as described below. Moreover, the activated CD3+ selected human
cells were used to prepare a cDNA library, as described in Example
6, below.
[0214] D. Testing Supernatant from Activated CD3+ Selected Human
Cells for zalpha11 Ligand Using BaF3/Zalpha11 cells and an Alamar
Blue Proliferation Assay
[0215] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0216] Proliferation of the BaF3/Zalpha11 cells was assessed using
conditioned media from activated CD3+ selected human cells (see
Example 5C) diluted with mIL-3 free media to 50%, 25%, 12.5%,
6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations. 100 .mu.l of
the diluted conditioned media was added to the BaF3/Zalpha11 cells.
The total assay volume is 200 .mu.l. The assay plates were
incubated and assayed as described in Example 5B.
[0217] Results confirmed the proliferative response of the
BaF3/Zalpha11 cells to a factor present in the activated CD3+
selected human Cell conditioned media. The response, as measured,
was approximately 10-fold over background at the 50% concentration.
The untransfected BaF3 cells did not proliferate in response to
this factor, showing that this factor is specific for the Zalpha11
receptor. Moreover soluble alpha 11 receptor blocked this
proliferative activity in the BaF3/Zalpha11 cells (see, Example
11).
Example 6
[0218] Cloning of Human zalpha11 Ligand from a Human CD3+ Selected
Cell Library
[0219] Screening of a primary human activated CD3+ selected cell
cDNA library revealed an isolated cDNA that is a novel member of
the four-helix bundle cytokine family. This cDNA encoded the
zalpha11 Ligand. The cDNA was identified by screening for activity
of the zalpha11 Ligand using the zalpha11 receptor.
[0220] A. The Vector for CD3+ Selected Library Construction
[0221] The vector for CD3+ selected library construction was
pZP7NX. The pZP7NX vector was constructed as follows: The coding
region for the DHFR selective marker in vector pZP7 was removed by
DNA digestion with NcoI and PstI restriction enzymes (Boehringer
Mannheim). The digested DNA was run on 1% agarose gel, cut out and
gel purified using the Qiagen Gel Extraction Kit (Qiagen) as per
manufacturer's instructions. A DNA fragment representing the coding
region of Zeocin selective marker was amplified by PCR method with
primers ZC13,946 (SEQ ID NO:25) and ZC13,945 (SEQ ID NO:26), and
pZeoSV2(+) as a template. There are additional PstI and BclI
restriction sites in primer ZC13,946 (SEQ ID NO:25), and additional
NcoI and SfuI sites in primer ZC13,945 (SEQ ID NO:26). The PCR
fragment was cut with PstI and NcoI restriction enzymes and cloned
into pZP7 vector prepared by cleaving with the same two enzymes and
subsequent gel purification. This vector was named pZP7Z. Then the
Zeocin coding region was removed by DNA digestion of vector pZP7Z
with BclI and SfuI restriction enzymes. The digested DNA was run on
1% agarose gel, cut out and gel purified, and then ligated with a
DNA fragment of Neomycin coding region cut from pZem228 vector
(deposited at the American Type Culture Collection (ATCC),
Manassas, Va.; ATCC Deposit No. 69446) with the same restriction
enzymes (BclI and SfuI).
[0222] This new vector was named pZP7N, in which the coding region
for DHFR selective marker was replaced by the coding region for a
Neomycin selective marker from vector pZem228. A stuffer fragment
including an Xho1 site was added to pZP7N to create a vector
suitable for high efficiency directional cloning of cDNA; this new
vector was called pZP7NX. To prepare the vector for cDNA,
20.quadrature.g of pZP7NX was digested with 20 units of EcoR1 (Life
Technologies Gaithersberg, Md.) and 20 units of Xho1 (Boehringer
Mannheim Indianapolis, Ind.) for 5 hours at 37.quadrature.C, then
68.quadrature.C for 15 minutes. The digest was then run on a 0.8%
low melt agarose 1.times.TAE gel to separate the stuffer from the
vector. The vector band was excised and digested with
"beta-Agarase" (New England Biolabs, Beverly, Mass.) following the
manufacturer's recommendations. After ethanol precipitation the
digested vector was resuspended in water to 45 ng/ml in preparation
for ligation of CD3+ selected cDNA library described below.
[0223] B. Preparation of Primary Human Activated CD3+ Selected Cell
cDNA Library
[0224] Approximately 1.5.times.108 primary human CD3+ selected
cells stimulated in ionomycin/PMA were isolated by centrifugation
after culturing at 37.degree. C. for 13 hours (Example 5C). Total
RNA was isolated from the cell pellet using the "RNeasy Midi" kit
from Qiagen, Inc. (Valencia, Calif.). mRNA was isolated from 225
micrograms of total RNA using the "MPG mRNA purification kit" from
CPG Inc. (Lincoln Park, N.J.). 3.4 micrograms of mRNA was isolated
and converted to double stranded cDNA using the following
procedure.
[0225] First strand cDNA from stimulated human CD3+ selected cells
was synthesized as follows. Nine .mu.l Oligo d(T)-selected poly(A)
CD3+ RNA at a concentration of 0.34 .mu.g/.mu.l and 1.0 .mu.l of 1
.mu.g/.mu.l first strand primer ZC18,698 (SEQ ID NO:27) containing
an XhoI restriction site were mixed and heated at 65.degree. C. for
4 minutes and cooled by chilling on ice. First strand cDNA
synthesis was initiated by the addition of 9 .mu.l of first strand
buffer (5.times. SUPERSCRIPT.RTM. buffer; (Life Technologies), 4
.mu.l of 100 mM dithiothreitol and 2 .mu.l of a deoxynucleotide
triphosphate solution containing 10 mM each of DATP, dGTP, dTTP and
5-methyl-dCTP (Pharmacia Biotech Inc.) to the RNA-primer mixture.
The reaction mixture was incubated at 45.degree. C. for 4 minutes
followed by the addition of 8 .mu.l of 200 U/.mu.l
Superscript.RTM., RNase H--reverse transcriptase (Life
technologies). The reaction was incubated at 45.degree. C. for 45
minutes followed by an incubation ramp of 1.degree. C. every 2
minutes to 50.degree. C. where the reaction was held for 10
minutes. To denature any secondary structure and allow for
additional extension of the cDNA the reaction was then heated to
70.degree. C. for 2 minutes then dropped to 55.degree. C. for 4
minutes after which 2 .mu.l of SuperscriptII.RTM. RT was added and
incubated an additional 15 minutes followed by a ramp up to
70.degree. C. 1 minute/1.degree. C. Unincorporated nucleotides were
removed from the cDNA by twice precipitating in the presence of 2
.mu.g of glycogen carrier, 2.0 M ammonium acetate and 2.5 volume
ethanol, followed by a 100 .mu.l wash with 70% ethanol. The cDNA
was resuspended in 98 .mu.l water for use in second strand
synthesis.
[0226] Second strand synthesis was performed on the first strand
cDNA under conditions that promoted first strand priming of second
strand synthesis resulting in DNA hairpin formation. The second
strand reaction contained 98 .mu.l of the first strand cDNA, 30
.mu.l of 5.times. polymerase I buffer (100 mM Tris: HCl, pH 7.5,
500 mM KCl, 25 mM MgCl.sub.2, 50 mM (NH.sub.4).sub.2SO.sub.4), 2
.mu.l of 100 mM dithiothreitol, 6 .mu.l of a solution containing 10
mM of each deoxynucleotide triphosphate, 5 .mu.l of 5 mM b-NAD, 1
.mu.l of 3 U/.mu.l E. coli DNA ligase (New England Biolabs Inc.)
and 4 .mu.l of 10 U/.mu.l E. coli DNA polymerase I (New England
Biolabs Inc.). The reaction was assembled at room temperature and
was incubated at room temperature for 2 minutes followed by the
addition of 4 .mu.l of 3.8 U/.mu.l RNase H (Life Technologies). The
reaction was incubated at 15.degree. C. for two hours followed by a
15 minute incubation at room temperature. 10 .mu.l of 1M TRIS pH7.4
was added to the reaction and extracted twice with
phenol/chloroform and once with chloroform, the organic phases were
then back extracted with 50 .mu.l of TE (10 mM TRIS pH 7.4, 1 mM
EDTA), pooled with the other aqueous and ethanol precipitated in
the presence of 0.3 M sodium acetate. The pellet was washed with
100 .mu.l 70% ethanol air dried and resuspended in 40 .mu.l
water.
[0227] The single-stranded DNA of the hairpin structure was cleaved
using mung bean nuclease. The reaction mixture contained 40 .mu.l
of second strand cDNA, 5 .mu.l of 10.times. mung bean nuclease
buffer (Life technologies), 5 .mu.l of mung bean nuclease
(Pharmacia Biotech Corp.) diluted to 1 U/.mu.l in 1.times. mung
bean nuclease buffer. The reaction was incubated at 37.degree. C.
for 45 minutes. The reaction was terminated by the addition of 10
.mu.l of 1 M Tris: HCl, pH 7.4 followed by sequential
phenol/chloroform and chloroform extractions as described above.
Following the extractions, the cDNA was ethanol precipitated in the
presence of 0.3 M sodium acetate. The pellet was washed with 100
.quadrature.l 70% ethanol air dried and resuspended in 38 .mu.l
water.
[0228] The resuspended cDNA was blunt-ended with T4 DNA polymerase.
The cDNA, which was resuspended in 38 .quadrature.l of water, was
mixed with 12 .mu.l 5.times. T4 DNA polymerase buffer (250 mM
Tris:HCl, pH 8.0, 250 mM KCl, 25 mM MgCl2), 2 .mu.l 0.1 M
dithiothreitol, 6 .mu.l of a solution containing 10 mM of each
deoxynucleotide triphosphate and 2 .mu.l of 1 U/.mu.l T4 DNA
polymerase (Boehringer Mannheim Corp.). After an incubation of 45
minutes at 15.degree. C., the reaction was terminated by the
addition of 30 .mu.l TE followed by sequential phenol/chloroform
and chloroform extractions and back extracted with 20 .mu.l TE as
described above. The DNA was ethanol precipitated in the presence
of 2 .mu.l Pellet Paint.TM. (Novagen) carrier and 0.3 M sodium
acetate and was resuspended 11 .mu.l of water.
[0229] Eco RI adapters were ligated onto the 5' ends of the cDNA
described above to enable cloning into an expression vector. 11
.mu.l of cDNA and 4 .mu.l of 65 pmole/.mu.l of Eco RI
hemiphophorylated adaptor (Pharmacia Biotech Corp) were mixed with
5 .quadrature.l 5.times. ligase buffer (Life Technologies), 2 .mu.l
of 10 mM ATP and 3 .mu.l of 1 U/.mu.l T4 DNA ligase (Life
Technologies), 1 .mu.l 10.times. ligation buffer (Promega Corp), 9
.mu.l water. The extra dilution with 1.times. buffer was to prevent
the pellet paint from precipitating. The reaction was incubated 9
hours in a water bath temperature ramp from 10.degree. C. to
22.degree. C. over 9 hours, followed by 45 minutes at 25.degree. C.
The reaction was terminated by incubation at 68.degree. C. for 15
minutes.
[0230] To facilitate the directional cloning of the cDNA into an
expression vector, the cDNA was digested with XhoI, resulting in a
cDNA having a 5' Eco RI cohesive end and a 3' XhoI cohesive end.
The XhoI restriction site at the 3' end of the cDNA had been
previously introduced using the ZC18698 (SEQ ID NO:27) primer.
Restriction enzyme digestion was carried out in a reaction mixture
containing 35 .mu.l of the ligation mix described above, 6 .mu.l of
10.times. H buffer (Boehringer Mannheim Corp.), 1 .mu.l of 2 mg/ml
BSA (Biolabs Corp.), 17 .mu.l water and 1.0 .mu.l of 40 U/.mu.l
XhoI (Boehringer Mannheim). Digestion was carried out at 37.degree.
C. for 1 hour. The reaction was terminated by incubation at
68.degree. C. for 15 minutes followed by ethanol precipitation,
washing drying as described above and resuspension in 30 .mu.l
water.
[0231] The resuspended cDNA was heated to 65.degree. C. for 5
minutes and cooled on ice, 4 .mu.l of 5.times. gel loading dye
(Research Genetics Corp.) was added, the cDNA was loaded onto a
0.8% low melt agarose 1.times.TAE gel (SEA PLAQUE GTG.TM. low melt
agarose; FMC Corp.) and electrophoresed. The contaminating adapters
and cDNA below 0.6 Kb in length were excised from the gel. The
electrodes were reversed, molten agarose was added to fill in the
wells, the buffer was changed and the cDNA was electrophoresed
until concentrated near the lane origin. The area of the gel
containing the concentrated cDNA was excised and placed in a
microfuge tube, and the agarose was melted by heating to 65.degree.
C. for 15 minutes. Following equilibration of the sample to
45.degree. C., 2 .mu.l of 1 U/.mu.l Beta-agarase I (Biolabs, Inc.)
was added, and the mixture was incubated for 90 min. at 45.degree.
C. to digest the agarose. After incubation, 1 tenth volume of 3 M
Na acetate was added to the sample, and the mixture was incubated
on ice for 15 minutes. The sample was centrifuged at 14,000.times.g
for 15 minutes at room temperature to remove undigested agarose,
the cDNA was ethanol precipitated, washed in 70% ethanol, air-dried
and resuspended in 40 .mu.l water.
[0232] To determine the optimum ratio of cDNA to vector several
ligations were assembled and electroporated. Briefly, 2 .mu.l of
5.times. T4 ligase buffer (Life Technologies), 1 .mu.l of 10 mM
ATP, 1 .mu.l pZP7NX digested with EcoR1-Xho1, 1 ll T4 DNA ligase
diluted to 0.25 u/.mu.l (Life Technologies) water to 10 .mu.l and
0.5, 1, 2 or 3 .mu.l of cDNA were mixed in 4 separate ligations,
incubated at 22.degree. C. for 4 hours, 68.degree. C. for 20
minutes, sodium acetate-ethanol precipitated, washed, dried and
resuspended in 10 .mu.l. A single microliter of each ligation was
electroporated into 40 .mu.l DH10b ElectroMax.TM. electrocompetent
bacteria (Life Technologies) using a 0.1 cm cuvette (Biorad) and a
Genepulser, pulse controller.quadrature. (Biorad) set to 2.5 KV, 25
.mu.F, 200 ohms. These cells were immediately resuspended in 1 ml.
SOC broth (Manniatis, et al. supra.) followed by 500 .mu.l of 50%
glycerol-SOC as a preservative. These "glycerol stocks" were frozen
in several aliquots at -70.degree. C. An aliquot of each was thawed
and plated serially on LB-agar plates supplemented with ampicillin
at 100 .mu.g/ml. Colony numbers indicated that the optimum ratio of
CD3+ cDNA to pZP7NX vector was 1 .mu.l to 45 ng; such a ligation
yielded 4.5 million primary clones.
[0233] For the purpose of screening this library using a
BaF3-zalpha11 based proliferation assay (Example 5) glycerol stocks
from above were diluted into liquid cultures of 100 or 250 clones
per pool in deep well microtiter plates, grown 24 hours at
37.degree. C. with shaking and plasmid isolated using a Qiagen kit
following the manufacturer's instructions. Such DNA was
subsequently transfected into BHK cells, media conditioned 72
hours, harvested and placed on 5K BaF3-zalpha11 cells for 72 hours
after which proliferation was assessed using an "Alamar blue"
fluorescence assay (Example 5B and Example 2B)
[0234] For the purpose of screening the library by secretion trap
cloning, a complex, amplified form of the library was needed to
transfect COS-7 cells. About 4.8 million clones were plated on 110
15 cm LB-agar plates supplemented with 100 .mu.g/ml ampicillin, 10
.mu.g/ml methicillin. After growing the plates overnight at
37.degree. C. the bacteria were harvested by scraping and pelleted.
Plasmid DNA was extracted from the pelleted bacteria using a
Nucleobond-giga.TM. (Clonetech) following the manufacturer's
instructions. This plasmid was then used to transfect COS-7 cells
(ATCC No. CRL 1651) on slides and screened using the secretion trap
technique described below (Example 12).
Example 7
[0235] Expression Cloning of Human zalpha11 Ligand
[0236] The glycerol stocks from the activated human CD3+ selected
cell library (Example 6) were added to Super Broth II.TM. (Becton
Dickinson, Cockeysville, Md.) +0.1 mg/ml ampicillin (amp) at a
concentration of 250 cells per 800 microliters. The E. coli were
allowed to equilibrate for 24 hours at room temperature. At the
time of inoculation, 400 microliters was plated on LB+ amp plates
to determine the actual titer of the inoculation. After 24 hours
the plates were counted and then the final concentration of the
SuperBrothII.TM. +E. coli was adjusted so that the final
concentration was 250 cells per 1.2 ml. Three times 2 liters were
inoculated for a total of 6 liters. The media were then plated into
96-well deep well blocks (Qiagen). Plating was done on the
8-channel Q-Fill2.TM. dispenser (Genetix, Christchurch, Dorset,
UK). The E. coli were grown overnight at 37.degree. C. shaking at
250 rotations/min. on a New Brunswick Scientific Innova 4900
multi-tier environment shaker. The E. coli were spun out of
solution at 3000 rpm, using a Beckman GS-6KR centrifuge. These E.
coli pellets were frozen at -20.degree. C. or used fresh before
miniprepping the plasmid DNA. Each pellet contains approximately
250 cDNA clones from the human CD3+ selected cell library.
[0237] These pools of 250 cDNA clones were then mini-prepped using
QIAprep.TM. 96 Turbo Miniprep kit (Qiagen). Plasmid DNA was eluted
using 125 .mu.l of TE (10 mM Tris pH 8, 1 mM EDTA). This plasmid
DNA was then used to transfect BHK cells.
[0238] BHK Transfection
[0239] BHK cells were plated in 96-well tissue culture plates at a
density of 12,000 cells per well in a volume of 100 .mu.l. per
well. Culture media was DMEM (GibcoBRL), 5% heat-inactivated fetal
bovine serum, 2 mM L-glutamine (GibcoBRL), 1.times.PSN (GibcoBRL),
1 mM NaPyruvate (GibcoBRL).
[0240] The following day, BHK cells were washed once with 100 .mu.l
SFA. SFA is serum-free media which is DMEM/F12 (Gibco/BRL), 2 mM
GlutaMax.TM. (Gibco/BRL), 1 mM NaPyruvate, 10 .mu.g/ml transferrin,
5 .mu.g/ml insulin, 10 .mu.g/ml fetuin, 2 .mu.g/ml selenium, 25 mM
HEPES (Gibco/BRL), 100 .mu.M non-essential amino acids
(Gibco/BRL).
[0241] A DNA/Lipofectamine.TM. mix is made as follows: 2.2 .mu.l
Lipofectamine.TM. reagent (Gibco/BRL) is combined with 102.8 .mu.l
SFA at room temperature; approximately 5 .mu.l of the plasmid DNA
(200 ng/.mu.l) is then added to the Lipofectamine.TM./SFA to form
the DNA/Lipofectamine.TM. mixture, which is incubated at room
temperature for 30 minutes. The SFA was removed from the BHK cells
and the cells were incubated with 50 .mu.l of the
DNA/lipofectamine.TM. mix for 5 hours at 37.degree. C. with 5% CO2.
Fifty .mu.l of the DNA/Lipofectamine.TM. mixture was added to each
of two wells of the BHK cells, so that transfections were done in
duplicate.
[0242] After BHK cells were incubated with DNA/Lipofectamine.TM.
mix for 5 hours, the DNA/Lipofectamine.TM. mix was removed and 100
.mu.l culture media was added. Cells were incubated overnight, the
media was removed and replaced with 100 .mu.l. culture media. After
culturing cells for 72 hours, conditioned media was removed, frozen
at -80.degree. C. for a minimum of 20 minutes, thawed, and then 50
.mu.l was assayed in the zalpha11/BaF3 proliferation assay,
described in Examples 2B and Example 5, to identify pools of 250
clones with ligand activity.
[0243] Thirty-five 96-well plates were screened in a single assay.
This represented approximately 250 cDNAs/well or 840,000 cDNAs
total. Of these, conditioned media from 54 wells (representing 250
cDNAs per well) tested positive in the proliferation assay. The
conditioned media from these positive pools was re-tested in a
second assay (secretion trap) with and without the soluble receptor
(see, Example 12). The zalpha11CEE soluble receptor (Example 10A)
was used at a final concentration of about 1 .mu.g/ml. For all 54
positive pools, essentially all of the activity was neutralized by
addition of the soluble zalpha11 receptor, indicating that these
pools contained a cDNA from the zalpha11 Ligand. Four of these
positive pools were chosen to break-down and isolate a single cDNA
that would encode the zalpha11 Ligand. These were 45C5, 46G11,
40H12, and 60A1.
[0244] For each of these 4 pools, 1 .mu.l. of DNA was used to
transform ElectroMax.TM. DH10B cells (Gibco/BRL) by
electroporation. The transformants were plated on LB+ amp (100
.mu.g/ml)+methicillin (10 .mu.g/ml) plates to give single colonies.
For each electroporated pool, 960 individual colonies were
toothpicked into ten 96-well plates containing 1.2 ml of
SuperBrothII.TM. per well. These plates were numbered #1-10 for
each of the breakdown pools (45C5, 46G11, 40H12, and 60A1). These
were cultured overnight and the plasmid DNA miniprepped as above.
For 46G11, 40H12, and 60A1, plasmid DNA from the breakdown plates
was transfected into BHK cells as above.
[0245] For 45 C5, a "fast track" protocol was utilized to
accelerate the identification of the zalpha11 Ligand cDNA. BHK
cells were transfected with plasmid DNA from the breakdown plates
as above, DNA/Lipofectamine.TM. mix was removed after a 5 hour
incubation, and culture media was added. Since the transfections
were done in duplicate, the culture media was harvested the
following day after 24 hours from one of the transfected BHK
plates, and harvested the following day after 48 hours from the
remaining transfected plate. The 24 hour conditioned media was
assayed as above for zalpha11 Ligand activity using the
proliferation assay as described herein.
[0246] Plasmid DNA was pooled from 45C5 breakdown plates #1-4 and
assayed for binding of zalpha11 soluble receptor to its ligand by
the "secretion-trap" protocol (see, Example 12, below). Eight
positive clones were identified from a total of 384 sequences.
Results from the proliferation assay confirmed activity of the
zalpha11 Ligand and correlated with results of the secretion trap
assay (see Example 12). Concurrently, plasmid DNA miniprepped from
plates #1-4 of the 45C5 pool breakdown was sequenced to determine
the DNA sequence of each of the 384 clones.
[0247] Several clones that were positively identified in the
proliferation and secretion trap assays were also sequenced using
the following primers: ZC14,063 (SEQ ID NO:28), ZC7,764a (SEQ ID
NO:38), ZC7,764b (SEQ ID NO:39), ZC22,034 (SEQ ID NO:40), and
ZC22,035 (SEQ ID NO:41). The polynucleotide sequence of zalpha11
Ligand was full-length (SEQ ID NO:1) and its corresponding amino
acid sequence is shown (SEQ ID NO:2).
Example 8
[0248] Construction of Mammalian Expression Vectors that Express
zalpha11 Soluble Receptors: zalpha11CEE zalpha11CFLG zalpha11CHIS
and zalpha11-Fc4
[0249] A. Construction of zalpha11 Mammalian Expression Vector
Containing zalpha11CEE, zalpha11CFLG and zalpha11CHIS
[0250] An expression vector was prepared for the expression of the
soluble, extracellular domain of the zalpha11 polypeptide,
pC4zalpha11CEE, wherein the construct is designed to express a
zalpha11 polypeptide comprised of the predicted initiating
methionine and truncated adjacent to the predicted transmembrane
domain, and with a C-terminal Glu-Glu tag (SEQ ID NO:29).
[0251] A 700 bp PCR generated zalpha11 DNA fragment was created
using ZC19,931 (SEQ ID NO:30) and ZC19,932 (SEQ ID NO:31) as PCR
primers to add Asp718 and BamHI restriction sites. A plasmid
containing the zalpha11 receptor cDNA (SEQ ID NO:7) was used as a
template. PCR amplification of the zalpha11 fragment was performed
as follows: Twenty five cycles at 94C for 0.5 minutes; five cycles
at 94.degree. C. for 10 seconds, 50.degree. C. for 30 seconds,
68.degree. C. for 45 seconds, followed by a 4.degree. C. hold. The
reaction was purified by chloroform/phenol extraction and
isopropanol precipitation, and digested with Asp718 and BamHI
(Gibco BRL) following manufacturer's protocol. A band of the
predicted size, 700 bp, was visualized by 1% agarose gel
electrophoresis, excised and the DNA was purified using a
QiaexII.TM. purification system (Qiagen) according the
manufacturer's instructions.
[0252] The excised DNA was subcloned into plasmid pC4EE which had
been cut with BamHI and Asp718. The pC4zalpha11CEE expression
vector uses the native zalpha11 signal peptide and attaches the
Glu-Glu tag (SEQ ID NO:29) to the C-terminus of the extracellular
portion of the zalpha11 polypeptide-encoding polynucleotide
sequence. Plasmid pC4EE, is a mammalian expression vector
containing an expression cassette having the mouse
metallothionein-1 promoter, multiple restriction sites for
insertion of coding sequences, a stop codon and a human growth
hormone terminator. The plasmid also has an E. coli origin of
replication, a mammalian selectable marker expression unit having
an SV40 promoter, enhancer and origin of replication, a DHFR gene
and the SV40 terminator.
[0253] About 30 ng of the restriction digested zalpha11 insert and
about 12 ng of the digested vector were ligated overnight at
16.degree. C. One microliter of each ligation reaction was
independently electroporated into DH10B competent cells (GIBCO BRL,
Gaithersburg, Md.) according to manufacturer's direction and plated
onto LB plates containing 50 mg/ml ampicillin, and incubated
overnight. Colonies were screened by restriction analysis of DNA
prepared from 2 ml liquid cultures of individual colonies. The
insert sequence of positive clones was verified by sequence
analysis. A large scale plasmid preparation was done using a
QIAGEN.RTM. Maxi prep kit (Qiagen) according to manufacturer's
instructions.
[0254] The same process was used to prepare the zalpha11 soluble
receptors with a C-terminal his tag, composed of 6 His residues in
a row; and a C-terminal flag (SEQ ID NO:37) tag, zalpha11CFLAG. To
prepare these constructs, the aforementioned vector has either the
HIS or the FLAG.RTM. tag in place of the glu-glu tag (SEQ ID
NO:29).
[0255] B. Mammalian Expression Construct of Soluble zalpha11
Receptor zalpha11-Fc4
[0256] An expression plasmid containing all or part of a
polynucleotide encoding zalpha11 was constructed via homologous
recombination. The extracellular domain of the zalpha11 receptor
was fused to the Fc region derived from human IgG, called "Fc4"
(SEQ ID NO:33) which contains a mutation so that it no longer binds
the Fc receptor. A fragment of zalpha11 cDNA was isolated using PCR
that includes the polynucleotide sequence from extracellular domain
of the zalpha11 receptor. The two primers used in the production of
the zalpha11 fragment were: (1) The primers for PCR each include
from 5' to 3' end: 40 bp of the vector flanking sequence (5' of the
insert) and 17 bp corresponding to the 5' end of the zalpha11
extracellular domain (SEQ ID NO:32); and (2) 40 bp of the 5' end of
the Fc4 polynucleotide sequence (SEQ ID NO:33) and 17 bp
corresponding to the 3' end of the zalpha11 extracellular domain
(SEQ ID NO:34). The fragment of Fc-4 for fusion with the zalpha11
was generated by PCR in a similar fashion. The two primers used in
the production of the Fc4 fragment were: (1) a 5' primer consisting
of 40 bp of sequence from the 3' end of zalpha11 extracellular
domain and 17 bp of the 5' end of Fc4 (SEQ ID NO:35); and (2) a 3'
primer consisting of 40 bp of vector sequence (3' of the insert)
and 17 bp of the 3' end of Fc4 (SEQ ID NO:36).
[0257] PCR amplification of the each of the reactions described
above was performed as follows: one cycle at 94.degree. C. for 2
minutes; twenty-five cycles at 94.degree. C. for 30 seconds,
60.degree. C. for 30 seconds, 72.degree. C. for 1 minute; one cycle
at 72.degree. C. for 5 minutes; followed by a 4.degree. C. hold.
Ten .mu.l of the 100 .mu.l PCR reaction was run on a 0.8% LMP
agarose gel (Seaplaque GTG) with 1.times.TBE buffer for analysis.
The remaining 90 .mu.l of the PCR reaction is precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol. The
expression vector used was derived from the plasmid pCZR199 derived
from pZP9 (ATCC Deposit No. 98668), and was cut with SmaI (BRL).
The expression vector was derived from the plasmid pCZR199, and is
a mammalian expression vector containing an expression cassette
having the CMV immediate early promoter, a consensus intron from
the variable region of mouse immunoglobulin heavy chain locus,
multiple restriction sites for insertion of coding sequences, a
stop codon and a human growth hormone terminator. The expression
vector also has an E. coli origin of replication, a mammalian
selectable marker expression unit having an SV40 promoter, enhancer
and origin of replication, a DHFR gene and the SV40 terminator. The
expression vector used was constructed from pCZR199 by the
replacement of the metallothionein promoter with the CMV immediate
early promoter.
[0258] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l containing approximately 1
.mu.g each of the zalpha11 and Fc4 inserts, and 100 ng of SmaI
(BRL) digested expression vector and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixtures were electropulsed
at 0.75 kV (5 kV/cm), "infinite" ohms, 25.degree. F. To each
cuvette is added 600 .mu.l of 1.2 M sorbitol and the yeast was
plated in two 300 .mu.l aliquots onto two URA-D plates and
incubated at 30.degree. C.
[0259] After about 48 hours, the Ura+ yeast transformants from a
single plate were resuspended in 1 ml H2O and spun briefly to
pellet the yeast cells. The cell pellet was 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 was
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, followed by a 5 minute spin in a
Eppendorf centrifuge at maximum speed. Three hundred microliters of
the aqueous phase was transferred to a fresh tube, and the DNA
precipitated with 600 .mu.l ethanol (EtOH), followed by
centrifugation for 10 minutes at 4.degree. C. The DNA pellet was
resuspended in 100 .mu.l H2O.
[0260] Transformation of electrocompetent E. coli cells (DH10B,
GibcoBRL) is done with 0.5-2 ml yeast DNA prep and 40 .mu.l of
DH10B cells. The cells were electropulsed at 2.0 kV, 25 mF and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto.TM. Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was
plated in 250 .mu.l aliquots on four LB AMP plates (LB broth
(Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0261] Individual clones harboring the correct expression construct
for zalpha11-Fc4 were identified by restriction digest to verify
the presence of the zalpha11-Fc4 insert and to confirm that the
various DNA sequences have been joined correctly to one another.
The insert of positive clones were subjected to sequence analysis.
Larger scale plasmid DNA is isolated using the Qiagen Maxi kit
(Qiagen) according to manufacturer's instructions.
Example 9
[0262] Transfection and Expression of Zalpha11 Soluble Receptor
Polypeptides
[0263] A. Mammalian Expression of Soluble zalpha11 Receptor
zalpha11CEE, zalpha11CFLG and zalpha11CHIS
[0264] BHK 570 cells (ATCC No. CRL-10314), passage 27, were plated
at 1.2.times.10.sup.6 cells/well (6-well plate) in 800 .mu.l of
serum free (SF) DMEM media (DMEM, Gibco/BRL High Glucose) (Gibco
BRL, Gaithersburg, Md.). The cells were transfected with expression
plasmids containing zalpha11CEE, zalpha11CFLG, or zalpha11CHIS
described above (see, Example 8), using Lipofectin.TM. (Gibco BRL),
in serum free (SF) DMEM. Three micrograms of zalpha11CEE,
zalpha11CFLG, or zalpha11CHIS each were separately diluted into 1.5
ml tubes to a total final volume of 100 .mu.l SF DMEM. In separate
tubes, 15 .mu.l of Lipofectin.TM. (Gibco BRL) was mixed with 100
.mu.l of SF DMEM. The Lipofectin.TM. mix was incubated at room
temperature for 30-45 minutes then the DNA mix was added and
allowed to incubate approximately 10-15 minutes at room
temperature.
[0265] The entire DNA: Lipofectin.TM. mixture was added to the
plated cells and distributed evenly over them. The cells were
incubated at 37.degree. C. for approximately five hours, then
transferred to separate 150 mm MAXI plates in a final volume of 30
ml DMEM/5% fetal bovine serum (FBS) (Hyclone, Logan, Utah). The
plates were incubated at 37.degree. C., 5% CO.sub.2, overnight and
the DNA: Lipofectin.TM. mixture was replaced with selection media
(5% FBS/DMEM with 1 .mu.M methotrexate (MTX)) the next day.
[0266] Approximately 10-12 days post-transfection, the plates were
washed with 10 ml SF DMEM. The wash media was aspirated and
replaced with 7.25 ml serum-free DMEM. Sterile Teflon meshes
(Spectrum Medical Industries, Los Angeles, Calif.) pre-soaked in SF
DMEM were then placed over the clonal cell colonies. A sterile
nitrocellulose filter pre-soaked in SF DMEM was then placed over
the mesh. Orientation marks on the nitrocellulose were transferred
to the culture dish. The plates were then incubated for 5-6 hours
in a 37.degree. C., 5% CO.sub.2 incubator.
[0267] Following incubation, the filters/meshes were removed, and
the media aspirated and replaced with 5% FBS/DMEM with 1 .mu.M MTX.
The filters were then blocked in 10% nonfat dry milk/Western A
buffer (Western A: 50 mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150
mM NaCl and 0.25% gelatin) for 15 minutes at room temperature on a
rotating shaker. The filters were then incubated with an
anti-Glu-Glu, anti-FLAG.RTM., or anti-HIS antibody-HRP conjugates,
respectively, in 2.5% nonfat dry milk/Western A buffer for one hour
at room temperature on a rotating shaker. The filters were then
washed three times at room temperature with Western A for 5-10
minutes per wash. The filters were developed with ultra ECL reagent
(Amersham Corp., Arlington Heights, Ill.) according the
manufacturer's directions and visualized on the Lumi-Imager (Roche
Corp.)
[0268] Positive expressing clonal colonies were mechanically picked
to 12-well plates in one ml of 5% FCS/DMEM with 5 .mu.M MTX, then
grown to confluence. Conditioned media samples were then tested for
expression levels via SDS-PAGE and Western analysis. The three
highest expressing clones for each construct were picked; two out
of three were frozen down as back up and one was expanded for
mycoplasma testing and large-scale factory seeding.
[0269] B. Mammalian Expression of Soluble zalpha11 Receptor
zalpha11-Fc4
[0270] BHK 570 cells (ATCC NO: CRL-10314) were 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 DMEM/FBS
media (DMEM, Gibco/BRL High Glucose, (Gibco BRL, Gaithersburg,
Md.), 5% fetal bovine serum (Hyclone, Logan, Utah), 1 mM
L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate
(Gibco BRL)). The cells were then transfected with the plasmid
containing zalpha11-Fc4 (see, Example 8), using Lipofectamine.TM.
(Gibco BRL), in serum free (SF) media formulation (DMEM, 10 mg/ml
transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%
sodium pyruvate). The plasmid containing zalpha11-Fc4 was diluted
into 15 ml tubes to a total final volume of 640 ml with SF media.
35 ml of Lipofectamine.TM. (Gibco BRL) was mixed with 605 ml of SF
medium. The Lipofectamine.TM. mix was added to the DNA mix and
allowed to incubate approximately 30 minutes at room temperature.
Five milliliters of SF media was added to the DNA:Lipofectamine.TM.
mixture. The cells were rinsed once with 5 ml of SF media,
aspirated, and the DNA:Lipofectamine.TM. mixture is added. The
cells were incubated at 37.degree. C. for five hours, then 6.4 ml
of DMEM/10% FBS, 1% PSN media was added to each plate. The plates
were incubated at 37.degree. C. overnight and the
DNA:Lipofectamine.TM. mixture was replaced with fresh 5% FBS/DMEM
media the next day. On day 2 post-transfection, the cells were
split into the selection media (DMEM/FBS media from above with the
addition of 1 .mu.M methotrexate (Sigma Chemical Co., St. Louis,
Mo.)) in 150 mm plates at 1:10, 1:20 and 1:50. The media on the
cells was replaced with fresh selection media at day 5
post-transfection. Approximately 10 days post-transfection, two 150
mm culture dishes of methotrexate resistant colonies from each
transfection were trypsinized and the cells are pooled and plated
into a T-162 flask and transferred to large scale culture.
Example 10
[0271] Purification of zalpha11 Soluble Receptors from BHK 570
cells
[0272] A. Purification of zalpha11CEE Polypeptide from BHK 570
[0273] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal GluGlu (EE) tags. Thirty
liters of cell factory conditioned media was concentrated to 1.6
liters with an Amicon S10Y3 spiral cartridge on a ProFlux A30. A
Protease inhibitor solution was added to the concentrated 1.6
liters of cell factory conditioned media from transfected BHK 570
cells (see, Example 9) to final concentrations of 2.5 mM
ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.
Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,
Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim). Samples were removed for analysis and the
bulk volume was frozen at -80.degree. C. until the purification was
started. Total target protein concentrations of the concentrated
cell factory conditioned media was determined via SDS-PAGE and
Western blot analysis with the anti-EE HRP conjugated antibody.
[0274] A 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated cell
factory conditioned media was thawed, 0.2 micron sterile filtered,
pH adjusted to 7.4, then loaded on the column overnight with 1
ml/minute flow rate. The column was washed with 10 column volumes
(CVs) of phosphate buffered saline (PBS, pH 7.4), then plug eluted
with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide
(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has
the sequence EYMPME (SEQ ID NO:29). The column was washed for 10
CVs with PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The
pH of the glycine-eluted column was adjusted to 7.0 with 2 CVs of
5.times.PBS, then equilibrated in PBS (pH 7.4). Five ml fractions
were collected over the entire elution chromatography and
absorbance at 280 and 215 nM were monitored; the pass through and
wash pools were also saved and analyzed. The EE-polypeptide elution
peak fractions were analyzed for the target protein via SDS-PAGE
Silver staining and Western Blotting with the anti-EE HRP
conjugated antibody. The polypeptide elution fractions of interest
were pooled and concentrated from 60 ml to 5.0 ml using a 10,000
Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0275] To separate zalpha11CEE from other co-purifying proteins,
the concentrated polypeptide elution pooled fractions were
subjected to a POROS HQ-50 (strong anion exchange resin from
PerSeptive BioSystems, Framingham, Mass.) at pH 8.0. A
1.0.times.6.0 cm column was poured and flow packed on a BioCad
Sprint. The column was counter ion charged then equibrated in 20 mM
TRIS pH 8.0 (Tris (Hydroxymethyl Aminomethane)). The sample was
diluted 1:13 (to reduce the ionic strength of PBS) then loaded on
the Poros HQ column at 5 ml/minute. The column was washed for 10
CVs with 20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20
mM Tris/1 M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml
fractions were collected over the entire chromatography and
absorbance at 280 and 215 nM were monitored. The elution peak
fractions were analyzed via SDS-PAGE Silver staining. Fractions of
interest were pooled and concentrated to 1.5-2 ml using a 10,000
Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0276] To separate zalpha11CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to chromatography on a 1.5.times.90 cm
Sephadex S200 (Pharmacia, Piscataway, N.J.) column equilibrated and
loaded in PBS at a flow rate of 1.0 ml/min using a BioCad Sprint.
1.5 ml fractions were collected across the entire chromatography
and the absorbance at 280 and 215 nM were monitored. The peak
fractions were characterized via SDS-PAGE Silver staining, and only
the most pure fractions were pooled. This material represented
purified zalpha11CEE polypeptide.
[0277] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0278] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the zalpha11CEE polypeptide was one major band of an
apparent molecular weight of about 50,000 Daltons. The mobility of
this band was the same on reducing and non-reducing gels.
[0279] The protein concentration of the purified material was
performed by BCA analysis (Pierce, Rockford, Ill.) and the protein
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. On IEF (isoelectric focusing) gels the protein
runs with a PI of less than 4.5. The concentration of zalpha11CEE
polypeptide was 1.0 mg/ml.
[0280] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with
100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene
0.45 micron filter unit. The gel was washed with 6.0 volumes of 200
mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an
equal volume of EE antibody solution containing 900 mg of antibody
was added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution
at
[0281] B. Purification of zalpha11CFLAG Polypeptide from BHK
570
[0282] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal FLAG.RTM. (FLG)
(Sigma-Aldrich Co.) tags. Thirty liters of cell factory conditioned
media was concentrated to 1.7 liters with an Amicon S10Y3 spiral
catridge on a ProFlux A30. A Protease inhibitor solution was added
to the 1.7 liters of concentrated cell factory conditioned media
from transfected BHK 570 cells (see, Example 9) to final
concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA,
Sigma Chemical Co. St. Louis, Mo.), 0.003 mM leupeptin
(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim).
Samples were removed for analysis and the bulk volume was frozen at
-80.degree. C. until the purification was started. Total target
protein concentrations of the cell factory conditioned media was
determined via SDS-PAGE and Western blot analysis with the
anti-FLAG.RTM. (Kodak) HRP conjugated antibody. A 125 ml column of
anti-FLAG.RTM. M2-Agarose affinity gel (Sigma-Aldrich Co.) was
poured in a Waters AP-5, 5 cm.times.10 cm glass column. The column
was flow packed and equilibrated on a BioCad Sprint (PerSeptive
BioSystems, Framingham, Mass.) with phosphate buffered saline (PBS)
pH 7.4. The concentrated cell factory conditioned media was thawed,
0.2 micron sterile filtered, pH adjusted to 7.4, then loaded on the
column overnight with 1 ml/minute flow rate. The column was washed
with 10 column volumes (CVs) of phosphate buffered saline (PBS, pH
7.4), then plug eluted with 250 ml of PBS (pH 6.0) containing 0.5
mg/ml FLAG.RTM. (Sigma-Aldrich Co.) peptide at 5 ml/minute. The
FLAG.RTM. peptide used has the sequence DYKDDDDK (SEQ ID NO:37).
The column was washed for 10 CVs with PBS, then eluted with 5 CVs
of 0.2M glycine, pH 3.0. The pH of the glycine-eluted column was
adjusted to 7.0 with 2 CVs of 5.times.PBS, then equilibrated in PBS
(pH 7.4). Five ml fractions were collected over the entire elution
chromatography and absorbence at 280 and 215 nM were monitored; the
pass through and wash pools were also saved and analyzed. The
FLAG.RTM.-polypeptide elution peak fractions were analyzed for the
target protein via SDS-PAGE Silver staining and Western Blotting
with the anti-FLAG HRP conjugated antibody. The polypeptide elution
fractions of interest were pooled and concentrated from 80 ml to 12
ml using a 10,000 Dalton molecular weight cutoff membrane spin
concentrator (Millipore, Bedford, Mass.) according to the
manufacturer's instructions.
[0283] To separate zalpha11CFLG from other co-purifying proteins,
the polypeptide elution pooled fractions were subjected to a POROS
HQ-50 (strong anion exchange resin from PerSeptive BioSystems,
Framingham, Mass.) at pH 8.0. A 1.0.times.6.0 cm column was poured
and flow packed on a BioCad Sprint. The column was counter ion
charged then equilibrated in 20 mM TRIS pH 8.0 (Tris (Hydroxymethyl
Aminomethane)). The sample was diluted 1:13 (to reduce the ionic
strength of PBS) then loaded on the Poros HQ-50 column at 5
ml/minute. The column was washed for 10 column volumes (CVs) with
20 mM Tris pH 8.0 then eluted with a 40 CV gradient of 20 mM Tris/1
M sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions were
collected over the entire chromatography and absorbance at 280 and
215 nM were monitored. The elution peak fractions were analyzed via
SDS-PAGE Silver staining. Fractions of interest were pooled and
concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight
cutoff membrane spin concentrator (Millipore, Bedford, Mass.)
according to the manufacturer's instructions.
[0284] To separate zalpha11CFLG polypeptide from free FLAG.RTM.
peptide and any contaminating co-purifying proteins, the pooled
concentrated fractions were subjected to chromatography on a
1.5.times.90 cm Sephacryl S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified zalpha11CFLG polypeptide.
[0285] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0286] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the zalpha11CFLG polypeptide was one major band of
an apparent molecular weight of about 50,000 Daltons. The mobility
of this band was the same on reducing and non-reducing gels.
[0287] The protein concentration of the purified material was
performed by BCA analysis (Pierce, Rockford, Ill.) and the protein
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. On IEF (isoelectric focusing) gels the protein
runs with a PI of less than 4.5. The concentration of zalpha11CFLG
polypeptide was 1.2 mg/ml.
[0288] C. Purification of zalpha11-Fc4 Polypeptide from Transfected
BHK 570 Cells
[0289] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11 polypeptide containing C-terminal fusion to human IgG/Fc
(zalpha11-Fc4; Examples 8 and 9). 12,000 ml of conditioned media
from BHK 570 cells transfected with zalpha11-Fc4 (Example 9) was
filtered through a 0.2 mm sterilizing filter and then supplemented
with a solution of protease inhibitors, to final concentrations of,
0.001 mM leupeptin (Boerhinger-Mannheim, Indianapolis, Ind.), 0.001
mM pepstatin (Boerhinger-Mannheim) and 0.4 mM Pefabloc
(Boerhinger-Mannheim). A protein G sepharose (6 ml bed volume,
Pharmacia Biotech) was packed and washed with 500 ml PBS
(Gibco/BRL) The supplemented conditioned media was passed over the
column with a flow rate of 10 ml/minute, followed by washing with
1000 ml PBS (BRL/Gibco). zalpha11-Fc4 was eluted from the column
with 0.1 M Glycine pH 3.5 and 2 ml fractions were collected
directly into 0.2 ml 2M Tris pH 8.0, to adjust the final pH to 7.0
in the fractions.
[0290] The eluted fractions were characterized by SDS-PAGE and
western blotting with anti-human Fc (Amersham) antibodies. Western
blot analysis of reducing SDS-PAGE gels reveal an immunoreactive
protein of about 80,000 KDa in fractions 2-10. Silver stained
SDS-PAGE gels also revealed an 80,000 KDa zalpha11:Fc polypeptide
in fractions 2-10. Fractions 2-10 were pooled.
[0291] The protein concentration of the pooled fractions was
performed by BCA analysis (Pierce, Rockford, Ill.) and the material
was aliquoted, and stored at -80.degree. C. according to our
standard procedures. The concentration of the pooled fractions was
0.26 mg/ml.
Example 11
[0292] Assay Using zalpha11 Soluble Receptor zalpha11CEE,
zalpha11CFLG and zalpha11-Fc4 (Mutant) Soluble Receptors in
Competitive Inhibition Assay
[0293] BaF3/Zalpha11 cells were spun down and washed in mIL-3 free
media. The cells were spun and washed 3 times to ensure the removal
of the mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at 5000 cells per well in a volume
of 100 .mu.l per well using the mIL-3 free media.
[0294] Both conditioned media from the monkey spleen cell
activation and the human CD3+ selected cells, described in Example
5, were added in separate experiments at 50%, 25%, 12.5%, 6.25%,
3.125%, 1.5%, 0.75% and 0.375% concentrations, with or without
zalpha11 soluble receptors (CEE, C-flag, and Fc4 constructs; See,
Example 9 and 10) at 10 .mu.g/ml. The total assay volume was 200
.mu.l.
[0295] The assay plates were incubated 37.degree. C., 5% CO.sub.2
for 3 days at which time Alamar Blue (Accumed) was added at 20
.mu.l/well. Plates were again incubated at 37.degree. C., 5%
CO.sub.2 for 24 hours. Plates were read on the Fmax.TM. plate
reader (Molecular Devices) as described in Example 2. Results
demonstrated complete inhibition of cell growth from each of the
different zalpha11 soluble receptor constructs at 10 .mu.g/ml,
confirming that the factor in each sample was specific for the
zalpha11 receptor.
[0296] Titration curves, diluting out the soluble receptors, were
also run using the above stated assay. Both the zalpha11CEE and
zalpha11CFLG soluble zalpha11 receptors were able to completely
inhibit growth at concentrations as low as 20 ng/ml. The mutant
zalpha11-Fc4 soluble zalpha11 receptor was only as effective at 1.5
.mu.g/ml.
Example 12
[0297] Secretion Trap Assay
[0298] A secretion trap assay was used to identify the cDNA by for
the zalpha11 Ligand. The positive DNA pools obtained from the
expression cloning effort described in Example 7.
[0299] The DNA pools of 250 clones were transfected into BHK cells
in 96-well format, and the condition medium were put into the
proliferation assay using BaF3/zalpha11 cells described in Examples
4 and Example 5. Several DNA pools gave positive activities which
were repeated and neutralized with zalpha11 soluble receptors (see
Example 11).
[0300] One of the positive DNA pools, 45C5, was transfected into
COS cells in 12-well format, using the Lipofectamine.TM. method
described below. A secretion trap assay was then performed using
zalpha11 soluble receptors (C-terminal Glu-Glu tagged either with
or without biotinylation; C-terminal Flag tagged; or Fc4 zalpha11
soluble receptor fusions) (Example 9) to test the direct binding
between potential ligand of zalpha11 receptor in pool 45C5 and
zalpha11 soluble receptor (see below). The result was positive.
Thus, the DNA of pool 45C5 was electroporated into E. coli, and
single colonies were picked into ten 96-well plates. Plates were
shaken at 37.quadrature.C for 24 hours, and then DNA minipreps
(QiaPrep.TM. 96 Turbo Miniprep Kit; Qiagen) were prepared in
96-well format using a TomTech Quadra 9600. The plasmid DNA was
then pooled in the format of rows and columns, transfected into COS
cells, and then the positive pools were determined by secretion
trap as described below.
[0301] COS Cell Transfections
[0302] The COS cell transfection was performed as follows: Mix 3 ul
pooled DNA and 5 ul Lipofectamine.TM. in 92 ul serum free DMEM
media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mg transferrin,
2.5 mg insulin, 1 .mu.g selenium and 5 mg fetuin in 500 ml DMEM),
incubate at room temperature for 30 minutes and then add 400 ul
serum free DMEM media. Add this 500 ul mixture onto
1.5.times.10.sup.5 COS cells/well plated on 12-well tissue culture
plate and incubate for 5 hours at 37.degree. C. Add 500 ul 20% FBS
DMEM media (100 ml FBS, 55 mg sodium pyruvate and 146 mg
L-glutamine in 500 ml DMEM) and incubate overnight.
[0303] Secretion Trap Assay
[0304] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and then fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed with TNT (0.1M
Tris-HCL, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O), and
permeated with 0.1% Triton-X in PBS for 15 minutes, and again
washed with TNT. Cells were blocked for 1 hour with TNB (0.1M
Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance
TSA-Direct Kit) in H.sub.2O), and washed again with TNT. If using
the biotinylated protein, the cells were blocked for 15 minute
incubations with Avidin and then Biotin (Vector Labs) washing
in-between with TNT. Depending on which soluble receptor was used,
the cells were incubated for 1 hour with: (A) 1-3 .mu.g/ml zalpha11
soluble receptor zalpha11-Fc4 fusion protein (Example 10); (B) 3
.mu.g/ml zalpha11 soluble receptor C-terminal FLAG tagged,
zalpha11CFLG (Example 10); (C) 3 .mu.g/ml zalpha11 soluble receptor
C-terminal GluGlu tagged, zalpha11CEE (Example 10); or (D) 3
.mu.g/ml biotinylated zalpha11 soluble receptor zalpha11CEE in TNB.
Cells were then washed with TNT. Depending on which soluble
receptor was used, cells were incubated for another hour with: (A)
1:200 diluted goat-anti-human Ig-HRP (Fc specific); (B) 1:1000
diluted M2-HRP; (C) 1:1000 diluted anti-GluGlu antibody-HRP; or (D)
1:300 diluted streptavidin-HRP (NEN kit) in TNB. Again cells were
washed with TNT.
[0305] Positive binding was detected with fluorescein tyramide
reagent diluted 1:50 in dilution buffer (NEN kit) and incubated for
4-6 minutes, and washed with TNT. Cells were preserved with
Vectashield Mounting Media (Vector Labs Burlingame, Calif.) diluted
1:5 in TNT. Cells were visualized using a FITC filter on
fluorescent microscope.
Example 13
[0306] Chromosomal Assignment and Placement of the Gene for the
zalpha11 Ligand.
[0307] The gene for the zalpha11 Ligand was mapped to chromosome 4
using the commercially available version of the "Stanford G3
Radiation Hybrid Mapping Panel" (Research Genetics, Inc.,
Huntsville, Ala.). The "Stanford G3 RH Panel" contains DNAs from
each of 83 radiation hybrid clones of the whole human genome, plus
two control DNAs (the RM donor and the A3 recipient). A publicly
available WWW server (http://shgc-www.stanford.edu) allows
chromosomal localization of markers.
[0308] For the mapping of the zalpha11 Ligand gene with the
"Stanford G3 RH Panel", 20 .mu.l reactions were set up in a 96-well
microtiter plate (Stratagene, La Jolla, Calif.) and used in a
"RoboCycler Gradient 96" thermal cycler (Stratagene). Each of the
85 PCR reactions consisted of 2 .mu.l 10.times. KlenTaq PCR
reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.),
1.6 .mu.l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,
Calif.), 1 .mu.l sense primer, ZC 22,050 (SEQ ID NO:42), 1 .mu.l
antisense primer, ZC 22,051 (SEQ ID NO:43), 2 .mu.l "RediLoad"
(Research Genetics, Inc., Huntsville, Ala.), 0.4 .mu.l 50.times.
Advantage KlenTaq Polymerase Mix (Clontech Laboratories, Inc.), 25
ng of DNA from an individual hybrid clone or control and ddH2O for
a total volume of 20 .mu.l. The reactions were overlaid with an
equal amount of mineral oil and sealed. The PCR cycler conditions
were as follows: an initial 1 cycle 5 minute denaturation at
94.degree. C., 35 cycles of a 45 seconds denaturation at 94.degree.
C., 45 seconds annealing at 60.degree. C. and 1 minute AND 15
seconds extension at 72.degree. C., followed by a final 1 cycle
extension of 7 minutes at 72.degree. C. The reactions were
separated by electrophoresis on a 2% agarose gel (Life
Technologies, Gaithersburg, Md.).
[0309] The results showed linkage of the zalpha11 Ligand gene to
the IL2 framework marker SHGC-12342 with a LOD score of >12 and
at a distance of 6 cR.sub.--10000 (approximately 180 kb) from the
marker. The use of surrounding markers positions the zalpha11
Ligand gene in the 4q27 region on the integrated LDB chromosome 4
map.
Example 14
[0310] Identification and Cloning of Murine zalpha11 Ligand
[0311] Using an EST Sequence to Obtain Full-Length Murine zalpha11
Ligand
[0312] A. EST Sequence of Mouse zalpha11 Ligand
[0313] By searching the database with human zalpha11 Ligand cDNA
sequence (SEQ ID NO:1) as a query, a mouse EST (EST1483966) was
identified as potential partial sequence for mouse zalpha11 Ligand.
The EST1483966 represents a mouse genomic fragment, in which a
peptide sequence derived from two potential exons shared high
sequence identity with a peptide segment of the human zalpha11
Ligand (amino acid 13 (Ile) through amino acid 80 (Gln) of the SEQ
ID NO:2).
[0314] B. PCR Screen of Mouse Marathon cDNA Panel
[0315] Eleven mouse Marathon cDNA (Clontech) samples were screened
by PCR described below. The mouse marathon cDNA samples were
prepared from brain, pancreas, kidney, placenta, salivary gland,
skin, testis, uterus, embryo, and spleen tissues. They were made
in-house using a Marathon.quadrature. cDNA Amplification Kit
(Clontech) according to manufacturer's instructions. Based on the
EST sequence, two PCR primers, ZC22,056 (SEQ ID NO:44) and ZC22,057
(SEQ ID NO:45) were used to identify a source of mouse zalpha11
Ligand by PCR. The PCR reaction conditions were as follows:
94.degree. C. for 2 min.; 35 cycles at 94.degree. C. for 30 sec.,
68.degree. C. for 2 min.; followed by 68.degree. C. for 4 min.;
then a 10.degree. C. soak. The PCR products were run on a 1%
agarose gel. A strong 150 bp band representing an amplified cDNA
fragment was visualized. This indicated mouse spleen marathon cDNA
is the source for mouse zalpha11 Ligand cDNA cloning. The mouse
spleen marathon cDNA contained a positive cDNA which was
subsequently identified by sequence analysis as a partial cDNA for
mouse zalpha11 Ligand.
[0316] C. A composite Sequence for Mouse Full-Length cDNA was
Generated by 5'- and 3'-RACE
[0317] The 5' and 3' flanking sequences of the mouse zalpha11
Ligand partial cDNA sequence were obtained by 5' and 3' RACE
amplification. Two rounds of nested PCR amplification were
performed with additional gene-specific oligo primers ZC22,205 (SEQ
ID NO:46) and ZC22,206 (SEQ ID NO:47), ZC22,056 (SEQ ID NO:44) and
ZC22,057 (SEQ ID NO:45), and two adapter oligo primers ZC9,739 (SEQ
ID NO:48) and ZC9,719 (SEQ ID NO:49). The PCR reactions were run as
follows: 94.degree. C. for 2 min; 35 cycles at 94.degree. C. for 30
sec, 68.degree. C. for 2 min; followed by 68.degree. C. for 4 min;
then a 10.degree. C. soak. The PCR products were run on a 1%
agarose gel, and an approximately 300 bp 5' RACE product and an
approximately 800 bp 3' RACE product were identified. These
fragments were isolated using Qiaquick.TM. gel extraction kit
(Qiagen).
[0318] The purified PCR products were sequenced using the following
primers: ZC9,719 (SEQ ID NO:49), ZC22,205 (SEQ ID NO:46) and
ZC22,206 (SEQ ID NO:47). A preliminary composite full-length mouse
zalpha11 Ligand sequence was identified by combining the 5' and 3'
RACE fragments. The full length mouse clone was isolated as
described in Example 15 below.
Example 15
[0319] Isolation of Mouse zalpha11 cDNA Clone from an Activated
Mouse Spleen Library
[0320] A. Murine Primary Source Used to Isolate Mouse zalpha11
Ligand
[0321] Mouse spleens from Balb/C mice, were collected and mashed
between frosted-end slides to create a cell suspension. The
isolated primary mouse cell yield was 6.4.times.10.sup.8 cells
prior to selection described below.
[0322] The spleen cells were suspended in 9.6 ml MACS buffer (PBS,
0.5% EDTA, 2 mM EDTA). 1.6 ml of cell suspension was removed and
0.4 ml CD90 (Thy1.2) microbeads (Miltenyi Biotec) added. The
mixture was incubated for 15 min. at 4.degree. C. These cells
labeled with CD90 beads were washed with 30 ml MACS buffer, and
then resuspended in 2 ml MACS buffer.
[0323] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS.TM. magnetic field (Miltenyi). The column was
equilibrated with 5 ml MACS buffer. The isolated primary mouse
cells were then applied to the column. The CD3 negative cells were
allowed to pass through. The column was rinsed with 9 ml (3.times.3
ml) MACS buffer. The column was then removed from the magnet and
placed over a 15 ml falcon tube. CD90+ cells were eluted by adding
5 ml MACS buffer to the column and bound cells flushed out using
the plunger provided by the manufacturer. The incubation of the
cells with the CD90 magnetic beads, washes, and VS+ column steps
(incubation through elution) above were repeated once more. The
resulting CD90+fractions from the 2 column separations were pooled.
The yield of CD90+ selected mouse spleen cells were
1.times.10.sup.8 total cells.
[0324] A sample of the pooled CD90+ selected mouse cells was
removed for staining and sorting on a fluorescent antibody cell
sorter (FACS) to assess their purity. A PE-conjugated hamster
anti-mouse CD3+ antibody (PharMingen) was used for staining and
sorting the CD90+ selected cells. The mouse CD90+ selected cells
were 93% CD3+ cells, suggesting the cells were 93% T-cells.
[0325] The murine CD90+ selected cells were activated by incubating
3.times.10.sup.6 cells/ml in RPMI+5% FBS+PMA 10 ng/ml and Ionomycin
0.5 .mu.g/ml (Calbiochem) for overnight at 37.quadrature. C. The
supernatant from these activated CD90+ selected mouse cells was
tested for zalpha11 Ligand activity as described below. Moreover,
the activated CD90+ selected mouse cells were used to prepare a
cDNA library, as described in Example 16, below.
Example 16
[0326] Cloning of Mouse zalpha11 Ligand from a Mouse CD90+ Selected
Cell Library
[0327] Screening of a primary mouse activated CD90+ selected cell
cDNA library revealed an isolated cDNA that is a novel member of
the four-helix bundle cytokine family. This cDNA encoded the mouse
ortholog of the human zalpha11 Ligand. The cDNA was identified by
hybridization screening.
[0328] A. The Vector for CD90+ Selected Library Construction
[0329] The vector, pZP7N was used for CD3+ selected library
construction (See Example 6A)
[0330] B. Preparation of Primary Mouse Activated CD90+ Selected
Cell cDNA Library
[0331] Approximately 1.5.times.10.sup.8 primary mouse CD90+
selected cells stimulated in ionomycin/PMA (Example 15) were
isolated by centrifugation. Total RNA was isolated from the cell
pellet, and converted to double stranded cDNA as described in
Example 6B. This DNA was subsequently transfected into BHK cells,
as described in Example 6B, and proliferation was assessed using an
"Alamar blue" fluorescence assay (Example 2B).
[0332] For the purpose of screening the library by secretion trap
cloning, a complex, amplified form of the library was needed to
transfect COS-7 cells. 4.8 million clones were plated on 110 15 cm
LB-agar plates supplemented with 100 .mu.g/ml ampicillin, 10
.mu.g/ml methicillin. After growing the plates overnight at
37.degree. C. the bacteria were harvested by scraping and pelleted.
Plasmid DNA was extracted from the pelleted bacteria using a
Nucleobond-giga.TM. (Clonetech) following the manufacturer's
instructions. This plasmid was then used to transfect COS-7 cells
on slides and screened using the secretion trap technique described
below (Example 17).
[0333] C. Screening the Activated Mouse cDNA Library
[0334] Approximately 5.times.10.sup.5 clones were plated on 10
LB/Amp Maxi plates. The colonies were lifted, denatured,
neutralized, and cross-linked using the standard procedure
(Sambrook, J. et al. supra.). Fifty nanograms of the 300 bp 5' RACE
PCR fragment (Example 14) was labeled with 32P using Prime-Itr RmT
random primer labeling kit (Stratagene). The 10 filters were
hybridized with this labeled probe at 65.degree. C. overnight using
ExpressHyb.TM. Hybridization Solution (Clontech). The filters were
then washed sequentially at 60.degree. C. for 1 hour three times
with 0.2.times.SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.0), 0.1%
SDS; and then at 65.degree. C. for 1 hour. The filters were exposed
at -80.degree. C. overnight, and the X-ray film were developed.
Agar plugs containing the positive colonies were pulled, and the
clones plated on 10-cm LB/Amp plates. The colonies were then
filter-lifted and hybridized again following the same procedure
described above.
[0335] One DNA clone, named M11L/pZP7, was isolated and sequenced
using the following primers: ZC14,063 (SEQ ID NO:50), ZC5,020 (SEQ
ID NO:51), ZC22,421 (SEQ ID NO:52), ZC22,604 (SEQ ID NO:53), and
ZC22,641 (SEQ ID NO:54). The polynucleotide sequence of this clone
is full-length mouse zalpha11 Ligand (SEQ ID NO:55) and consistent
with the composite sequence obtained from 5' and 3' RACE products.
The corresponding amino acid sequence for the mouse zalpha11 Ligand
is shown in SEQ ID NO:56.
Example 17
[0336] Mouse zalpha11 Ligand Binds to Human zalpha11 Soluble
Receptor in Secretion Trap Assay
[0337] The DNA for mouse clone M11L/pZP7 was transfected into COS
cells, and the binding of human zalpha11 soluble receptor
zalpha11-Fc4 (Example 10C) to the transfected COS cells was tested
by a secretion trap assay (Example 12). The assay confirmed that
the mouse zalpha11 Ligand binds to human zalpha11 soluble
receptor.
[0338] The COS cell transfection was performed as per example 12
using 0.7 .quadrature.g M11L/pZP7 DNA (Example 16) in 3 .mu.l.
[0339] The secretion trap was performed as per example 12 using 1
.mu.g/ml zalpha11 soluble receptor Fc4 fusion protein (Example 10C)
in TNB, and 1:200 diluted goat-anti-human Ig-HRP (Fc specific) in
TNB for the detectable antibody. Positive binding of the soluble
human zalpha11 receptor to the prepared fixed cells was detected
with fluorescein tyramide reagent as per Example 12. Cells were
preserved and visualized according to Example 12.
[0340] The positive result indicated the mouse zalpha11 Ligand
binds to human zalpha11 soluble receptor.
Example 18
[0341] Expression of Mouse zalpha11 Ligand in Mammalian Cells
[0342] A. Construction of Expression Vector M11L/pZP9
[0343] An expression vector was prepared for the expression of the
mouse zalpha11 Ligand in mammalian cells. A 500 bp PCR generated
zalpha11 Ligand DNA fragment was created using ZC22,283 (SEQ ID
NO:57) and ZC22,284 (SEQ ID NO:58) as PCR primers to amplify the
coding region of mouse zalpha11 Ligand and add XhoI and XbaI
restriction sites. The mouse zalpha11 Ligand clone M11L/pZP7
(Example 16) was used as a template. The PCR reaction conditions
were as follows: 94.degree. C. for 2 min.; 25 cycles at 94.degree.
C. for 30 sec., 68.degree. C. for 2 min.; followed by 68.degree. C.
for 4 min.; then a 10.degree. C. soak. A band of the predicted
size, about 500 bp, was visualized by 1% agarose gel
electrophoresis, excised and the DNA was purified using a
QiaexII.TM. purification system (Qiagen) according to the
manufacturer's instructions. The purified DNA was digested with
XhoI and XbaI (Boehringer Mannheim) at 37.degree. C. for 2 hours.
Then the DNA was gel isolated and purified following the above
procedure.
[0344] The excised DNA was subcloned into plasmid pZP9 which was
cut with XhoI and XbaI (Boehringer Mannheim). Plasmid pZP9 is a
mammalian expression vector containing an expression cassette
having the mouse metallothionein-1 (MT-1) promoter, multiple
restriction sites for insertion of coding sequences, and a human
growth hormone terminator. The plasmid also has an E. coli origin
of replication, a mammalian selective marker expression unit having
an SV40 promoter, enhancer and origin of replication, a DHFR gene,
and the SV40 terminator.
[0345] About 30 ng of the restriction digested mouse zalpha11
Ligand fragment and about 10 ng of the digested pZP9 vector were
ligated at room temperature for 2 hours. Two .mu.g of ligation
reaction was transformed into INVaF' competent cells (Invitrogen)
according to manufacturer's protocol and plated onto LB plates
containing 50 .mu.g/ml ampicillin, and incubated at 37.degree. C.
overnight. Colonies were screened by restriction analysis using
XhoI and XbaI (Boerhinger Mannheim) of DNA prepared from liquid
cultures of individual colonies. The insert sequence of positive
clones was verified by sequence analysis to be the mouse zalpha11
Ligand sequence. A large scale plasmid preparation was done using a
Qiagen.RTM. Maxi prep kit (Qiagen) according to manufacturer's
instruction. The expression vector that contains mouse zalpha11
Ligand was named M11L/pZP9.
[0346] B. Mammalian Expression of Mouse zalpha11 Ligand
[0347] BHK 570 cells (ATCC No: CRL-10314) were plated in 10 cm
tissue culture dishes and allowed to grow to approximately 20%
confluence overnight at 37.degree. C., 5% CO.sub.2, in DMEM/FBS
media (DMEM, Gibco/BRL High Glucose media; Gibco BRL, Gaithersburg,
Md.), 5% fetal bovine serum (Hyclone, Logan, Utah), 1 mM
L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate
(Gibco BRL)). The cells were then transfected with the plasmid
M11L/pZP9 (Example 18A) using a mammalian stable CaPO.sub.4
transfection kit (Stratagene) according to the manufacturer's
instructions.
[0348] One day after transfection, the cells were split 1:10 and
1:20 into the selection media (DMEM/FBS media with the addition of
1 .mu.M methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150
mm plates. The media on the cells was replaced with fresh selection
media at day 5 post-transfection. Approximately 10 days
post-transfection, methotrexate resistant colonies were trypsinized
and the cells pooled and plated into large scale culture flasks.
Once the cells were grown to approximately 90% confluence, they
were rinsed with PBS three times, and cultured with serum-free
ESTEP2 media (DMEM (Gibco BRL), 0.11 g/l Na Pyruvate, 3.7 g/l
NaHCO.sub.3, 2.5 mg/l insulin, 5 mg/l transferrin, pH7.0)
conditioned media. The conditioned media were collected three days
later, and put into a BaF3 proliferation assay using Alamar Blue,
described in Example 19 below.
Example 19
[0349] Mouse zalpha11 Ligand Activates Human zalpha11 Receptor in
BaF3 Assay Using Alamar Blue
[0350] Proliferation of BaF3/zalpha11 cells (Example 4, and 5B) was
assessed using serum-free conditioned media from BHK cells
expressing mouse zalpha11 Ligand (Example 18).
[0351] BaF3/Zalpha11 cells were spun down, washed and plated in
mIL-3 free media as described in Example 5B.
[0352] Proliferation of the BaF3/Zalpha11 cells was assessed using
serum-free conditioned media from BHK cells expressing mouse
zalpha11 Ligand (Example 18). Conditioned media was diluted with
mIL-3 free media to: 50%, 25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75%
and 0.375% concentrations. The proliferation assay was performed as
per Example 5B.
[0353] Results confirmed the proliferative response of the
BaF3/Zalpha11 cells to mouse zalpha11 Ligand. The response, as
measured, was approximately 5-fold over background at the 50%
concentration.
Example 20
[0354] Zalpha11 Ligand Activates Human zalpha11 Receptor in
Luciferase Assay
[0355] A. Construction of BaF3/KZ134/zalpha11 Cell Line
[0356] The KZ134 plasmid was constructed with complementary
oligonucleotides ZC12,749 (SEQ ID NO:59) and ZC12,748 (SEQ ID
NO:60) that contain STAT transcription factor binding elements from
4 genes. A modified c-fos S is inducible element (m67SIE, or hSIE)
(Sadowski, H. et al., Science 261:1739-1744, 1993), the p21 SIE1
from the p21 WAFI gene (Chin, Y. et al., Science 272:719-722,
1996), the mammary gland response element of the
.quadrature.-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.
11:3745-3755, 1991), and a STAT inducible element of the Fcg RI
gene, (Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045,
1995). These oligonucleotides contain Asp718-XhoI compatible ends
and were ligated, using standard methods, into a recipient firefly
luciferase reporter vector with a c-fos promoter (Poulsen, L. K. et
al., J. Biol. Chem. 273:6229-6232, 1998) digested with the same
enzymes and containing a neomycin selectable marker. The KZ134
plasmid was used to stably transfect BaF3 cells, using standard
transfection and selection methods, to make the BaF3/KZ134 cell
line.
[0357] A stable BaF3/KZ134 indicator cell line, expressing the
full-length zalpha11 receptor was constructed as per Example 2A,
using about 30 .mu.g of the zalpha11 expression vector, described
in Example 3. Clones were diluted, plated and selected using
standard techniques. Clones were screened by luciferase assay (see
Example 20B, below) using the human zalpha11 Ligand conditioned
media as an inducer. Clones with the highest luciferase response
(via STAT luciferase) and the lowest background were selected. A
stable transfectant cell line was selected. The cell line was
called BaF3/KZ134/zalpha11.
[0358] B. Human and Mouse Zalpha11 Ligand Activates Human zalpha11
Receptor in BaF3/KZ134/Zalpha11 Luciferase Assay
[0359] BaF3/KZ134/Zalpha11 cells were spun down and washed in mIL-3
free media. The cells were spun and washed 3 times to ensure
removal of mIL-3. Cells were then counted in a hemacytometer. Cells
were plated in a 96-well format at about 30,000 cells per well in a
volume of 100 .mu.l per well using the mIL-3 free media. The same
procedure was used for untransfected BaF3/KZ134 cells for use as a
control in the subsequent assay.
[0360] STAT activation of the BaF3/KZ134/Zalpha11 cells was
assessed using conditioned media from (1) BHK570 cells transfected
with the human zalpha11 Ligand (Example 7) or (2) BHK570 cells
transfected with the mouse zalpha11 Ligand (Example 18), or (4)
mIL-3 free media to measure media-only control response.
Conditioned media was diluted with RPMI mIL-3 free media to 50%,
25%, 12.5%, 6.25%, 3.125%, 1.5%, 0.75% and 0.375% concentrations.
100 .mu.l of the diluted conditioned media was added to the
BaF3/KZ134/Zalpha11 cells. The assay using the conditioned media
was done in parallel on untransfected BaF3/KZ134 cells as a
control. The total assay volume was 200 .mu.l. The assay plates
were incubated at 37.degree. C., 5% CO.sub.2 for 24 hours at which
time the cells were pelleted by centrifugation at 2000 rpm for 10
min., and the media was aspirated and 25 .mu.l of lysis buffer
(Promega) was added. After 10 minutes at room temperature, the
plates were measured for activation of the STAT reporter construct
by reading them on a luminometer (Labsystems Luminoskan, model RS)
which added 40 .mu.l of luciferase assay substrate (Promega) at a
five second integration.
[0361] Results confirmed the STAT reporter response of the
BaF3/KZ134/Zalpha11 cells to the human zalpha11 Ligand. The
response, as measured, was approximately 50 fold over media-only
control at the 50% concentration. STAT activation in response to
human zalpha11 Ligand was absent in the untransfected BaF3/KZ134
control cells, showing that the response is mediated through the
Zalpha11 receptor.
[0362] Results also confirmed the STAT reporter response of the
BaF3/KZ134/Zalpha11 cells to the mouse zalpha11 Ligand. The
response, as measured, was approximately 40 fold over media-only
control at the 50% concentration. Moreover, STAT activation in
response to mouse zalpha11 Ligand was evident (about 5-fold) on the
untransfected BaF/KZ134 control cells, suggesting that the murine
BaF3 cells may have endogenous mouse receptor.
Example 21
[0363] Mouse zalpha11 Ligand is Active in Mouse Bone Marrow
Assay
[0364] A. Isolation of Non-adherent Low Density Marrow Cells:
[0365] Fresh mouse femur aspirate (marrow) was obtained from 6-10
week old male Balb/C or C57BL/6 mice. The marrow was then washed
with RPMI+10% FBS (JRH, Lenexa Kans.; Hyclone, Logan Utah) and
suspended in RPMI+10% FBS as a whole marrow cell suspension. The
whole marrow cell suspension was then subjected to a density
gradient (Nycoprep, 1.077, Animal; Gibco BRL) to enrich for low
density, mostly mononuclear, cells as follows: The whole marrow
cell suspension (About 8 ml) was carefully pipeted on top of about
5 ml Nycoprep gradient solution in a 15 ml conical tube, and then
centrifuged at 600.times.g for 20 minutes. The interface layer,
containing the low density mononuclear cells, was then removed,
washed with excess RPMI+10% FBS, and pelleted by centrifugation at
400.times.g for 5-10 minutes. This pellet was resuspended in
RPMI+10% FBS and plated in a T-75 flask at approximately 10.sup.6
cells/ml, and incubated at 37.degree. C. 5% CO.sub.2 for
approximately 2 hours. The resulting cells in suspension were
Non-Adherent Low Density (NA LD) Marrow Cells.
[0366] B. 96-Well Assay
[0367] NA LD Mouse Marrow Cells were plated at 25,000 to 45,000
cells/well in 96 well tissue culture plates in RPMI+10% FBS+1 ng/mL
mouse Stem Cell Factor (mSCF) (R&D Systems, Minneapolis,
Minn.), plus 5% conditioned medium from one of the following: (1)
BHK 570 cells expressing mouse zalpha11 Ligand (Example 18), (2)
BHK 570 cells expressing human zalpha11 Ligand (Example 7), or (3)
control BHK 570 cells containing vector and not expressing either
Ligand. These cells were then subjected to a variety of cytokine
treatments to test for expansion or differentiation of
hematopoietic cells from the marrow. To test, the plated NA LD
mouse marrow cells were subjected to human Interleukin-15 (hIL-15)
(R&D Systems), or one of a panel of other cytokines (R&D
Systems). Serial dilution of hIl-15, or the other cytokines, were
tested, with 2-fold serial dilution from about 50 ng/ml down to
about 6025 ng/ml concentration. After 8 to 12 days the 96-well
assays were scored for cell proliferation by Alamar blue assay as
described in Example 5B.
[0368] C. Results from the 96-Well NA LD Mouse Marrow Assay
[0369] Conditioned media from the BHK cells expressing both mouse
and human zalpha11 Ligand acted in synergy with hIL-15 to promote
the expansion of a population of hematopoietic cells in the NA LD
mouse marrow. This expansion of hematopoietic cells was not shown
with control BHK conditioned medium plus IL-15. The population
hematopoietic cells expanded by the mouse zalpha11 Ligand with
hIL-15, and those hematopoietic cells expanded by the human
zalpha11 Ligand with hIL-15, were further propagated in cell
culture. These hematopoietic cells were stained with a
Phycoerythrin labeled anti-Pan NK cell antibody (Pharmingen) and
subjected to flow cytometry analysis, which demonstrated that the
expanded cells stained positively for this natural killer (NK) cell
marker.
[0370] The same 96-well assay was run, using fresh human marrow
cells bought from Poietic Technologies, Gaithersburg, Md. Again, in
conjunction with IL-15, the mouse and human zalpha11Ligand expanded
a hematopoietic cell population that stained positively for the NK
cell marker using the antibody disclosed above.
Example 22
[0371] Constructs for Generating Human zalpha11 Ligand Transgenic
Mice
[0372] A. Construct for Expressing Human zalpha11 Ligand from the
Liver-Specific MT-1 Promoter
[0373] Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the human zalpha11 Ligand
coding region. These oligonucleotides were designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into (a) pMT12-8, our standard transgenic vector, or (b)
pKFO51, a lymphoid-specific transgenic vector (Example 22B).
[0374] PCR reactions were carried out with 200 ng human zalpha11
Ligand template (Example 7) and oligonucleotides ZC22,143 (SEQ ID
NO:61) and ZC22,144 (SEQ ID NO:62). PCR reaction conditions were as
follows: 95.degree. C. for 5 minutes, wherein Advantage.TM. cDNA
polymerase (Clontech) was added; 15 cycles of 95.degree. C. for 60
seconds, 60.degree. C. for 60 seconds, and 72.degree. C. for 90
seconds; and 72.degree. C. for 7 minutes. PCR products were
separated by agarose gel electrophoresis and purified using a
QiaQuick* (Qiagen) gel extraction kit. The isolated, 488 bp, DNA
fragment was digested with FseI and AscI (Boerhinger-Mannheim),
ethanol precipitated and ligated into pMT12-8 previously digested
with FseI and AscI. The pMT12-8 plasmid, designed for expressing a
gene of interest in liver and other tissues in transgenic mice,
contains an expression cassette flanked by 10 kb of MT-1 5' DNA and
7 kb of MT-1 3' DNA. The expression cassette comprises the MT-1
promoter, the rat insulin II intron, a polylinker for the insertion
of the desired clone, and the human growth hormone (hGH) poly A
sequence.
[0375] About one microliter of each ligation reaction was
electroporated into DH10B ElectroMax* competent cells (GIBCO BRL,
Gaithersburg, Md.) according to manufacturer's direction and plated
onto LB plates containing 100 .mu.g/ml ampicillin, and incubated
overnight. Colonies were picked and grown in LB media containing
100 .mu.g/ml ampicillin. Miniprep DNA was prepared from the picked
clones and screened for the human zalpha11Ligand insert by
restriction digestion with EcoRI alone, or FseI and AscI combined,
and subsequent agarose gel electrophoresis. Maxipreps of the
correct pMT-human zalpha11Ligand were performed. A SalI fragment
containing with 5' and 3' flanking sequences, the MT-1 promoter,
the rat insulin II intron, human zalpha11 Ligand cDNA and the hGH
poly A sequence was prepared to be used for microinjection into
fertilized murine oocytes. Microinjection and production of
transgenic mice were produced as described in Hogan, B. et al.
Manipulating the Mouse Embryo, 2nd ed., Cold Spring Harbor
Laboratory Press, NY, 1994.
[0376] B. Construct for Expressing Human zalpha11 Ligand from the
Lymphoid-Specific E.quadrature.LCK Promoter
[0377] Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the human zalpha11 Ligand
coding region. These oligonucleotides were designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into pKFO51, a lymphoid-specific transgenic vector.
[0378] PCR reactions were carried out with 200 ng human zalpha11
Ligand template (Example 7) and oligonucleotides ZC22,143 (SEQ ID
NO:61) and ZC22,144 (SEQ ID NO:62). A PCR reaction was performed
using Advantage.TM. cDNA polymerase (Clontech) under the following
conditions: 95.degree. C. for 5 minutes; 15 cycles of 95.degree. C.
for 60 seconds, 60.degree. C. for 60 seconds, and 72.degree. C. for
90 seconds; and 72.degree. C. for 7 minutes. PCR products purified
as described above. The isolated, 488 bp, DNA fragment was digested
with FseI and AscI (Boerhinger-Mannheim), ethanol precipitated and
ligated into pKFO51 previously digested with FseI and AscI. The
pKFO51 transgenic vector is derived from p1026X (Iritani, B. M., et
al., EMBO J. 16:7019-31, 1997) and contains the T cell-specific lck
proximal promoter, the B/T cell-specific immunoglobulin .mu. heavy
chain enhancer, a polylinker for the insertion of the desired
clone, and a mutated hGH gene that encodes an inactive growth
hormone protein (providing 3' introns and a polyadenylation
signal).
[0379] About one microliter of each ligation reaction was
electroporated, plated, clones picked and screened for the human
zalpha11 Ligand insert by restriction digestion as described above.
A correct clone of pKFO51-zalpha11 Ligand was verified by
sequencing, and a maxiprep of this clone was performed. A NotI
fragment, containing the lck proximal promoter and immunoglobulin
11 enhancer (E.mu.LCK), zalpha11 Ligand cDNA, and the mutated hGH
gene was prepared to be used for microinjection into fertilized
murine oocytes.
Example 23
[0380] Mouse Zalpha11 Ligand Tissue Distribution
[0381] Murine Multiple Tissue Northern Blots (Mouse, Mouse Embryo,
Clontech; MB1010, MB1012 Origene) were probed to determine the
tissue distribution of murine zalpha11 Ligand expression. An
approximately 484 bp PCR derived probe was amplified using the
plasmid M11L/pZP7 (Example 16) as a template and oligonucleotide
ZC22283 (SEQ ID NO:57) and ZC22284 (SEQ ID NO:58) as primers. The
PCR amplification was carried out as follows: 1 cycle at 94.degree.
C. for 1.0 minutes; 35 cycles of 94.degree. C. for 30 seconds,
50.degree. C. for 30 seconds, and 72.degree. C. for 30 seconds;
followed by 1 cycle at 72.degree. C. for 10 minutes. The PCR
products were visualized by agarose gel electrophoresis and the
approximately 484 bp PCR product was purified using a Gel
Extraction Kit (Qiagen) according to manufacturer's instructions.
The probe was radioactively labeled using the REDIPRIME.TM.
labeling kit (Amersham) according to the manufacturer's
instructions. The probe was purified using a NUCTRAP.TM. push
column (Stratagene). EXPRESSHYB.TM. (Clontech) solution was used
for prehybridization and as a hybridizing solution for the Northern
blots. Hybridization took place overnight at 65.degree. C. using
10.sup.6 cpm/ml of labeled probe. The blots were then washed three
times in 2.times.SSC and 0.1% SDS at room temperature, followed by
2 washes in 0.1.times.SSC and 0.1% SDS at 55.degree. C. Two
transcripts of approximately 1.2 and 3.5 kb were seen in testis.
The upper transcript only was seen in thymus.
[0382] A murine RNA Master Dot Blot (Clontech) that contained RNAs
from various tissues that were normalized to 8 housekeeping genes
was also probed and hybridized as described above. Expression was
seen in testis.
Example 24
[0383] Purification of Untagged Human and Murine zalpha11 Ligand
from BHK 570
[0384] Unless other wise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying human
and murine zalpha11 Ligand from conditioned media from BHK 570
cells transfected with a construct expressing either the human
zalpha11 Ligand (Example 25) or the mouse zalpha11 Ligand
(M11L/pZP9) (Example 18). The conditioned media was concentrated by
standard techniques. Concentrated conditioned media (CM) was
sterile filtered through 0.45 and 0.22 micron filters. The media
was then diluted to low ionic strength (<2 mS) in 0.01 M HEPES
(JRH Biosciences, Lenexa, Kans.) at pH 7.0. The low ionic strength
diluted CM was then loaded onto a 10.times.66 mm (6 ml) Poros HS 50
column (PerSeptive BioSystems, Framingham, Mass.) overnight at 4
ml/min using a BioCAD SPRINT (Perceptive BioSystems). The column
was washed for 10-20 column volumes (CV) with 0.01 M HEPES pH7.0.
The bound proteins were then step eluted with 1 M NaCl
(Mallinckrodt, Paris, Ky.) in 0.01 M HEPES pH 7.0 at 5 ml/min; two
ml fractions were collected over the entire chromatography and
absorbence at 280 and 215 nM were monitored. Peak absorbence
fractions were analyzed by bioassay and by SDS-PAGE Silver (Geno
Technology, St. Louis, Mo.) and Coomassie (Sigma, St. Louis, Mo.)
staining. Peak fractions were pooled, sterile filtered and diluted
to <19 mS with Phosphate buffered saline (PBS, Gibco BRL) at pH
7.2.
[0385] The diluted sample was then loaded at 2 ml/min using a
BioCad SPRINT, onto either a 0.8 ml Poros AL column that had
zalpha11CFLAG soluble receptor (Example 10B) or zalpha11-Fc4 fusion
soluble receptor (Example 10C) immobilized on the resin (see,
below). The column was then washed with at least 20 CV of PBS at 10
ml/min. The column was then rapidly eluted with a 600 .mu.l
injection of 0.1 M glycine (Aminoacetic Acid; Glycocol, Spectrum,
Gardena, Calif.) pH 2.5 at a flow rate of 10 ml/min with PBS on a
BioCAD 700E. The 1 ml fractions were collected for 6 seconds each
and immediately pH neutralized with 55 .mu.l of 2 M TRIS (Tris
(Hydroxymethyl) Aminomethane, EM Science, Gibbstown, N.J.) pH 8.8.
The absorbence at 280 and 215 nM were monitored over the entire
chromatography.
[0386] The peak fractions were analyzed by bioassay and by SDS-PAGE
Silver (Geno Technology) and Coomassie (Sigma) staining. Two bands,
approximately 24 kD and 18 kD, were seen on both Silver and
Coomassie gels for mouse zalpha11 Ligand. A single band, at
approximately 18 kD, was seen on both Silver and Coomassie gels for
human zalpha11 Ligand.
[0387] Immobilization of Human zalpha11 Soluble Receptor
Polypeptides on POROS AL Media
[0388] Poros AL columns having immobilized zalpha11CFLAG soluble
receptor (Example 10B) or zalpha11-Fc4 fusion soluble receptor
(Example 10C) were prepared. Approximately 3 mg of zalpha11CFLAG
soluble receptor and approximately 10 mg of zalpha11-Fc4 fusion
soluble receptor were used. All operations were carried out at room
temperature on a BioCAD 700E. A 4.5.times.50 mm column with the
POROS AL media was flow packed in 2 M NaCl according to
manufactures specifications. The column was then equilibrated in
1.1 M Na.sub.2SO.sub.4/50 mM NaPhosphate pH 7.2. The receptor was
concentrated to 4 mg/ml using a Millipore 30 MWKO spin concentrator
then diluted 1:1 in 1.1 M Na.sub.2SO.sub.4/50 mM NaPhosphate pH
7.2. The column was flowed at 2 ml/min in 1.1 M Na.sub.2SO.sub.4/50
mM NaPhosphate pH 7.2 and 100 .mu.l injections of the diluted
ligand were made ever 9 CVs until a steady state of saturation, or
break through, was reached. A 62 CV gradient was then run from 1.1
M Na.sub.2SO.sub.4/50 mM NaPhosphate pH 7.2, to 550 mM
Na.sub.2SO.sub.4/50 mM NaPhosphate pH 7.2/5 mg/ml Sodium
Cyanoborohydride. The column was then held for about 2 hours to
complete the immobilization chemistry. The column was then
equilibrated in 0.2 M TRIS pH 7.2/5 mg/ml Sodium Cyanoborohydride
and allowed to rest for about 1 hour to cap the column. Finally the
column was equilibrated in PBS/0.02% Sodium Azide, and stored at
4.degree. C. until needed. Prior to use, the column was pre-eluted
with 0.1 M glycine to ensure that non-specific proteins were
removed and that the column was not leaching the immobilized human
zalpha11 soluble receptor.
Example 25
[0389] Expression of Human zalpha11 Ligand in Mammalian Cells
[0390] A. Construction of Expression Vector PZMP11/zalpha11Lig
[0391] An expression plasmid containing all or part of a
polynucleotide encoding human zalpha11 Ligand was constructed via
homologous recombination. A fragment of human zalpha11 Ligand cDNA
(SEQ ID NO:63) was isolated using PCR. Two primers were used in the
production of the human zalpha11 Ligand fragment in a PCR reaction:
(1) Primer ZC22,052 (SEQ ID NO:64), containing 40 bp of the vector
flanking sequence and 17 bp corresponding to the amino terminus of
the human zalpha11 Ligand, and (2) primer ZC22,053 (SEQ ID NO:65),
containing 40 bp of the 3' end corresponding to the flanking vector
sequence and 17 bp corresponding to the carboxyl terminus of the
human zalpha11 Ligand. The PCR Reaction conditions were as follows:
1 cycle at 94.degree. C. for 2.0 minutes; 25 cycles of 94.degree.
C. for 30 seconds, 60.degree. C. for 30 seconds, and 72.degree. C.
for 30 seconds; followed by 1 cycle at 72.degree. C. for 5 minutes;
4.degree. C. soak. Ten .mu.l of the 100 .mu.l PCR reaction was run
on a 0.8% LMP agarose gel (Seaplaque GTG) with 1.times.TBE buffer
for analysis, and the expected approximately 560 bp fragment seen.
The remaining 90 .mu.l of PCR reaction was precipitated with the
addition of 5 .mu.l 1 M NaCl and 250 .mu.l of absolute ethanol to
be used for recombining onto the recipient vector pZMP11 as
described below. The recipient plasmid pZMP11 was previously cut
with SmaI.
[0392] Plasmid pZMP11 was derived from the plasmid pCZR199
(described herein, e.g., Example 8). The plasmid pCZR199 is a
mammalian expression vector containing an expression cassette
having the CMV immediate early promoter, a consensus intron from
the variable region of mouse immunoglobulin heavy chain locus,
multiple restriction sites for insertion of coding sequences, a
stop codon and a human growth hormone terminator. The plasmid also
has an E. coli origin of replication, a mammalian selectable marker
expression unit having an SV40 promoter, enhancer and origin of
replication, a DHFR gene and the SV40 terminator. The vector pZMP11
was constructed from pCZR199 and includes the replacement of the
metallothionein promoter with the CMV immediate early promoter, and
Kozac sequences at the 5' end of the open reading frame.
[0393] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l of a mixture containing
approximately 1 .mu.g of the human zalpha11 Ligand insert, and 100
ng of SmaI digested pZMP11 vector, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixtures were electropulsed
at 0.75 kV (5 kV/cm), infinite ohms, 25.degree. F. To each cuvette
was added 600 .mu.l of 1.2 M sorbitol and the yeast was then plated
in two 300 .mu.l aliquots onto two URA-D plates and incubated at
30.degree. C.
[0394] After about 48 hours, the Ura+ yeast transformants from a
single plate were resuspended in 1 ml H.sub.2O and spun briefly to
pellet the yeast cells. The cell pellet was resuspended in 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 was
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, followed by a 5 minute spin in a
Eppendorf centrifuge at maximum speed. Three hundred microliters of
the aqueous phase was transferred to a fresh tube, and the DNA
precipitated with 600 .mu.l ethanol (EtOH), followed by
centrifugation for 10 minutes at 4.degree. C. The DNA pellet was
resuspended in 100 .mu.l H.sub.2O.
[0395] Transformation of electrocompetent E. coli cells (DH10B,
GibcoBRL) was done with 0.5-2 ml yeast DNA prep and 40 .mu.l of
DH10B cells. The cells were electropulsed at 2.0 kV, 25 mF and 400
ohms. Following electroporation, 1 ml SOC (2% Bacto.TM. Tryptone
(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was
plated in 250 .mu.l aliquots on four LB AMP plates (LB broth
(Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L Ampicillin).
[0396] Individual clones harboring the correct expression construct
for human zalpha11 Ligand were identified by restriction digest to
verify the presence of the insert and to confirm that the various
DNA sequences have been joined correctly to one another. The insert
of positive clones were subjected to sequence analysis. Larger
scale plasmid DNA was isolated using the Qiagen Maxi kit (Qiagen)
according to manufacturer's instruction
[0397] B. Mammalian Expression of Human zalpha11 Ligand
[0398] BHK 570 cells (ATCC NO: CRL-10314) were 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, (Gibco BRL, Gaithersburg,
Md.), 5% fetal bovine serum (Hyclone, Logan, Utah), 1 mM
L-glutamine (JRH Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate
(Gibco BRL). The cells were then transfected with the plasmid
PZMP11/zalpha11Lig (Example 25A), using Lipofectamine.TM. (Gibco
BRL), in serum free (SF) media formulation (DMEM, 10 mg/ml
transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%
sodium pyruvate). Zalpha11-Fc4/pZMP6 (Example 8B) was diluted into
15 ml tubes to a total final volume of 640 .mu.l with SF media. 35
.mu.l of Lipofectamine.TM. (Gibco BRL) was mixed with 605 .mu.l of
SF medium. The Lipofectamine.TM. mix was added to the DNA mix and
allowed to incubate approximately 30 minutes at room temperature.
Five milliliters of SF media was added to the DNA:Lipofectamine.TM.
mixture. The cells were rinsed once with 5 ml of SF media,
aspirated, and the DNA:Lipofectamine.TM. mixture was added. The
cells were incubated at 37.degree. C. for five hours, then 6.4 ml
of DMEM/10% FBS, 1% PSN media was added to each plate. The plates
were incubated at 37.degree. C. overnight and the
DNA:Lipofectamine.TM. mixture was replaced with fresh 5% FBS/DMEM
media the next day. On day 5 post-transfection, the cells were
split into T-162 flask in selection medium (DMEM/5% FBS, 1% L-GLU,
1% NaPyr). Approximately 10 days post-transfection, two 150 mm
culture dishes of methotrexate resistant colonies from each
transfection were trypsinized and the cells are pooled and plated
into a T-162 flask and transferred to large scale culture.
Conditioned media from large scale culture was used to purify human
zalpha11 Ligand polypeptide as described in Example 24.
Example 26
[0399] Construct for Generating Mouse zalpha11 Ligand Transgenic
Mice
[0400] A. Construct for Expressing Mouse zalpha11 Ligand from the
Lymphoid-Specific E.mu.LCK Promoter
[0401] Oligonucleotides were designed to generate a PCR fragment
containing a consensus Kozak sequence and the mouse zalpha11 Ligand
coding region. These oligonucleotides were designed with an FseI
site at the 5' end and an AscI site at the 3' end to facilitate
cloning into: (a) pKFO51, a lymphoid-specific transgenic vector, or
(b) pTG12-8, our standard transgenic vector.
[0402] PCR reactions were carried out with 200 ng mouse zalpha11
Ligand template (SEQ ID NO:55; Example 16) and oligonucleotides
ZC23,115 (SEQ ID NO:66) and ZC23,116 (SEQ ID NO:67). A PCR reaction
was performed using Advantage.TM. cDNA polymerase (Clontech) under
the PCR conditions described in Example 22B. PCR product was
isolated as described in Example 22B. The isolated, 440 bp DNA
fragment was digested and ligated into pKFO51 previously digested
with FseI and AscI, as described in Example 22B.
[0403] About one microliter of each ligation reaction was
electroporated, plated, clones picked and screened for the human
zalpha11 Ligand insert by restriction digestion as described in
Example 22. A correct pKFO51-zalpha11 Ligand clone was verified by
sequencing, and a maxiprep of this clone was performed. A NotI
fragment, containing the lck proximal promoter, immunoglobulin .mu.
enhancer, zalpha11 Ligand cDNA, and the mutated hGH gene was
prepared to be used for microinjection into fertilized murine
oocytes.
[0404] B. Construct for Expressing Mouse zalpha11 Ligand from the
Liver-Specific MT-1 Promoter
[0405] This same mouse zalpha11 Ligand insert from Example 26A, was
subcloned into the pTG12-8 vector, as described in Example 22A. For
this construct, about 10 mg of the pKFO51-zalpha11 Ligand maxiprep
DNA was digested with FseI and AscI combined, ethanol precipitated,
and the mouse zalpha11 Ligand fragment was purified as described in
Example 22. This fragment was then ligated into pTG12-8 which had
been previously digested with FseI and AscI, as described in
Example 22A. Electroporation, screening of clones and a maxiprep
was performed as described in Example 22. A SalI fragment
containing 5' and 3' flanking sequences, the MT-1 promoter, the rat
insulin II intron, mouse zalpha11 Ligand cDNA and the hGH poly A
sequence was prepared to be used for microinjection into fertilized
murine oocytes.
Example 27
[0406] Mouse zalpha11-Ligand Polyclonal Antibodies
[0407] Polyclonal antibodies were prepared by immunizing 2 female
New Zealand white rabbits with the purified recombinant protein
muzalpha11L/MBP-6H (Example 32). The rabbits were each given an
initial intraperitoneal (ip) injection of 200 mg of purified
protein in Complete Freund's Adjuvant followed by booster ip
injections of 100 mg peptide in Incomplete Freund's Adjuvant every
three weeks. Seven to ten days after the administration of the
second booster injection (3 total injections), the animals were
bled and the serum was collected. The animals were then boosted and
bled every three weeks.
[0408] The muzalpha11L/MBP-6H specific rabbit serum was
pre-adsorbed of anti-MBP antibodies using a CNBr-SEPHAROSE 4B
protein column (Pharmacia LKB) that was prepared using 10 mg of
purified recombinant maltose binding protein (MBP) per gram of
CNBr-SEPHAROSE. Recombinant MBP was made and purified on an amylose
column in house, using methods well known in the art. The
muzalpha11-ligand-specific polyclonal antibodies were affinity
purified from the rabbit serum using a CNBr-SEPHAROSE 4B protein
column (Pharmacia LKB) that was prepared using 10 mg of the
specific antigen purified recombinant protein muzalpha11L/MBP-6H
(Example 32) followed by 20.times. dialysis in PBS overnight.
Muzalpha11-ligand-specific antibodies were characterized by ELISA
using 1 ug/ml of the purified recombinant proteins
muzalpha11L/MBP-6H (Example 32) or huzalpha11L-MBP/6H (Example 32)
as antibody targets. The lower limit of detection (LLD) of the
rabbit anti-muzalpha11L/MBP-6H affinity purified antibody is 100
pg/ml on its specific purified recombinant antigen
muzalpha11L/MBP-6H and 500 pg/ml on purified recombinant
huzalpha11L-MBP/6H.
Example 28
[0409] Construction of Mammalian Expression Vector and Large-Scale
Human zalpha11 Ligand Expression in CHO DG44 Cells
[0410] A mammalian expression vector for human zalpha11 Ligand (SEQ
ID NO:1) designed to add a SalI site at the 5' end and a PmeI site
to the 3' end of the cDNA, was constructed via amplification by PCR
from a plasmid containing human zalpha11 Ligand (Example 7) with
oligonucleotide primers, ZC22,054 (SEQ ID NO:70) and ZC22,055 (SEQ
ID NO:71). The PCR reaction conditions were as follows: 94.degree.
C. for 4 min.; 25 cycles of 94.degree. C. for 45 sec., 50.degree.
C. for 45 seconds, and 72.degree. C. for 3 min.; and 72.degree. C.
for 10 minutes. The PCR product was isolated as described herein,
and cut with SalI and PmeI then ligated to plasmid pDC312
previously cut at the appropriate restriction sites in the
polylinker, using standard methods described herein. The plasmid
pDC312 is described in Morris, A. et al., "Expression Augmenting
Sequence Element (EASE) isolated from Chinese Hamster Ovary Cells,"
in Animal Cell Technology, Carrondo, M J T et al (eds.) (1997)
Kluwer Academic Publishers, The Netherlands, p. 529-534.
[0411] The ligated vector was transfected into suspension-adapted
CHO DG44 (in house Novo Nordisk, Denmark) cells by lipofection
using LipofectaminePlus.TM. reagent (Gibco/BRL) according to
manufacturer's instructions. Transfectants were selected on PFCHO
medium (JRH, Lenexa, Kans.) free of thymidine, and hypoxanthine,
followed by selection on 200 nM methotrexate (Sigma, St. Louis,
Mo.). The methotrexate resistant cells were cloned by dilution and
assayed for production of zalpha11 Ligand by a BaF3 activity assay
(Example 5B).
[0412] A productive clone was scaled up and grown in a 7 to 20
liter bioreactor (Applikon Bioreactors, Schiedam, The Netherlands)
in PFCHO medium to produce material for purification (Example
29).
Example 29
[0413] Large-Scale Purification of Untagged Human and Murine
zalpha11 Ligand from BHK and CHO Mammalian Expression Cell
Lines.
[0414] A. CHO Expressed Human zalpha11 Ligand
[0415] Unless otherwise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying human
zalpha11 Ligand from at least 30 liters of CHO conditioned media
(see, Example 28). Concentrated or non-concentrated conditioned
media (CM) was sterile filtered through 0.45 and 0.22 micron
filters. The conditioned media was then buffered with 0.01 M MES
(Fluka BioChemika, Switzerland)) and the pH adjusted to 6.0, and
then loaded onto a 50.times.100 mm (196 ml) Poros 50 HS column
(strong cation exchanger from PerSeptive BioSystems, Framingham,
Mass.) overnight at 4-10 ml/min. using a BioCAD SPRINT (Perceptive
BioSystems). The column was washed for 10-20 column volumes (CV)
with 0.01 M MES/0.130 M NaCl (Mallinckrodt, Paris, Ky.) pH 6.0. The
bound proteins were then eluted with a 0.130 M to 1 M NaCl 10 CV
gradient in 0.01 M MES pH 6.0 at 30 ml/min.; 25 ml fractions were
collected over the entire chromatography and absorbence at 280 and
215 nM were monitored. Peak absorbence fractions were analyzed by
SDS-PAGE Silver (Geno Technology, St. Louis, Mo.), Coomassie
(Sigma, St. Louis, Mo.) staining and Western immunological blotting
using antibodies against the human zalpha11 Ligand (Example 33 and
Example 34).
[0416] Peak fractions were pooled then concentrated in a stirred
cell concentrator on a YM10 membrane (Millipore/Amicon, Bedford,
Mass.) to a nominal volume (1-10 ml). The sample was then loaded on
an appropriate Sephacryl S-200 (Pharmacia, Uppsala, Sweden) high
resolution size exclusion column (52-600 ml) equilibrated in PBS
(Gibco BRL) at flow rates 1-2 ml/ml; 1-2 ml fractions were
collected over the entire chromatography and absorbence at 280 and
215 nM were monitored. Peak fractions were analyzed by SDS-PAGE
Silver (Geno Technology, St. Louis, Mo.), and Coomassie (Sigma, St.
Louis, Mo.) staining.
[0417] The fractions of interest were pooled and concentrated with
Millipore 5 kD MWKO centrifugal spin concentrators to a minimal
volume. The final product was then analyzed by SDS PAGE Coomassie
staining (Sigma, St. Louis, Mo.), Western Immunological blotting,
N-terminal sequencing, Amino Acid Analysis, and BCA (Pierce,
Rockford, Ill.) for protein purity and concentration.
[0418] B. BHK 570 Expressed Murine zalpha11 Ligand
[0419] Unless otherwise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying murine
zalpha11 Ligand from BHK conditioned media (Example 18).
Concentrated or non-concentrated conditioned media (CM) was sterile
filtered through 0.45 and 0.22 micron filters. The media was then
buffered with 0.01 M MES (Fluka BioChemika, Switzerland)) and the
pH adjusted to 6.0. The CM was analyzed, loaded and eluted on an AS
column as described in Example 29A.
[0420] Fractions of interest were pooled then concentrated in a
stirred cell concentrator as in Example 29A, to a volume of 20-30
ml. The pH was adjusted to 7.0 then the sample was loaded onto
either a 0.8 ml Poros AL column that had about 3 mg of
zalpha11CFLAG tagged soluble receptor (Example 10B) or one with
about 10 mg of zalpha11-Fc4 fusion receptor (Example 10C)
immobilized on the resin (see method below) at 1 ml/min on a BioCAD
SPRINT. The column was then washed with at least 20 CV of 0.3 M
NaCl/PBS (Gibco BRL)/0.01 M MES at 10 ml/min. The column was then
rapid eluted with a 600 .mu.l injection of 0.1 M glycine
(Aminoacetic Acid; Glycocol, Spectrum, Gardena, Calif.) pH 2.5 at a
flow rate of 10 ml/min with PBS on a BioCAD SPRINT. The 1 ml
fractions were collected for 6 seconds each and immediately pH
neutralized with 55 .mu.l of 2 M TRIS pH 8.8 (Tris (Hydroxymethyl)
Aminomethane, EM Science, Gibbstown, N.J.). The absorbence at 280
and 215 nM were monitored over the entire chromatography. The peak
fractions were analyzed SDS-PAGE Silver (Geno Technology, St.
Louis, Mo.), Coomasise (Sigma, St. Louis, Mo.) staining and Western
Immunological blotting as above.
[0421] Peak fractions were pooled then concentrated in a stirred
cell concentrator as in Example 29A to a minimal volume (1-10 ml).
The sample was then loaded, equilibrated and analyzed as in Example
29A, on an appropriate Sephacryl S-200 (Pharmacia) high resolution
size exclusion column. Peak fractions were analyzed by SDS-PAGE
Silver (Geno Technology, St. Louis, Mo.), and Coomasise (Sigma, St.
Louis, Mo.) staining. The fractions of interest were pooled and
concentrated and analyzed as in Example 29A.
[0422] C. Protein Immobilization on POROS AL Media
[0423] All operations were carried out at room temperature on a
BioCAD 700E. Flow packed a 4.5.times.50 mm column with the POROS AL
media in 2 M NaCl according to manufactures specifications. The
column was then equilibrated in 1.1 M Na.sub.2SO.sub.4 and 50 mM
NaPhosphate pH 7.2. The receptor was concentrated to 4 mg/ml using
a Millipore 30 kD MWKO centrifugal spin concentrator then diluted
1:1 in 1.1 M Na.sub.2SO.sub.4 and 50 mM NaPhosphate pH 7.2. The
column was flowed at 2 ml/min in 1.1 M Na.sub.2SO.sub.4 and 50 mM
NaPhosphate pH 7.2 and 100 .mu.l injections of the diluted ligand
were made ever 9 CVs until a steady state of saturation or break
through was reached. A 62 CV gradient was then ran from 1.1 M
Na.sub.2SO.sub.4 and 50 mM NaPhosphate pH 7.2 to 550 mM
Na.sub.2SO.sub.4 and 50 mM NaPhosphate pH 7.2 with 5 mg/ml Sodium
Cyanoborohydride. Column was held for 2 hr. to complete the
immobilization chemistry. The column was then equilibrated in 0.2 M
TRIS pH 7.2 with 5 mg/ml Sodium Cyanoborohydride and allowed to
rest for 1 hr. Finally the column was equilibrated in PBS with
0.02% Sodium Azide, then stored at 4.degree. C. until needed. Prior
to use, the column was pre-eluted with 0.1 M glycine to ensure that
non-specific proteins were removed and the column is not leaching
the immobilized receptor.
Example 30
[0424] Expression Vector Construction Expression and Purification
of Untagged Human and Murine Zalpha11 Ligand from Baculovirus.
[0425] A. Construct for Expressing Human zalpha11 Ligand in
Baculovirus
[0426] An expression vector, pzalpha11L, was prepared to express
Human zalpha11 Ligand polypeptides in insect cells. A 517 bp
fragment containing sequence for Human zalpha11 Ligand and encoded
BamH1 and XhoI restriction sites on the 5' and 3' ends
respectively, was generated by PCR amplification from a plasmid
containing human zalpha11 Ligand cDNA (Example 7) using primers
ZC23,444 (SEQ ID NO:74) and ZC23,445 (SEQ ID NO:75). The PCR
reaction conditions were as follows: 1 cycle of 94.degree. C. for 4
minutes, followed by 25 cycles of 94.degree. C. for 45 seconds,
50.degree. C. for 45 seconds, and 72.degree. C. for 2 minutes; 1
cycle at 72.degree. C. for 10 min; followed by a 4.degree. C. soak.
The fragment was visualized by gel electrophoresis (1% SeaPlaque/1%
NuSieve). The band was excised, diluted to 0.5% agarose with 2 mM
MgCl.sub.2, melted at 65.degree. C., digested with BamH1 and XhoI
(Boerhinger Mannheim), and ligated into an BamH1/XhoI digested
baculovirus expression vector, pZBV3L. The pZBV3L vector is a
modification of the pFastBac1.TM. (Life Technologies) expression
vector, where the polyhedron promoter has been removed and replaced
with the late activating Basic Protein Promoter. About 14 nanograms
of the restriction digested zalpha11 Ligand insert and about 40 ng
of the corresponding vector were ligated overnight at 16.degree.
C.
[0427] The ligation mix was diluted 3 fold in TE (10 mM Tris-HCl,
pH 7.5 and 1 mM EDTA) and about 4 fmol of the diluted ligation mix
was transformed into DH5* Library Efficiency competent cells (Life
Technologies) according to manufacturer's direction by heat shock
for 45 seconds in a 42.degree. C. waterbath. The transformed DNA
and cells were diluted in 450 .mu.l of SOC media (2% Bacto.TM.
Tryptone, 0.5% Bacto.TM. Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl,
10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) and plated
onto LB plates containing 100 .mu.g/ml ampicillin. Clones were
analyzed by restriction digests and 1 .mu.l of the positive clone
was transformed into 20 .mu.l DH10Bac Max Efficiency competent
cells (GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's
instruction, by heat shock as described above. The transformed
cells were then diluted in 980 .mu.l SOC media (2% Bacto.TM.
Tryptone, 0.5% Bacto.TM. Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl,
10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) out grown in
shaking incubator at 37.degree. C. for four hours and plated onto
Luria Agar plates containing 50 .mu.g/ml kanamycin, 7 .mu.g/ml
gentamicin (Life Technologies), 10 .mu.g/ml tetracycline, IPTG
(Pharmacia Biotech) and Bluo-Gal (Life Technologies). The plated
cells were incubated for 48 hours at 37.degree. C. A color
selection was used to identify those cells having Human zalpha11
Ligand encoding donor insert that had incorporated into the plasmid
(referred to as a "bacmid"). Those colonies, which were white in
color, were picked for analysis. Human zalpha11 Ligand Bacmid DNA
was isolated from positive colonies using the QiaVac Miniprep8
system (Qiagen) according the manufacturer's directions. Clones
were screened for the correct insert by amplifying DNA using
primers to the transposable element in the bacmid via PCR using
primers ZC447 (SEQ ID NO:76) and ZC976 (SEQ ID NO:77). The PCR
reaction conditions were as follows: 35 cycles of 94.degree. C. for
45 seconds, 50.degree. C. for 45 seconds, and 72.degree. C. for 5
minutes; 1 cycle at 72.degree. C. for 10 min.; followed by
4.degree. C. soak. The PCR product was run on a 1% agarose gel to
check the insert size. Those clones having the correct insert were
used to transfect Spodoptera frugiperda (Sf9) cells.
[0428] B. Expression and Generation of Material for Purification of
Human zalpha11 Ligand from Baculovirus
[0429] Sf9 cells were seeded at 5.times.10.sup.6 cells per 35 mm
plate and allowed to attach for 1 hour at 27.degree. C. Five
microliters of human zalpha11 Ligand bacmid DNA (above) was diluted
with 100 .mu.l Sf-900 II SFM (Life Technologies). Six .mu.l of
CellFECTIN Reagent (Life Technologies) was diluted with 100 .mu.l
Sf-900 II SFM. The bacmid DNA and lipid solutions were gently mixed
and incubated 30-45 minutes at room temperature. The media from one
plate of cells were aspirated, the cells were washed 1.times. with
2 ml fresh Sf-900 II SFM media. Eight hundred microliters of Sf-900
II SFM was added to the lipid-DNA mixture. The wash media was
aspirated and the DNA-lipid mix added to the cells. The cells were
incubated at 27.degree. C. for 4-5 hours. The DNA-lipid mix was
aspirated and 2 ml of Sf-900 II media was added to each plate. The
plates were incubated at 27.degree. C., 90% humidity, for 96 hours
after which the virus was harvested.
[0430] For Primary Amplification Sf9 cells were grown in 50 ml
Sf-900 II SFM in a 125 ml shake flask to an approximate density of
0.41-0.52.times.105 cells/ml. They were then infected with 150
.mu.l of the virus stock from above and incubated at 27.degree. C.
for 3 days after which time the virus was harvested according to
standard methods known in the art. A 500 .mu.l sample submitted for
activity in a BaF3 assay (Example 5) to show that it was
biologically active.
[0431] For Secondary Amplification Sf9 cells were grown in 1 L of
Sf-900 II SFM in a 2800 ml shake flask to an approximate density of
0.5.times.10.sup.5 cells/ml. It was infected with 500 .mu.l of the
Primary viral stock from above and incubated at 27.degree. C. for 4
days after which time the virus was harvested according to standard
methods known in the art. Virus was titered and grown up large
scale for purification of the baculovirus-produced human zalpha11
Ligand (huzalpha11L-Bv), as described in Example 30C and Example
30D, below.
[0432] C. Large-Scaled Purification of Baculovirus Expressed
Human/Murine zalpha11 Ligand
[0433] Unless otherwise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying human
zalpha11 Ligand (huzalpha11L-Bv) from BV conditioned media (Example
30B). Conditioned media (CM) was sterile filtered through 0.45 and
0.22 micron filters, then buffered with 0.01 M MES (Fluka
BioChemika, Switzerland)) and the pH adjusted to 6.0 The CM was
then loaded onto a POROS 50 HS column and run, fractions collected,
analyzed, as described in Example 29A.
[0434] The above peak fractions were pooled, concentrated run on a
high resolution size exclusion column, and analyzed as described in
Example 29A.
[0435] The fractions of interest from the size exclusion column
were pooled and concentrated with 5 kD MWCO Millipore centrifugal
spin concentrators to a minimal volume. The final product was then
analyzed by SDS-PAGE Coomassie (Sigma, St. Louis, Mo.), Western
immunological blotting, N-terminal sequencing, Amino Acid Analysis,
and CB (Pierce, Rockford, Ill.) for protein purity and
concentration as described in Example 29A. Bulk protein was stored
at -80.degree. C.
[0436] D. Small Scale (<2 mg) Purification of
Baculovirus-Expressed Human/Murine zalpha11 Ligand
[0437] Unless other wise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying <2
mg of human or murine zalpha11 Ligand from BV conditioned media.
The CM was filtered, buffered and pH adjusted as in Example 30C.
The CM was then loaded, eluted and the POROS 50 HS chromatography
was analyzed as in Example 30C.
[0438] Fractions were pooled then concentrated via diafiltration in
a stirred cell concentrator on a YM10 membrane (10 kD MWCO)
(Millipore/Amicon, Bedford, Mass.) to a nominal volume (20-30 ml).
The pH was adjusted to 7.0 then the sample was loaded onto either a
0.8 ml Poros AL column that had about 3 mg of zalpha11CFLAG soluble
receptor (Example 10B) or one with about 10 mg of zalpha11-Fc4
fusion soluble receptor (Example 10C) immobilized on the resin (see
method in example 29C) at 1 ml/min on a BioCad SPRINT. The column
was then washed with at least 20 CV of 0.3 M NaCl/PBS (Gibco
BRL)/0.01 M MES at 10 ml/min. The column was then rapid eluted with
a 600 .mu.l injection of 0.1 M glycine (Aminoacetic Acid; Glycocol,
Spectrum, Gardena, Calif.) pH 2.5 at a flow rate of 10 ml/min with
PBS on a BioCAD SPRINT. The 1 ml fractions were collected for 6
seconds each and immediately pH neutralized with 55 .mu.l of 2 M
TRIS (Tris (Hydroxymethyl) Aminomethane, EM Science, Gibbstown,
N.J.) pH 8.8. The absorbence at 280 and 215 nM were monitored over
the entire chromatography. Fractions were analyzed as above.
[0439] Peak fractions were pooled then concentrated via
diafiltration in a stirred cell concentrator on a YM10 membrane (10
kD MWCO) (Millipore/Amicon, Bedford, Mass.) to 1-2 ml. The sample
was then loaded on an appropriate Sephacryl S-200 (Pharmacia,
Uppsala, Sweden) high resolution size exclusion column equilibrated
in PBS (Gibco BRL) at an optimal flow rate; fractions were
collected over the entire chromatography and absorbence at 280 and
215 nM were monitored. Fractions were analyzed as above.
[0440] The fractions of interest were pooled and concentrated with
5 Kd MWCO Millipore centrifugal spin concentrators to a nominal
volume. The final product was then analyzed by SDS-PAGE Coomassie
(Sigma, St. Louis, Mo.), Western immunological blotting, N-terminal
sequencing, Amino Acid Analysis, and BCA (Pierce, Rockford, Ill.)
for protein purity and concentration. Bulk protein stored as
described above.
[0441] E. Construct for Expressing mouse zalpha11 Ligand in
Baculovirus: pzalpha11lig.M
[0442] An expression vector, pzalpha11LM, was prepared to express
Murine zalpha11 Ligand polypeptides in insect cells. A 413 bp
fragment containing sequence for Murine zalpha11 Ligand and encoded
BspE1 and Xba1 restriction sites on the 5' and 3' ends,
respectively, was generated by PCR amplification from a plasmid
containing murine zalpha11 Ligand cDNA (Example 16) using primers
ZC25,970 (SEQ ID NO: 109) and ZC25,969 (SEQ ID NO:110) utilizing
the Expand High Fidelity PCR System (Boerhinger Mannheim) as per
manufacturer's instructions. The PCR conditions were as follows: 1
cycle of 94.degree. C. for 2 minutes, followed by 35 cycles of
94.degree. C. for 15 seconds, 50.degree. C. for 30 seconds, and
72.degree. C. for 2 minutes; 1 cycle at 72.degree. C. for 10 min;
followed by a 4.degree. C. soak. A small portion of the PCR product
was visualized by gel electrophoresis (1% NuSieve agarose). The
remainder of the fragment was purified using the Qiagen PCR
purification kit as per manufacturer's instructions and eluted into
30 .mu.l of H.sub.2O. The fragment was then digested with Bspe1 and
XbaI (Boerhinger Mannheim) restriction enzymes at 37C for about 2
h, then run on an agarose gel as described above. The band was
excised, purified and eluted using the Qiagen gel extraction kit as
per manufacturers instructions. The purified fragment was ligated
into an Bspe1/XbaI digested baculovirus expression vector, pZBV37L.
The pZBV37L vector is a modification of the pFastBac1* (Life
Technologies) expression vector, where the polyhedron promoter has
been removed and replaced with the late activating Basic Protein
Promoter followed by the secretory signal sequence from Ecdysteroid
UDP-Glucosyltransferase (EGT). About 5 .mu.l of the restriction
digested Murine zalpha11 Ligand insert and about 100 ng of the
corresponding cut vector were ligated overnight at 16.degree. C. in
about 20 .mu.l. Five .mu.l of the ligation mix was electroporated
into 50 .mu.l Life Technologies DH12S electrocompetent bacterial
cells utilizing a 2 mm cuvette with 2 kV, 25 .mu.F and 400 ohms
settings. The electroporated cells were rescued in 1 ml of SOC
media (2% Bacto.TM. Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M
NaCl, 1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM
glucose (Gibco BRL)) outgrown at 37.degree. C. for about 1 hr and
plated onto LB plates containing 100 .mu.g/ml ampicillin. DNA from
clones was isolated and analyzed by restriction digests to identify
positive clones. About 5 ng DNA from a positive clone was
transformed into 20 .mu.l DH10Bac Max Efficiency competent cells
(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's
instruction, by heat shock for 45 seconds in a 42.degree. C. water
bath. The transformed cells were then diluted and grown as
described in example 30A. Bacmids containing the murine zalpha11
Ligand insert were identified and isolated as described in Example
30A. Clones were screened for the correct insert by amplifying DNA
using primers to the transposable element in the bacmid via PCR
using primers ZC447 (SEQ ID NO:76) and ZC976 (SEQ ID NO:77). The
PCR reaction conditions were as follows: 35 cycles of 94.degree. C.
for 45 seconds, 50.degree. C. for 45 seconds, and 72.degree. C. for
5 minutes; 1 cycle at 72.degree. C. for 10 min.; followed by
4.degree. C. soak. The PCR product was run on a 1% agarose gel to
check the insert size. Those having the correct insert were used to
transfect Spodoptera frugiperda (Sf9) cells.
[0443] F. Expression and Generation of Material for Purification of
Mouse zalpha11 from Baculovirus
[0444] Sf9 cells were seeded at 1 million cells per 35 mm plate and
allowed to attach for 1 hour at 27.degree. C. The murine zalpha11
Ligand bacmid DNA was transfected as described in Example 30B and
the virus was harvested.
[0445] For primary amplification, Sf9 cells were seeded as above
and 500 .mu.l of 72 hr post transfection supernatant was added and
cultures were allowed to proceed for 96 hr. after which time the
virus was harvested according to standard methods.
[0446] For Secondary amplification, Sf9 cells were seeded as above
and 200 .mu.l of the Primary viral stock was added. Cultures were
incubated at 27.degree. C. for 72 hr., after which time the virus
was harvested according to standard methods.
[0447] For Tertiary amplification, 10 .mu.l of Secondary Amplified
virus stock was placed on SF9s at 500,000 cells per well in 50 ml
of SF90011 media in a 250 ml vol. shake flask for 6 days and virus
was harvested as above. Virus was titered and grown up large scale
for purification of the baculovirus-produced murine zalpha11 Ligand
(muzalpha11L-Bv), as described in Example 30C and Example 30D.
[0448] Presence of predicted molecular weight protein in the
supernatant was determined by western analysis using an
anti-muzalpha11L/MBP-6H polyclonal antibody (Example 27). BaF3
based proliferation assay analysis (Example 5) also showed that the
secreted ligand was active.
Example 31
[0449] Expression of Human and Mouse zalpha11 Ligand in E. coli
[0450] A. Construction of Human zalpha11 Ligand-MBP Fusion
Expression Vector PTAP98/zalpha11 Ligand
[0451] An expression plasmid containing a polynucleotide encoding
part of the human zalpha11 Ligand fused N-terminally to maltose
binding protein (MBP) was constructed via homologous recombination.
A fragment of human zalpha11 Ligand cDNA (SEQ ID NO: 1) was
isolated using PCR. Two primers were used in the production of the
human zalpha11 Ligand fragment in a PCR reaction: (1) Primer
ZC22,128 (SEQ ID NO:78), containing 40 bp of the vector flanking
sequence and 26 bp corresponding to the amino terminus of the human
zalpha11 Ligand, and (2) primer ZC22,127 (SEQ ID NO:79), containing
40 bp of the 3' end corresponding to the flanking vector sequence
and 28 bp corresponding to the carboxyl terminus of the human
zalpha11 Ligand. The PCR reaction conditions were as follows: 25
cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds, and 72.degree. C. for 1 minute; followed by 4.degree. C.
soak, run in duplicate. Two .mu.l of the 100 .mu.l PCR reaction
were run on a 1.0% agarose gel with 1.times.TBE buffer for
analysis, showing the expected band of approximately 472 bp. The
remaining 90 .mu.l of PCR reaction was combined with the second PCR
tube precipitated with 400 .mu.l of absolute ethanol to be used for
recombining into the Sma1 cut recipient vector pTAP98 to produce
the construct encoding the MBP-zalpha11 Ligand fusion, as described
below.
[0452] Plasmid pTAP98 was derived from the plasmids pRS316 and
pMAL-c2. The plasmid pRS316 is a Saccharomyces cerevisiae shuttle
vector (Hieter P. and Sikorski, R., Genetics 122:19-27, 1989).
pMAL-C2 (NEB) is an E. coli expression plasmid. It carries the tac
promoter driving MalE (gene encoding MBP) followed by a His tag, a
thrombin cleavage site, a cloning site, and the rrnB terminator.
The vector pTAP98 was constructed using yeast homologous
recombination. 100 ng of EcoR1 cut pMAL-c2 was recombined with 1
.mu.g Pvul cut pRS316, 1 .mu.g linker, and 1 .mu.g Sca1/EcoR1 cut
pRS316. The linker consisted of oligos ZC19,372 (SEQ ID NO:80) (100
pmol): ZC19,351 (SEQ ID NO:81) (1 pmol): ZC19,352 (SEQ ID NO:82) (1
pmol), and ZC19,371 (SEQ ID NO:83) (100 pmol) combined in a PCR
reaction. Conditions were as follows: 10 cycles of 94.degree. C.
for 30 seconds, 50.degree. C. for 30 seconds, and 72.degree. C. for
30 seconds; followed by 4.degree. C. soak. PCR products were
concentrated via 100% ethanol precipitation.
[0453] One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 10 .mu.l of a mixture containing
approximately 1 .mu.g of the human zalpha11 Ligand PCR product, and
100 ng of SmaI digested pTAP98 vector, and transferred to a 0.2 cm
electroporation cuvette. The yeast/DNA mixture was electropulsed at
0.75 kV (5 kV/cm), infinite ohms, 25 .mu.F. To each cuvette was
added 600 .mu.l of 1.2 M sorbitol. The yeast was then plated in two
300 .mu.l aliquots onto two-URA D plates and incubated at
30.degree. C.
[0454] After about 48 hours, the Ura+ yeast transformants from a
single plate were resuspended in 1 ml H.sub.2O and spun briefly to
pellet the yeast cells. The cell pellet was resuspended in 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 was
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, followed by a 5 minute spin in a
Eppendorf centrifuge at maximum speed. Three hundred microliters of
the aqueous phase was transferred to a fresh tube, and the DNA
precipitated with 600 .mu.l ethanol (EtOH), followed by
centrifugation for 10 minutes at 4.degree. C. The DNA pellet was
resuspended in 100 .mu.l H.sub.2O.
[0455] Transformation of electrocompetent E. coli cells (MC1061,
Casadaban et. al. J. Mol. Biol. 138, 179-207) was done with 1 .mu.l
yeast DNA prep and 40 .mu.l of MCI 061 cells. The cells were
electropulsed at 2.0 kV, 25 .mu.F and 400 ohms. Following
electroporation, 0.6 ml SOC (2% Bacto.TM. Tryptone (Difco, Detroit,
Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was plated in one
aliquot on LB AMP plates (LB broth (Lennox), 1.8% Bacto.TM. Agar
(Difco), 100 mg/L Ampicillin).
[0456] Individual clones harboring the correct expression construct
for human zalpha11 Ligand were identified by expression. Cells were
grown in Superbroth II (Becton Dickinson) with 100 .mu.g/ml of
ampicillin overnight. 50 .mu.l of the overnight culture was used to
inoculate 2 ml of fresh Superbroth II +100 .mu.g/ml ampicillin.
Cultures were grown at 37.degree. C., shaking for 2 hours. 1 ml of
the culture was induced with 1 mM IPTG. 2-4 hours later the 250
.mu.l of each culture was mixed with 250 .mu.l acid washed glass
beads and 250 .mu.l Thorner buffer with 5% .beta.ME and dye (8M
urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples
were vortexed for one minute and heated to 65.degree. C. for 5-10
minutes. 20 .mu.l were loaded per lane on a 4%-12% PAGE gel
(NOVEX). Gels were run in 1.times.MES buffer. The positive clones
were designated pTAP126 and subjected to sequence analysis. The
polynucleotide sequence of MBP- human zalpha11 Ligand fusion within
pTAP126 is shown in SEQ ID NO:84, and the corresponding polypeptide
in SEQ ID NO:85.
[0457] B. Bacterial Expression of Human zalpha11 Ligand.
[0458] One microliter of sequencing DNA was used to transform
strain W3110 (ATCC). The cells were electropulsed at 2.0 kV,
25.degree. F. and 400 ohms. Following electroporation, 0.6 ml SOC
(2% Bacto.TM. Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract
(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM
MgSO.sub.4, 20 mM glucose) was plated in one aliquot on LB AMP
plates (LB broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L
Ampicillin).
[0459] Individual were expressed. Cells were grown in Superbroth II
(Becton Dickinson) with 100 .mu.g/ml of ampicillin overnight. 50
.mu.l of the overnight culture was used to inoculate 2 ml of fresh
Superbroth II +100 .mu.g/ml ampicillin. Cultures were grown at
37.degree. C., shaking for 2 hours. 1 ml of the culture was induced
with 1 mM IPTG. 2-4 hours later the 250 .mu.l of each culture was
mixed with 250 .mu.l acid washed glass beads and 250 .mu.l Thorner
buffer with 5% DME and dye (8M urea, 100 mM Tris pH7.0, 10%
glycerol, 2 mM EDTA, 5% SDS). Samples were vortexed for one minute
and heated to 65.degree. C. for 10 minutes. 20 .mu.l were loaded
per lane on a 4%-12% PAGE gel (NOVEX). Gels were run in 1.times.MES
buffer. The positive clones were used to grow up for protein
purification of the huzalpha11L/MBP-6H fusion protein (Example 32,
below).
[0460] C. Construction of Mouse zalpha11 Ligand-MBP Fusion
Expression Vector pTAP98/Mouse zalpha11 Ligand
[0461] An expression plasmid containing a polynucleotide encoding
part of the mouse zalpha11 Ligand fused N-terminally to maltose
binding protein (MBP) was constructed via homologous recombination,
as described in Example 31A. A fragment of mouse zalpha11 Ligand
cDNA (SEQ ID NO:55) was isolated using PCR. Two primers were used
in the production of mouse zalpha11 Ligand fragment in a PCR
reaction: (1) Primer ZC22,849 (SEQ ID NO:86), containing 40 bp of
the vector flanking sequence and 24 bp corresponding to the amino
terminus of the mouse zalpha11 Ligand, and (2) primer ZC22,850 (SEQ
ID NO:87), containing 40 bp of the 3' end corresponding to the
flanking vector sequence and 21 bp corresponding to the carboxyl
terminus of the mouse zalpha11 Ligand. The PCR reaction conditions
were as per above. The approximately 450 bp fragment was cloned
into pTAP98 as described above. Clones were transformed, identified
and grown up as described above. The positive clones were
designated pTAP134 and subjected to sequence analysis. The
polynucleotide sequence of MBP-mouse zalpha11 Ligand fusion within
pTAP134 is shown in SEQ ID NO:88, and the corresponding polypeptide
sequence shown in SEQ ID NO:89. The positive clones were used to
grow up in E. coli as described above for protein purification of
the muzalpha11L/MBP-6H fusion protein (Example 32).
Example 32
[0462] Purification of zalpha11-MBP Ligand or zalpha11-MBP
Receptor
[0463] Unless otherwise stated, all operations were carried out at
4.degree. C. The following procedure was used for purifying
zalpha11-MBP Ligand fusions for human zalpha11-MBP Ligand
(huzalpha11L/MBP-6H) or murine zalpha11-MBP Ligand
(muzalpha11L/MBP-6H) from E. coli. Human or mouse zalpha11-MBP
receptor fusions were carried out using the same method. Pre-spun
frozen E. coli paste was thawed and diluted into 2 liters of Buffer
B (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M
2-mercapto-ethanol (EM Science); pH 8.0; with 5 mg/l Pepstatin A
(Boerhinger Mannheim); 5 mg/l Aprotinin (Boerhinger Mannheim); and
1 mg/l PMSF (Fluka)) plus 1-2 ml of an anti-foaming reagent AF289
antifoam (Sigma). The mixture was processed in a pre-chilled French
Press cell disrupter (Constant Systems LTD) with 20-30 kPSI.
[0464] The lysate was then centrifuged at 18,000.times.g for 45
minutes at 4.degree. C.; retained the supernatant. A 200 ml slurry
of Amylose resin (New England BioLabs), pre-equilibrated in Buffer
A (0.02 M TRIS (EM Science); 0.2 M NaCl (Mallincrodt); 0.01 M
2-mercapto-ethanol (EM Science); pH 8.0), was added to the lysate
supernatant and incubated overnight in 21 roller bottles to allow
for maximum batch absorption of the MBP fusion protein. The resin
was washed in batch column format for >5 column volumes with
Buffer A, then batch eluted with Buffer C (Buffer A with 0.02 M
Maltose (Sigma)). Crude fractions were collected and monitored by
absorbence 280 nm.
[0465] The eluted protein was analyzed by SDS NuPAGE (NOVEX)
Coomassie (Sigma) staining. Sample and bulk protein were stored at
-80.degree. C.
Example 33
[0466] Human zalpha11 Ligand Polyclonal Antibodies
[0467] Polyclonal antibodies to Human zalpha11 Ligand were prepared
by immunizing 2 female New Zealand white rabbits with the purified
recombinant protein huzalpha11L/MBP-6H (Example 32) or the purified
CHO recombinant protein huzalpha11L-CHO (Example 29). The rabbits
were each given an initial intraperitoneal (ip) injection of 200 mg
of purified protein in Complete Freund's Adjuvant followed by
booster ip injections of 100 mg purified protein in Incomplete
Freund's Adjuvant every three weeks. Seven to ten days after the
administration of the second booster injection (3 total
injections), the animals were bled and the serum was collected. The
animals were then boosted and bled every three weeks.
[0468] The rabbit serum raised to huzalpha11L/MBP-6H was
pre-adsorbed of anti-MBP antibodies using a CNBr-SEPHAROSE 4B
protein column (Pharmacia LKB) that was prepared using 10 mg of
purified recombinant MBP per gram of CNBr-SEPHAROSE (Pharmacia).
Recombinant MBP was made and purified on an amylose column in
house, using methods well known in the art. The
huzalpha11-ligand-specific polyclonal antibodies were affinity
purified from the rabbit serum using a CNBr-SEPHAROSE 4B protein
column (Pharmacia LKB) that was prepared using 10 mg of the
specific antigen purified recombinant protein huzalpha11L/MBP-6H or
10 mg of the purified CHO recombinant protein huzalpha11L-CHO per
gram of CNBr-SEPHAROSE, followed by 20.times. dialysis in PBS
overnight. Huzalpha11-ligand-specific antibodies were characterized
by ELISA using 1 .mu.g/ml of the purified recombinant proteins
huzalpha11L/MBP-6H (Example 32), human zalpha11 Ligand
(huzalpha11L-CHO) (Example 29), or muzalpha11L-MBP/6H (Example 32)
as antibody targets.
[0469] The lower limit of detection (LLD) of the rabbit
anti-huzalpha11L/MBP-6H affinity purified antibody was 10 ng/ml on
its specific purified recombinant antigen huzalpha11L/MBP-6H, 500
pg/ml on purified recombinant huzalpha11L-CHO, and 100 pg/ml on
purified recombinant muzalpha11L/MBP-6H (Example 32). The LLD of
the rabbit anti-huzalpha11L-CHO affinity purified antibody was 20
pg/ml on its specific purified recombinant antigen huzalpha11L-CHO,
500 pg/ml on purified recombinant huzalpha11L/MBP-6H, and 50 ng/ml
on purified recombinant muzalpha11L/MBP-6H.
Example 34
[0470] Human zalpha11 Ligand Anti-Peptide Antibodies
[0471] Polyclonal human zalpha11 Ligand anti-peptide antibodies
were prepared by immunizing 2 female New Zealand white rabbits with
the human zalpha11 Ligand peptide, huzalpha11L-1 (SEQ ID NO:72) or
huzalpha11L-3 (SEQ ID NO:73). The peptides were synthesized using
an Applied Biosystems Model 431A peptide synthesizer (Applied
Biosystems, Inc., Foster City, Calif.) according to manufacturer's
instructions. The peptides were then conjugated to the carrier
protein keyhole limpet hemocyanin (KLH) with maleimide-activation.
The rabbits were each given an initial intraperitoneal (ip)
injection of 200 mg of peptide in Complete Freund's Adjuvant
followed by booster ip injections of 100 mg peptide in Incomplete
Freund's Adjuvant every three weeks. Seven to ten days after the
administration of the second booster injection (3 total
injections), the animals were bled and the serum was collected. The
animals were then boosted and bled every three weeks.
[0472] The rabbit sera raised to the human zalpha11 Ligand peptides
were characterized by an ELISA titer check using 1 .mu.g/ml of the
respective peptide used to make the antibody (SEQ ID NO:72 or SEQ
ID NO:73) as an antibody target. The 2 rabbit seras to the
huzalpha11L-1 peptide had titer to their specific peptide at a
dilution of 1:5,000,000 (1:5E6). The 2 rabbit seras to the
huzalpha11L-3 peptide had titer to their specific peptide at a
dilution of 1:5E6.
[0473] Human zalpha11 Ligand peptide-specific polyclonal antibodies
were affinity purified from the rabbit serum using CNBR-SEPHAROSE
4B protein columns (Pharmacia LKB) that were prepared using 10 mg
of the respective specific peptide (SEQ. ID. NO:72 or SEQ. ID.
NO:73) per gram CNBr-SEPHAROSE, followed by 20.times. dialysis in
PBS overnight. Huzalpha11-ligand-specific antibodies were
characterized by an ELISA titer check using 1 .mu.g/ml of the
appropriate purified peptide antigen or purified recombinant
full-length proteins as antibody targets.
[0474] The lower limit of detection (LLD) of the rabbit
anti-huzalpha11L-1 affinity purified antibody is 500 pg/ml on its
specific peptide antigen (huzalpha11L-1; SEQ ID NO:72), 500 pg/ml
on purified recombinant huzalpha11L/MBP-6H (Example 32), and 500
pg/ml on purified CHO recombinant huzalpha11L-CHO (Example 29). No
cross-reactivity was seen to the purified recombinant
muzalpha11L/MBP-6H (Example 32). The LLD of the rabbit
anti-huzalpha11L-3 affinity purified antibody is 50 pg/ml on its
specific peptide antigen (huzalpha11L-1; SEQ ID NO:73), 50 pg/ml on
purified recombinant huzalpha11L/MBP-6H, 500 pg/ml on purified CHO
recombinant huzalpha11 L-CHO (Example 29), and 100 pg/ml on
purified Baculovirus recombinant huzalpha11L-Bv (Example 30).
Cross-reactivity was seen to the purified recombinant
muzalpha11L/MBP-6H (Example 32) with an LLD of 5 ng/ml.
Example 35
[0475] Human zalpha11 Receptor Monoclonal Antibodies
[0476] Zalpha11 receptor Monoclonal antibodies were prepared by
immunizing 5 male BalbC mice (Harlan Sprague Dawley, Indianapolis,
Ind.) with the purified recombinant soluble receptor protein,
zalpha11CEE (huzalpha11-CEE-BHK) (Example 10A). The mice were each
given an initial intraperitoneal (IP) injection of 20 mg of
purified protein in Complete Freund's Adjuvant (Pierce, Rockford,
Ill.) followed by booster IP injections of 10 mg purified protein
in Incomplete Freund's Adjuvant every two weeks. Seven to ten days
after the administration of the third booster injection, the
animals were bled and the serum was collected.
[0477] The mouse sera samples raised to the huzalpha11-CEE-BHK were
characterized by an ELISA titer check using purified recombinant
CHO huzalpha11-Fc protein (Example 10C) as an antibody target. One
mouse serum sample had titer to the specific antibody target at a
dilution of 1:1,000,000 (1:1E6). Four mouse serum samples had titer
to the specific antibody target at a dilution of 1:100,000
(1:1E5).
[0478] Splenocytes were harvested from the 4 high-titer mice and
fused to murine SP2/0 myeloma cells using PEG 1500 (Boerhinger
Mannheim, UK) in two separate fusion procedures using a 4:1 fusion
ratio of splenocytes to myeloma cells (Antibodies: A Laboratory
Manual, E. Harlow and D. Lane, Cold Spring Harbor Press). Following
10 days growth post-fusion, specific antibody-producing hybridomas
were identified by ELISA using purified recombinant BHK human
zalpha11-Fc4 protein (Example 10C) as an antibody target and by
FACS using Baf3 cells expressing the huzalpha11 sequence (Example
4, and Example 2) as an antibody target. The resulting 4 hybridomas
positive by both methods were cloned three times by limiting
dilution. The antibodies were designated: 249.28.2.1.2.2;
247.10.2.15.4.6; 249.19.2.2.3.5; and 249.15.2.4.2.7.
Example 36
[0479] Zalpha11 Ligand Transgenic Mice
[0480] A. Generation of Transgenic Mice Expressing Human and Mouse
zalpha11 Ligand
[0481] DNA fragments from transgenic vectors (Example 22 and
Example 26) containing 5' and 3' flanking sequences of the
respective promoter (MT-1 liver-specific promoter (mouse zalpha11
Ligand (Example 26B) or lymphoid specific LCK promoter (mouse and
human zalpha11 Ligand (Examples 26A and 22B), the rat insulin II
intron, zalpha11 Ligand cDNA and the human growth hormone poly A
sequence were prepared and used for microinjection into fertilized
B6C3fl (Taconic, Germantown, N.Y.) murine oocytes, using a standard
microinjection protocol. See, Hogan, B. et al., Manipulating the
Mouse Embryo. A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1994.
[0482] Eight transgenic mice expressing human zalpha11 Ligand from
the lymphoid-specific E.mu.LCK promoter were identified among 44
pups. Four of these were pups that died and 4 grew to adulthood.
Expression levels were fairly low in these animals. Twenty
transgenic mice expressing mouse zalpha11 Ligand from the
lymphoid-specific E.mu.LCK promoter were identified among 77 pups.
All 20 grew to adulthood. Expression levels were fairly low in
these animals. Three transgenic mice expressing mouse zalpha11
Ligand from the liver-specific MT-1 promoter were identified among
60 pups. Two of these pups died and 1 grew to adulthood. Expression
levels were fairly low in these animals. Tissues were prepared and
histologically examined as describe below.
[0483] B. Microscopic Evaluation of Tissues from Transgenic
Mice
[0484] Spleen, thymus, and mesenteric lymph nodes were collected
and prepared for histologic examination from transgenic animals
expressing human and mouse zalpha11 Ligand (Example 36A). Other
tissues which were routinely harvested included the following:
Liver, heart, lung, kidney, skin, mammary gland, pancreas, stomach,
small and large intestine, brain, salivary gland, trachea,
espohogus, adrenal, pituitary, reproductive tract, accessory male
sex glands, skeletal muscle including peripheral nerve, and femur
with bone marrow. The tissues were harvested from a neonatal pup
which died unexpectedly, and several adult transgenic mice, as
described below. Samples were fixed in 10% buffered formalin,
routinely processed, embedded in paraffin, sectioned at 5 microns,
and stained with hematoxylin and eosin. The slides were examined
and scored as to severity of tissue changes (0=none, 1=mild,
2=moderate, 3=severe) by a board certified veterinary pathologist
blinded to treatment.
[0485] The pup and 2 female adult mice expressing the human
zalpha11 Ligand, and 3 of the 6 male adult mice expressing the
mouse zalpha11 Ligand showed inflammatory infiltrates in many of
the tissues examined. The organs affected varied somewhat from
mouse to mouse. The inflammatory infiltrate was composed primarily
of neutrophils and macrophages in varying numbers and proportions
and was generally mild to moderate degree in severity. Moreover,
these animals showed changes in lymphoid organs, including moderate
to severe lymphopenia in the spleen and thymus (human and mouse
zalpha11 Ligand transgenics); and severe lymphopenia (human
zalpha11 Ligand transgenics), or mild to severe suppurative to
pyogranulomatous lymphadenitis (mouse zalpha11 Ligand transgenics)
in lymph nodes. In addition, increased extramedullary hematopoiesis
was evident in the spleens. These changes were not observed in
age-matched control mice.
[0486] C. Flow Cytometric Analysis of Tissues from Transgenic Mice
Over Expressing zalpha11 Ligand
[0487] Transgenic animals over expressing either human or mouse
zalpha11 ligand (Example 36A) were sacrificed for flow cytometric
analysis of peripheral blood, thymus, lymph node, bone marrow, and
spleen.
[0488] Cell suspensions were made from spleen, thymus and lymph
nodes by teasing the organ apart with forceps in ice cold culture
media (500 ml RPMI 1640 Medium (JRH Biosciences. Lenexa, Kans.); 5
ml 100.times. L-glutamine (Gibco BRL. Grand Island, N.Y.); 5 ml
100.times. Na Pyruvate (Gibco BRL); 5 ml 100.times. Penicillin,
Streptomycin, Neomycin (PSN) (Gibco BRL) and then gently pressing
the cells through a cell strainer (Falcon, VWR Seattle, Wash.).
Peripheral blood (200 ml) was collected in heparinized tubes and
diluted to 10 mls with HBSS containing 10 U Heparin/ml.
Erythrocytes were removed from spleen and peripheral blood
preparations by hypotonic lysis. Bone marrow cell suspensions were
made by flushing marrow from femurs with ice cold culture media.
Cells were counted and tested for viability using Trypan Blue
(GIBCO BRL, Gaithersburg, Md.). Cells were resuspended in ice cold
staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) at
a concentration of ten million per milliliter. Blocking of Fc
receptor and non-specific binding of antibodies to the cells was
achieved by adding 10% normal goat sera and Fc Block (Pharmingen,
La Jolla, Calif.) to the cell suspension.
[0489] Cell suspensions were mixed with equal volumes of
fluorochrome labeled monoclonal antibodies (PharMingen), incubated
on ice for 60 minutes and then washed twice with ice cold wash
buffer (PBS, 1% fetal bovine serum, 0.1% sodium azide) prior to
resuspending in 400 ml wash buffer containing 1 mg/ml 7-AAD
(Molecular Probes, Eugene, Oreg.) as a viability marker in some
samples. Flow data was acquired on a FACSCalibur flow cytometer (BD
Immunocytometry Systems, San Jose, Calif.). Both acquisition and
analysis were performed using CellQuest software (BD
Immunocytometry Systems).
[0490] The transgenic animals that expressed either the human or
mouse zalpha11 Ligand at the highest levels had dramatically
altered cell populations in all lymphoid organs analyzed. Changes
seen included complete loss of thymic cellularity, complete absence
of CD45R positive B cells and increased size and cellularity of
spleens. Both spleen and bone marrow had increased numbers of
myeloid sized cells, which was accounted for by increases in both
monocytes and neutrophils. The pan NK cell marker (DX5) was
increased in many populations. Moderate expressing founders had
less dramatic but still significant changes consistent with the
phenotype seen in the high expressers. Mice with the lowest level
of expression had neither a significant increase in myeloid cells
nor decrease in B cells numbers. They did show significant changes
in thymocyte populations with decreases in CD4+ CD8+ double
positive cells and increases in both CD4 and CD8 single positive
cells.
Example 37
[0491] Zalpha11 Ligand Purified Recombinant Human Protein
[0492] Dose-Response Study in Normal Mice
[0493] A. Summary
[0494] Normal six week old female C57B1/6 (Harlan Sprague Dawley,
Indianapolis, Ind.). mice were treated by intraperitoneal injection
once daily for either four or eight days with one of four dose
levels of purified recombinant human zalpha11 Ligand (Example 24)
at 0.1, 0.5, 5 or 50 .mu.g/mouse/day or with vehicle as a control.
Body weights and body temperatures were monitored daily. On either
day four or day nine, four of the eight mice from each protein
treatment group and five of the ten mice in the vehicle control
group were sacrificed. Blood, bone marrow and tissues were
harvested and analyzed. Potential perturbations in lymphoid tissues
were examined, as well as general physiologic and toxicological
parameters.
[0495] There was no evidence of toxicity of human zalpha11 Ligand
protein at any of the doses tested. Body weights and temperatures
were unchanged. There were no apparent changes in clinical
chemistry parameters. However, there were consistent findings
relating to increased percentages of myeloid lineage cells in bone
marrow, spleen and peripheral blood in mice treated with the
highest dose of zalpha11 Ligand compared to the vehicle control.
There was a statistically significant increase in myeloid lineage
sized cells identified by flow cytometric analysis of spleen
homogenate in the high-dose group. The spleens of the two highest
dose groups were statistically significantly larger than the other
groups. On histopathologic examination, however, only a marginal
increase in extramedullary hematopoiesis was seen in the highest
dose group. There was a statistically significant increase in the
myeloid to erythroid ratio of the bone marrow in the highest dose
group compared to the other groups. Finally, there were increases
seen in peripheral blood both in total white blood cell counts and
in the percentage of monocytes in the same group.
[0496] B. Dosing Solution Preparation
[0497] Purified recombinant human zalpha11 Ligand (Example 24) was
diluted into sterile phosphate buffered saline (GibcoBRL, Grand
Island, N.Y.) at concentrations to deliver 50, 5, 0.5 or 0.1
micrograms of protein in 0.1 ml of PBS vehicle. The doses for the
first four days were made on day 0 and frozen in a frosty
-20.degree. C. freezer prior to use. The doses for days five
through eight were made on day five and frozen as above. Aliquots
of the same PBS were similarly frozen for the vehicle treated
control group. On the day of administration the appropriate
aliquots were thawed and 0.1 ml of solution was injected
intraperitoneally into the mice each day for either four or eight
days.
[0498] C. Study Design
[0499] The mice were six weeks old at the start of the study. Each
treatment group consisted of eight mice, except for the vehicle
control group that included ten mice. One half of the mice in each
treatment group were sacrificed after four days of treatment and
the other half after eight days.
[0500] Before treatment each day, each mouse was weighed and her
body temperature recorded using the Portable Programmable Notebook
System (BMDS, Inc, Maywood, N.J.), by scanning the mouse for
identification number and body temperature from transponders
implanted subcutaneously (IPTT-100, BMDS, Maywood, N.J.).
[0501] At sacrifice, tissues harvested to assess white blood cell
populations by flow cytometric analysis included bone marrow,
thymus and spleen. FACS analysis of the lymphoid organs and bone
marrow was performed with the FACSCalibur, (Becton Dickinson,
Mansfield, Mass.). The tissues harvested for histologic examination
for signs of toxicity of the protein included: spleen, thymus,
liver, kidney, adrenal gland, heart and lungs. All tissues fixed
for histology were kept at 4.quadrature. C overnight in 10% Normal
Buffered Saline (NBF) (Surgipath, Richmond, Ill.). The following
day the NBF was replaced with 70% ethanol and the tissues returned
to 4.degree. C. until processing for histology.
[0502] The tissues were processed and stained for Hematoxylin and
Eosin in house, then sent to a contract pathologist for
histopathologic analysis. Blood was collected for complete blood
cell counts (CBC) and serum chemistry profiles. The CBC's were
analyzed in-house with the Cell Dyn 3500 Hematology Analyzer
(Abbott Diagnostics Division, Abbott Park, Ill.) and manual
differential white blood cell counts were analyzed at Phoenix
Central Laboratory, (Everett, Wash.). The serum was kept frozen at
-20.degree. C. until submission to Phoenix Central Laboratory for
complete serum chemistry panels. To assess myeloid:erythroid
ratios, the bone marrow from one femur was applied to CytoSpin
slides (CYTOSPIN 3 CYTOCENTRIFUGE and CYTO SLIDES, Shandon,
Pittsburgh, Pa.) and sent to Phoenix Central Laboratories for
analysis.
[0503] D. Study Results
[0504] There were no apparent clinical indications of physiologic
effects or of toxicity of human zalpha11 Ligand at doses of 50
.mu.g/day or lower. Body weights and temperatures remained normal
for the duration of the treatments. Serum chemistry parameters were
in normal ranges. Red blood cell and platelet counts appeared
normal. In the mice receiving 50 .mu.g/day for 8 days, manual
differential white blood cell counts showed that the percentage of
monocytes was elevated in the peripheral blood, and an apparent
increase in the total white blood cell counts. In bone marrow
flushed from a femur, myeloid to erythroid ratios were increased in
the 50 .mu.g dose group, and to a lesser degree the 5 .mu.g dose
group from the 8-day dose set. In a non-parametric multiple column
comparison using InStat (InStat MAC; GraphPad Software, Inc., San
Diego, Calif.), this difference was statistically significant
(p=0.0049). The difference between the highest dose group and
vehicle was also significant, (p=0.0286). The increased white blood
cells in peripheral blood and the significant increase in myeloid
precursors in the marrow may thus be related.
[0505] Histologic evaluation of the following tissues showed no
apparent evidence of cytologic or structural changes, mitotic
events or necrosis: thymus, liver, kidney, adrenal gland, duodenum,
pancreas, jejunum, caecum, colon, mesenteric lymph nodes, uterus,
ovary, salivary gland, heart, trachea, lung, and brain. There were
no apparent differences between the treatment groups in the weights
of the thymus, kidney, liver or brain. Of all the tissues examined,
only the spleen weights were significantly affected.
[0506] Each mouse spleen weight was normalized to her brain weight.
In the 50 .mu.g/day treatment group compared to the vehicle, 0.1
.mu.g and 0.5 .mu.g treatment groups, the average of the spleen
weights was nearly 50% greater after four days of treatment and
almost 100% greater after eight days than the average spleen
weights of the other three groups. In the four-day set, the 5
.mu.g/day group also tended to have larger spleens than the control
and low dose groups. The difference in the spleen/brain weights
with data from the four-day and the eight-day sets combined by
treatment group was statistically significant (p=0.0072) by
Kruskall-Wallace non-parametric ANOVA, multiple column comparison
test using the InStat program (GraphPad Software).
[0507] A marginal increase in extrameduallary hematopoiesis,
especially in the red pulp was seen in spleens of mice from the
highest dose group, even in the mice treated for four days. Flow
cytometric analysis of the spleens showed a significant increase in
the proportion of myeloid size cells in the highest dose group
(p=0.01, Student's t test), representing increases in both
monocytes and neutrophils. This effect may be related to the
increased peripheral blood mononuclear cell percentage, as well as
the apparent increase in myeloid precursors in the bone marrow,
described above. Moreover, the transgenic mice derived from
insertion of the human zalpha11 gene had increased extramedullary
hematopoiesis in their spleens compared to non-transgenic litter
mates.
[0508] Several changes were observed in the 50 .mu.g per day dose
group compared to the control group that implicate zalpha11 Ligand
in production or development of cells of the myeloid lineage. Taken
together, the observed changes suggest that zalpha11 may be useful
as a therapeutic protein in such medical specialties as cancer and
immunologic disorders described herein.
Example 38
[0509] Preliminary Elimination and Tissue Distribution Study of
[0510] Purified Recombinant Human Zalpha11 Ligand Protein
[0511] A. Summary
[0512] In order to elucidate tissue distribution and elimination
patterns of the purified rhzalpha11 Ligand, a preliminary
pharmacokinetic study was undertaken. Nine week old male C57B1/6
mice were given purified recombinant human zalpha11 Ligand protein
labeled with .sup.111Indium (.sup.111In) (NEN, Boston, Mass.) by
one of three routes. A single bolus injection was given to each
mouse by either the intravenous (IV), intraperitoneal (IP), or
subcutaneous route (SC). The mice injected by either the
subcutaneous or intraperitoneal route were sacrificed at either one
or three hours after injection. The mice injected intravenously
were sacrificed after either ten minutes or one hour following
injection. Blood, plasma and selected tissues were harvested at
various timepoints and counted by a gamma counter to estimate the
approximate half-life and tissue distribution of the exogenous
labeled protein. The tissues that were harvested for counting as
well as the intervals of sacrifice were selected based on reports
of the distribution of other cytokines labeled with
radionuclides.
[0513] At sacrifice, tissues harvested for counting of
radioactivity included thymus, spleen, kidney, a lobe of liver, a
lobe of lung, and urinary bladder. In the group receiving the
injection intraperitoneally, gut was also counted to assess
incidence of injection into the gut, and in the subcutaneously
dosed mice, skin with underlying structures in the area of
injection was counted. The cpm for whole liver and lung were
calculated from a section that was counted and a percentage of the
whole organ weight represented by the section.
[0514] After the end of the study the collected tissues, whole
blood and plasma were counted on the COBRA II AUTO-GAMMA gamma
counter (Packard Instrument Company, Meriden, Conn.). An aliquot of
the original labeled dosing solution was also counted at the end of
the study with the tissues. This allowed calculation of percent
total injected radioactivity for each mouse and simultaneous
correction of all counts for radioactive decay. Approximations of
remaining blood volume and organ weights indicated that the
majority of the counts administered were accounted for, and
therefore the percentage of counts per tissue were a reasonable
representation of distribution of the counts following labeled
zalpha11 Ligand administration by each route.
[0515] B. .sup.111Indium Labeling of zalpha11 Ligand
[0516] Purified recombinant human zalpha11 Ligand (Example 29) was
conjugated with a 10 fold molar excess of DTPA (Peirce, Rockford,
Il) by incubating 30 minutes at room temperature in PBS. Unreacted
DTPA and hydrolyzates were removed by buffer exchange on a
Biomax-5k NMWL (Ultrafree-15, Millipore, Bedford, Mass.). The void
volume protein peak was concentrated to 5 mg/ml and an aliquot
taken for testing in a bioassay (anti-CD40 stimulation of murine
B-cells (Example 44)). Upon confirming that the DTPA-conjugate
still had full bioactivity the conjugate was diluted to 0.5 mg/ml
with 1M Na Acetate pH6.0. Two mCi of .sup.111Indium was taken up in
0.5 ml 1M Na Acetate pH6.0 and mixed with the DTPA-human zalpha11
Ligand for 30 min. at room temperature. Unincorporated
.sup.111Indium was removed during buffer exchange to PBS on a PD-10
column (Pharmacia, Piscataway, N.J.). The radio-labeled material
was diluted with unlabeled human zalpha11 Ligand to give a specific
activity of 100 mCi/mg, sterile filtered and stored at 4.degree. C.
overnight. One hundred percent of the labeled protein was retained
on a Biomax-5k NMWL membrane (Millipore). The labeled
.sup.111In-human zalpha11 Ligand was administered to mice in the
elimination and pharmacokinetic studies. Fifty .mu.g human zalpha11
Ligand protein labeled with 5 .mu.Ci of labeled human zalpha11
Ligand in 0.1 ml of PBS vehicle was administered to each
animal.
[0517] C. Results of Preliminary Distribution Study
[0518] After one and three hours following administration by all
three routes, the highest concentration of .sup.111In-human
zalpha11 Ligand, was found in kidney and the second highest was in
urine and urinary bladder, as evinced by these tissues having the
highest cpm. The average counts recovered from kidneys were from 3
to 8 times higher than the whole liver counts, depending on the
route of injection and the sacrifice timepoint. For example, the
average kidney cpm at 60 minutes following IV injection was 4.5
times greater than the average counts calculated for whole liver
from the same group. In the group that was sacrificed ten minutes
after intravenous administration, the highest cpm was again in
kidney, and the second highest accumulation was equivalent in
liver, urinary bladder and urine.
[0519] D. Preliminary Pharmacokinetic Study
[0520] Blood and plasma collections were done at 10, 30 and 60
minutes following injection by all three routes. Following
injection by the IV route, a separate set of mice had blood and
plasma samples taken at two, five and ten minutes. Another set of
mice who received their injections by either the IP or SC route had
blood sampled at one, two and three hours. For the treatment groups
see Table 6. The short collection times bracket the reported
half-life of IL-2 following intravenous injection. The reported
T1/2 was in the range of 2.5 to 5.1 minutes. For reference to in
vivo administration to IL-2, see Donohue J H and Rosenberg S A J
Immunol, 130:2203, 1983. The long timepoints were chosen to outline
the anticipated elimination phase. TABLE-US-00006 TABLE 6 Route of
injection Bleed Times(min.) Sacrifice Time Intravenous Group 1 2,
5, 10 10 min. Intravenous Group 2 10, 30, 60 60 min.
Intraperitoneal Group 1 10, 30, 60 60 min. Intraperitoneal Group 2
60, 120, 180 180 min. Subcutaneous Group 1 10, 30, 60 60 min.
Subcutaneous Group 2 60, 120, 180 180 min.
[0521] Un-labeled IL-2 has been shown to be eliminated from the
serum with a half-life of approximately three minutes in mice after
IV injection. For reference see Donahue, J H and Rosenburg supra.
Following IP and SC injection of similar amounts of IL-2, the
duration of persistence of IL-2 activity in serum was prolonged
from 2 units/ml for less than 30 minutes following IV injection to
greater than 2 units/ml for 2 hours following IP and 6 hours
following SC injections. The principle route of clearance of IL-2
appears to be the kidney. Zalpha11 ligand has been shown to be
structurally similar to IL-2, as discussed herein. Preliminary
evaluation of the elimination of zalpha11 Ligand appears to be
consistent with the apparent clearance of IL-2 by the kidneys,
based on the accumulation of cpm predominantly in the kidneys,
followed by the urinary bladder and urine in the present study.
[0522] Estimations were made of pharmacokinetic parameters based on
non compartmental analysis of the cpm data obtained from the
plasma, using the PK analysis program WinNonLin, Version 1.1,
(Scientific Consulting Inc., Cary, N.C.). Plasma half-lives of
zalpha11 Ligand were estimated using the predicted terminal
elimination rate constants for intravenous, subcutaneous, and
intraperitoneal administration of a 50 .mu.g dose. The
pharmacokinetic results were estimations due to limited data points
in the terminal elimination region of the plasma concentration vs.
time profiles. Moreover, the fit of the terminal elimination phase
for SC and IP dosing required use of data from timepoints during
which absorption of the .sup.111In-human zalpha11 Ligand was
apparently still occurring. However, estimations of half-lives
following intravenous, subcutaneous, and intraperitoneal dosing
were 13.6 min., 18.8 min., and 34.3 min., respectively. Since a
dosing range was not evaluated it was not apparent whether
saturable or active elimination (Michaelis Menten kinetics) was
occurring. Therefore, these half-life calculations are
estimations.
[0523] Estimates of the bioavailability of the labeled protein were
made based on the area under the curve (AUC) following subcutaneous
or intraperitoneal dosing compared to that of intravenous dosing.
The estimated bioavailability following subcutaneous and
intraperitoneal injection were 35.8% and 63.9% respectively.
Because only one protein dose was studied, the bioavailability was
not evaluated as a function of dose. The estimated clearance and
volume of distribution (based on the data from the intravenous
injection) were 0.48 ml/min. and 6.1 ml, respectively.
[0524] Although the data are preliminary, the fate of zalpha11
Ligand administered IV was similar to that reported for IL-2,
another 4-helix bundle cytokine (Donahue, J H and Rosenburg, S A
supra.). Like 11-2, IV-administered zalpha11 Ligand had a plasma
half life of only minutes with the main clearance in the kidney.
Three hours after injection, the majority of the labeled material
extracted from kidney was still retained in a Biomax 5K NMLW
membrane (Millipore). Since it has previously been reported that
the indium remains associated with protein even during lysosomal
degradation (Staud, F. et al., J. Pharm. Sciences 88:577-585, 1999)
zalpha11 Ligand is accumulating and may be degraded in the kidney.
The current study also showed, as observed with many other
proteins, including IL-2 (Donahue, J H and Rosenburg, S A, supra.),
that IP and SC administration significantly prolonged the plasma
levels of zalpha11 Ligand.
Example 39
[0525] Isolation and Expansion of Fresh Human Bone Marrow MNC
CD34+Fraction Using zalpha11 Ligand for Assessment of NK
Activity
[0526] A. Selection and Isolation of CD34+ Cells from Human Bone
Marrow
[0527] Fresh human bone marrow mononuclear cells (MNC) were
prepared to enrich for cells having NK cell activity. Fresh human
MNCs were obtained from Poeitic Technologies (Gaithersburg, Md.).
10 ml alpha MEM (JRH, Lenexa, Kans.) containing 10% HIA FBS
(Hyclone, Logan, Utah) and the antibiotic 1% PSN (Gibco, BRL, Grand
Island, N.Y.) was added to the cell suspension and the cells were
passed through a 100 .mu.m sieve. The cells were then counted,
pelleted, washed with 10 ml PBS containing 2% FBS, then pelleted
again and resuspended in 1 ml PBS containing 2% FBS. Cells having a
CD34 cell surface marker (CD34+ cells) were magnetically separated
using a Detachabead kit with Dynabeads M-450 CD34 ((Dynal, Oslo,
Norway), as per manufacturer's instructions. Both the CD34+ cell
and the CD34- cell fractions were further analyzed below.
[0528] B. Expansion of CD34+ Cells Using zalpha11 Ligand
[0529] A CD34+ cell fraction was plated into four wells in a
24-well plate. 50,000 positively selected cells suspended in 1 ml
Alpha MEM (JRH) containing 10% HIA FBS (Hyclone) and 1% PSN
(Gibco/BRL), plus the various cytokines described below were plated
in each of the 4 wells (1-4). Various reagents were used to test
for zalpha11 Ligand-induced expansion of the CD34+ selected bone
marrow MNCs: Reagents included human flt3 (R&D, Minneapolis,
Minn.); purified human zalpha11 Ligand (Example 30C and Example
30D); human IL-15 (R&D). Reagents were combined as follows at
day 0: In well #1, 2 ng/ml human flt3 was added. In well #2, 2
ng/ml human flt3 and 15 ng/ml purified human zalpha11 Ligand were
added. In well #3, 2 ng/ml human flt3 and 20 ng/ml human IL15 were
added. In well #4, 2 ng/ml human flt3, 15 ng/ml purified human
zalpha11 Ligand, and 20 ng/ml human IL15 were added. After
incubating for 18 days, the suspension cells from each well were
pelleted, and then resuspended in 0.5 ml alpha MEM (JRH) containing
10% HIA FBS (Hyclone) and 1% PSN (Gibco/BRL), and counted to assess
proliferation of the CD34+ cell fraction. A low level of
proliferation was seen in the presence of flt3 alone (control well
#1), but the presence of IL-15 or zalpha11 in addition to flt3 had
not significant effect on the expansion (wells, #2 and #3).
However, expansion beyond the flt3 control was evident in well #4
which contained IL-15 and zalpha11 Ligand in addition to flt3. This
result suggested that zalpha11 and IL-15 act in synergy to expand
the human CD34+ cell population. Moreover, the results of this
experiment supported the results seen with the mouse zalpha11
Ligand in the mouse BM assay (Example 21).
[0530] All cell populations were then tested for NK activity and
subjected to flow cytometry analysis, as shown below (Example
41).
[0531] C. Expansion of CD34+ or CD34-Cells Using zalpha11 Ligand
with Delayed Addition of IL-15
[0532] Both CD34 positive and negative (CD34-) fractions were
plated separately into six 12 well plate wells (1-6). Each of six
wells contained 100,000 positively or negatively selected cells in
2 ml alpha MEM containing 10% HIA FBS and PSN, described above.
Reagents used were as described above. In well #1, 2 ng/ml human
flt3 was added at day 0. In well #2, 2 ng/ml human flt3 was added
at day 0, and after 5 days incubation 20 ng/ml human IL15 was
added. In well #3, 2 ng/ml human flt3 and 15 ng/ml human zalpha11
Ligand were added at day 0. In well #4, 2 ng/ml human flt3 and 15
ng/ml human zalpha11 Ligand were added at day 0, and after 5 days
incubation 20 ng/ml human IL 15 was added. In well #5, 2 ng/ml
human flt3 and 20 ng/ml human IL 15 were added at day 0. In well
#6, 2 ng/ml human flt3, 15 ng/ml human zalpha11 Ligand, and 20
ng/ml human IL15 were added at day 0. After incubating for a total
of 15 days from the start of the experiment, the cells from each
well were harvested and counted.
[0533] In the CD34+population a low level of proliferation was seen
in the presence of flt3 alone (control well #1), but the presence
of IL-15 or zalpha11 added at day 0 in addition to flt3 had no
significant effect on the expansion (wells, #3 and #5). Addition of
IL-15 after 5 days had some proliferative effect in comparison to
the flt3 control (well #2 compared to well #1) and a proliferative
effect in the presence of zalpha11 (well #4 compared to well #3).
However, the greatest expansion was evident in well #6 which
contained IL-15 and zalpha11 Ligand in addition to flt3 at day
0.
[0534] In the CD34-population, no proliferation was seen in the
presence of flt3 alone (control well #1), and in fact a decrease in
the cell population was evident. The presence of zalpha11 added at
day 0 in addition to flt3 (well #3) was similar to the flt3
control. The presence of IL-15 added at day 5 increased
proliferation effect of the cells in the presence (well #4) or
absence (well #2) of zalpha11 Ligand. Again, the greatest expansion
was evident in well #6 which contained IL-15 and zalpha11 Ligand in
addition to flt3 at day 0.
[0535] All cell populations were then tested for NK activity and
subjected to FACS analysis, as shown below (Example 41).
Example 40
[0536] Isolation and Expansion of Fresh mouse Cells Using Human and
Mouse zalpha11 Ligand for Assessment of NK Activity and NK Cell
Markers
[0537] A. Isolation and Expansion of Fresh Mouse Low Density Bone
Marrow Cells Using Human and Mouse zalpha11 Ligand
[0538] Fresh mouse marrow cells were isolated by clipping both ends
of mouse femurs, and flushing two to three milliliters of growth
medium (see below) through the inside of the bone into a collection
tube. The growth medium was 500 ml RPMI 1640 Medium (JRH
Biosciences. Lenexa, Kans.); 5 ml 100.times. L-glutamine (Gibco
BRL. Grand Island, N.Y.); 5 ml 100.times. Na Pyruvate (Gibco BRL);
5 ml 10.times. Penicillin, Streptomycin, Neomycin (PSN) (Gibco
BRL); and 50 ml heat-inactivated Fetal Bovine Serum (FBS) (Hyclone
Laboratories. Logan, Utah). The marrow cells were then broken-up by
pipeting the media up and down several times. The cells were then
pelleted and washed once with growth medium, and passed through a
70-micron sieve. The low-density mononuclear cells were then
isolated by subjecting the marrow cells to a density gradient.
Marrow cells in five to eight milliliters of growth medium were
carefully pipetted on top of five to eight milliliters of NycoPrep
1.077 Animal (Nycomed. Oslo, Norway) in a centrifuge tube. This
gradient was then centrifuged at 600.times.g for 20 minutes. The
low density mononuclear cells were harvested from the interface
layer between the NycoPrep and the medium. These cells were then
diluted to approximately 20 milliliters in growth medium, pelleted
and washed. The cells were then plated at approximately
0.5-1.5.times.106 cells per milliliter in growth medium in a
standard tissue culture flask and incubated at 370 C, 5% CO2 for
two hours.
[0539] The non-adherent, low density (NA LD) marrow cells were then
harvested and plated at 0.5-2.0.times.10.sup.5 cells per milliliter
in growth medium plus 2.5 nanograms per milliliter mouse flt3 (R
and D Systems. Minneapolis, Minn.) plus 25 to 50 nanograms per
milliliter human Interleukin 15 (IL-15) (R and D Systems) with or
without 50 to 150 nanograms per milliliter human zalpha11 Ligand;
or with or without 0.12 to 10 nanograms per milliliter mouse
zalpha11 Ligand.
[0540] There was no significant expansion without the addition of
the human or mouse zalpha11 Ligand. Non-adherent cells were
expanded in the cultures containing mouse zalpha11 Ligand as low as
0.12 ng/ml and in the cultures containing human zalpha11 Ligand as
low as 22 ng/ml. In cultures containing both the human and mouse
zalpha11 Ligand, non-adherent cell expansion increased with
increasing dose if zalpha11 Ligand, with the mouse ligand
saturating response at about 5-10 ng/ml and the human not reaching
a saturating response even at the highest dose of 200 ng/ml. Human
zalpha11 Ligand appeared to be approximately 20 to 100 fold less
potent on mouse cells as the mouse zalpha11 Ligand. After
approximately five to ten days the zalpha11 Ligand expanded mouse
cells were harvested and analyzed by flow cytometry (FACSCalibur;
Becton Dickinson, Mansfield, Mass.) to determine what percentage of
them were positive for NK cell antigens, where 46% were positive
for the PanNK cell marker DX5 (Pharmingen).
[0541] B. Isolation and Expansion of Fresh Lineage Depleted Mouse
Marrow Cells
[0542] Fresh mouse lineage depleted (lin-) marrow cells were
isolated from fresh mouse marrow cells by first incubating the
cells with the following antibodies: TER119, Gr-1, B220, MAC-1,
CD3e and I-Ab (Pharmingen. San Diego, Calif.). The lin+ cells were
then removed with Dynabeads M-450 sheep anti-rat IgG (Dynal, Lake
Success, N.Y.) as per manufacturer's instructions.
[0543] The negatively selected lin- marrow cells were then plated
as above in growth medium plus either 2.5 ng/mL flt3(R&D
Systems) and 25 ng/mL IL-15 (R&D Systems); or flt3, IL-15 and
mouse zalpha11 Ligand, 2 to 5% BHK mouse zalpha11 Ligand
conditioned medium. After six days of growth, the cultures were
harvested, counted and submitted to an NK cell activity assay
(Example 41). Cells grown with mouse zalpha11 Ligand were
approximately two to three times more effective at lysing NK cell
target cells (YAC-1 cells) as the cells grown without zalpha11
Ligand.
[0544] C. Isolation and Expansion of CD4-CD8-(Double Negative or
DN) Thymocytes
[0545] Fresh mouse thymocytes were isolated by chopping and sieving
thymuses from three to eight week old mice. CD4-CD8-(DN) cells were
then negatively selected by incubating the thymocytes with anti-CD4
and anti-CD8 antibodies (PharMingen), then removing the CD4+ CD8+
cells with Dynabeads M-450 sheep anti-rat IgG (Dynal) as per
manufacturer's instructions.
[0546] The DN mouse thymocytes were then grown in growth medium
plus 2.5 ng/mL flt3 (R&D Systems), 25 ng/mL IL-15 (R&D
Systems) and 10 ng/mL IL-7 (R&D Systems) with or without mouse
zalpha11 Ligand as above. Six days later the cells were harvested,
counted, analyzed by flow cytometry as described above, and also
submitted to an NK cell activity assay (Example 41).
[0547] The culture grown with mouse zalpha11 Ligand yielded
approximately 480,000 cells while the culture without zalpha11
Ligand yielded only approximately 160,000 cells. The culture grown
with mouse zalpha11 Ligand was found to be approximately 16.2%
positive for the NK cell antigen Pan NK, DX5 (PharMingen). The
culture grown without zalpha11 Ligand was 14.6% positive for DX5.
The cells grown with zalpha11 Ligand lysed NK cell target cells,
YAC-1, approximately two times better than the cells grown without
zalpha11 Ligand. The expanded cells did not lyse significantly a
negative control target cell line, EL4. These results suggested
that zalpha11 Ligand selectively expands lytic NK cells.
Example 41
[0548] Activity of Human and Mouse zalpha11 Ligand Expanded Cells
and Mature Murine Nk Cells in NK Cell Cytotoxicity Assays
[0549] A. NK Cell Assay
[0550] NK cell-mediated target cytolysis was examined by a standard
.sup.51Cr-release assay. Target cells (K562 cells (ATCC No.
CCL-243) in human assays, and YAC-1 cells (ATCC No. TIB-160) in
mouse assays) lack expression of major histocompatability complex
(MHC) molecules, rendering them susceptible to NK cell-mediated
lysis. A negative control target cell line in mouse assays is the
MHC+ thymoma EL4 (ATCC No. TIB-39). We grew K562, EL4, and YAC-1
cells in RP10 medium (standard RPMI 1640 (Gibco/BRL, Grand Island,
N.Y.) supplemented with 10% FBS (Hyclone, Logan, Utah), as well as
4 mM glutamine (Gibco/BRL), 100 I.U./ml penicillin+100 MCG/ml
streptomycin (Gibco/BRL), 50 .mu.M .beta.-mercaptoethanol
(Gibco/BRL) and 10 mM HEPES buffer (Gibco/BRL). On the day of
assay, 1-2.times.10.sup.6 target cells were harvested and
resuspended at 2.5-5.times.10.sup.6 cells/ml in RP10 medium. We
added 50-100 .mu.l of 5 mCi/ml .sup.51Cr-sodium chromate (NEN,
Boston, Mass.) directly to the cells and incubated them for 1 hour
at 37.degree. C., then washed them twice with 12 ml of PBS and
resuspended them in 2 ml of RP10 medium. After counting the cells
on a hemacytometer, the target cells were diluted to
0.5-1.times.10.sup.5 cells/ml and 100 .mu.l (0.5-1.times.10.sup.4
cells) were mixed with effector cells as described below.
[0551] In human assays, effector cells were prepared from selected
and expanded human CD34+BM cells (Example 39B) which were
harvested, washed, counted, mixed at various concentrations with
51Cr-labeled target cells in 96-well round bottomed plates, and
incubated for 4 hours at 37.degree. C. After co-incubation of
effector cells and the labeled target cells, half of the
supernatant from each well was collected and counted in a gamma
counter for 1 min/sample. The percentage of specific .sup.51Cr
release was calculated from the formula 100.times. (X-Y)/(Z-Y),
where X is .sup.51Cr release in the presence of effector cells, Y
is the spontaneous release in the absence of effectors, and Z is
the total .sup.51Cr release from target cells incubated with 0.5%
Triton X-100. Data were plotted as the % specific lysis versus the
effector-to-target ratio in each well.
[0552] B. Activity of Human zalpha11 Ligand Expanded Cells
[0553] Isolated CD34+human HPCs cultured with flt3 +/-zalpha11
Ligand and flt3 +IL-15+/-zalpha11 Ligand (Example 39), were
harvested the cells on day 15 to assess their capacity to lyse MHC-
K562 cells in a standard .sup.51Cr-release assay as described
above, and to analyze their surface phenotype by flow cytometry. As
expected from previous reports (Mrozek, E et al., Blood
87:2632-2640, 1996; and Yu, H et al., Blood 92:3647-3657, 1998),
simultaneous addition of IL-15 and flt3L did induce the outgrowth
of a small population of CD56+ cells. Interestingly, although BM
cells cultured simultaneously with zalpha11 Ligand and flt3L did
not expand significantly, there was a significant increase in total
cell numbers in cultures containing a combination of flt3L,
zalpha11 Ligand and IL-15 (see, Example 39).
[0554] For an assessment of the surface phenotype of these human BM
cultures, we stained small aliquots of the cells for 3-color flow
cytometric analysis with anti-CD3-FITC, anti-CD56-PE and
anti-CD16-CyChrome mAbs (all from PharMingen, San Diego, Calif.)
and analyzed them on a FACSCalibur using CellQuest software (Becton
Dickinson, Mountain View, Calif.). This flow cytometric analysis
confirmed that the cells growing out of these cultures were
differentiated NK cells, as they were large and granular and
expressed both CD56 and CD16, and were CD3-(Lanier, L L Annu. Rev.
Immunol. 16:359-393, 1998). Furthermore, these cells exhibited
significantly higher effector function than those cells grown with
IL-15 and flt3. More specifically, cells grown in all three
cytokines lysed more than 40% of the K562 targets at an
effector-to-target ratio (E:T) of 1.5, whereas cells grown in
IL-15+flt3L lysed fewer than 5% of the targets at an E:T of 2.
These data demonstrate that, in combination with IL-15, zalpha11
Ligand stimulates the differentiation of NK cells from CD34+BM
cells.
[0555] C. Activity of Mouse zalpha11 Ligand Expanded Cells
[0556] To test the effects of zalpha11 Ligand on murine
hematopoietic progenitor cells, purified Lineage-negative (Lin-)
bone marrow cells from C57B1/6 mice were expanded in
flt3+IL-15+/-zalpha11 Ligand, as described in Example 40B. On day 6
of culture, the cells ("effectors") were harvested and counted,
then resuspended in 0.4 ml of RP10 medium (Example 41A). Two
aliquots (0.15 ml each) of each sample expanded with or without
zalpha11 Ligand (Example 41A) were diluted serially 3-fold in
duplicate in 96-well round bottomed plates, for a total of 6 wells
of 100 .mu.l each. The remaining 100 .mu.l of cells were stained
for NK cell surface markers with FITC-anti-2B4 and PE-anti-DX5 mAbs
(PharMingen) and analyzed by flow cytometry. Each group of cells
exposed to flt3+IL-15 with or without the presence of zalpha11
Ligand had similar fractions of 2B4+DX5+ cells, ranging from 65-75%
positive for both NK markers.
[0557] For the NK lysis assay, target cells (YAC-1 and EL4) were
labeled with .sup.51Cr as described above. After counting the
target cells on a hemacytometer, the target cells were diluted to
0.5-1.times.105 cells/ml and 100 .mu.l of YAC-1 or EL4
(0.5-1.times.10.sup.4 cells) were mixed with 100 .mu.l effector
cells and incubated for 4 hours at 37.degree. C. Specific lysis was
determined for each well as described above.
[0558] We found that cells grown in the presence of
flt3+IL-15+zalpha11 Ligand exhibited enhanced lytic activity
(roughly 2-fold) against the YAC-1 targets (but did not kill the
MHC+ control cell line EL4). At an effector-to-target ratio (E:T)
of 5, NK cells generated in the presence of all 3 cytokines
(zalpha11 Ligand+flt3+IL-15) lysed 12% of the YAC-1 cells, whereas
those NK cells expanded with flt3+IL-15 lysed 6% of the YAC-1
targets. Subsequent experiments confirmed this trend.
[0559] In a second approach to determine the biological activity of
zalpha11 Ligand on murine NK cells, we isolated immature CD4-CD8-
("double negative", DN) mouse thymocytes as described in Example
40C and cultured them with IL-15+flt3+IL-7 or IL-15+flt3+IL-2, with
or without zalpha11 Ligand. On day 6 of culture, the cells were
harvested and assayed for NK lytic activity on YAC-1 and EL4 cells
as described above. We found that cells cultured in the presence of
zalpha11 Ligand had the greatest lytic activity in this assay, with
enhanced lytic activity over those cells cultured in the presence
of the other cytokines. Specifically, DN thymocytes grown with
IL-15+flt3+IL-7 killed 18% of the YAC-1 cells at E:T of 24 while
cells grown in the presence of IL-15+flt3+IL-7 plus zalpha11 Ligand
killed 48% of the targets at the same E:T. DN thymocytes grown in
IL-15+flt3+IL-2 killed 15% of the YAC-1 targets at an E:T of 6,
whereas cells grown with these 3 cytokines and zalpha11 Ligand
killed 35% of the YAC-1 cells at an E:T of 9. Flow cytometry was
performed on the cultured cells one day before the NK lysis assay.
As was true for the bone marrow cultures, despite the proliferative
effect of zalpha11 Ligand (cell numbers increase approximately
2-fold when zalpha11 Ligand is added), it did not significantly
enhance the fraction of DX5+ cells (17-20% of total cells in the
cultures with IL-7, and 35-46% of total in cultures with IL-2).
These data imply that zalpha11 Ligand, in combination with IL-15
and flt3, enhances the lytic activity of NK cells generated from
murine bone marrow or thymus.
[0560] D. Activity of Mouse zalpha11 Ligand on Mature Murine NK
Cells
[0561] In order to test the effects of mouse zalpha11 Ligand on
mature NK cells, we isolated spleens from four 5-week old C57B1/6
mice (Jackson Laboratories, Bar Harbor, Me.) and mashed them with
frosted-end glass slides to create a cell suspension. Red blood
cells were removed by hypotonic lysis as follows: cells were
pelleted and the supernatant removed by aspiration. We disrupted
the pellet with gentle vortexing, then added 900 .mu.l of sterile
water while shaking, followed quickly (less than 5 sec later) by
100 .mu.l of 10.times.HBSS (Gibco/BRL). The cells were then
resuspended in 10 ml of 1.times.HBSS and debris was removed by
passing the cells over a nylon mesh-lined cell strainer (Falcon).
These RBC-depleted spleen cells were then pelleted and resuspended
in MACS buffer (PBS+1% BSA+2 mM EDTA) and counted. We stained
300.times.10.sup.6 of the cells with anti-DX5-coated magnetic beads
(Miltenyi Biotec) and positively selected DX5+ NK cells over a MACS
VS+separation column, according to the manufacturer's instructions,
leading to the recovery of 8.4.times.10.sup.6 DX5+ cells and
251.times.10.sup.6 DX5- cells. Each of these groups of cells were
cultured in 24-well plates (0.67.times.10.sup.6 cells/well, 2 wells
per treatment condition) in RP10 medium (Example 41A) alone or with
1) 30 ng/ml mouse zalpha11 Ligand, 2) 30 ng/ml recombinant mouse
IL-2 (R&D Systems, Inc., Minneapolis, Minn.), 3) 30 ng/ml
recombinant human IL-15 (R&D), 4) 30 ng/ml each of mouse
zalpha11 Ligand and hIL-15, or 5) 30 ng/ml each of mIL-2 and
hIL-15. The cells were harvested after 21 hours, washed, and
resuspended in RP10 medium and counted. The cells were then assayed
for their ability to lyse .sup.51Cr-labeled YAC-1 or EL4 targets
cells, as described in Example 41A.
[0562] In general, there was little NK activity from the DX5-
(non-NK cells) groups, but the DX5- cells cultured with zalpha11
Ligand and hIL-15 did lyse 25% of the YAC-1 target cells at an E:T
of 82. By comparison, DX5- cells cultured with hIL-15 alone lysed
14% of the YAC-1 targets at an E:T of 110. This suggests that
zalpha11 Ligand and IL-15 are acting together on the residual
NK1.1+NK cells in this cell preparation. As for the DX5+ cell
preparation, treatment with mouse zalpha11 Ligand alone did not
significantly increase their effector function (their lysis of
YAC-1 cells was similar to the untreated group). As expected, both
IL-2 and IL-15 significantly improved NK activity. The highest
level of lysis, however, was detected in the group treated with
zalpha11 Ligand and hIL-15 (65% lysis of YAC-1 cells at an E:T of
3.3, vs. 45% lysis at an E:T of 4 for the hIL-15 treatment group).
Taken together, these results suggest that although zalpha11 Ligand
alone may not increase NK cell lysis activity, it does enhance NK
lysis activity of mature NK cells, when administered with
IL-15.
Example 42
[0563] Zalpha11 Ligand Proliferation of Human and Mouse T-cells in
a T-cell Proliferation Assay
[0564] A. Murine Zalpha11 Ligand Proliferation of Mouse T-Cells
[0565] T cells from C57B1/6 mice (Jackson Laboratories, Bar Harbor,
Me.) were isolated from pooled splenocytes and lymphocytes from
axillary, brachial, inguinal, cervical, and mesenteric lymph nodes
(LNs). Spleens were mashed with frosted-end glass slides to create
a cell suspension. LNs were teased apart with forceps and passed
through a cell strainer to remove debris. Pooled splenocytes and LN
cells were separated into CD8+ and CD4+ subsets using two
successive MACS magnetic separation columns, according to the
manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.).
Whole thymocytes were collected from the same mice.
[0566] Cells were cultured at 3.times.10.sup.5 cells/well
(thymocytes) or 10.sup.5 cells/well (mature T cells) with
increasing concentrations of purified murine zalpha11 Ligand (0-30
ng/ml) (Example 24 and Example 29) in 96-well flat bottomed plates
pre-coated overnight at 4.degree. C. with various concentrations of
anti-CD3 mAb 2C11 (PharMingen) for 3 days at 37.degree. C. The
anti-CD3 antibody served to activate the murine T-cells through the
T-cell receptor. Each well was pulsed with 1.mu. Ci
.sup.3H-thymidine on day 2 and plates were harvested and counted 16
hours later to assess proliferation.
[0567] When we tested zalpha11 Ligand in T cell proliferation
assays, we found that it co-stimulated anti-CD3-activated murine
thymocytes, leading to an accelerated outgrowth of CD8+ CD4- cells
(the majority of the thymocytes cultured with anti-CD3+ zalpha11
Ligand were CD8+ CD4- by day 3 of culture, while cells cultured
with anti-CD3 alone did not significantly skew to this phenotype
until day 5). We did not observe significant levels of
proliferation of thymocytes to zalpha11 Ligand in the absence of
anti-CD3.
[0568] Interestingly, when we assayed mature peripheral murine T
cells for their ability to respond to zalpha11 Ligand+anti-CD3, we
found that only the CD8+, but not the CD4+ subset, responded in a
dose-dependent manner to zalpha11 Ligand. We also observed weak but
reproducible proliferation of CD8+ cells (but not CD4+ cells) in
response to zalpha11 Ligand alone. Interestingly, this was not
observed for human T cells (see Example 42B, below).
[0569] B. Human Zalpha11 Ligand Proliferation of Human T-Cells
[0570] Human CD4+ and CD8+ T cells were isolated from PBMC as
described in Example 43 (below) Cells were cultured at about 105
cells/well with increasing concentrations of purified human
zalpha11 Ligand (0-50 ng/ml) (Example 24) in 96-well flat bottomed
plates pre-coated overnight at 4.degree. C. with various
concentrations of anti-human CD3 mAb UCHT1 (PharMingen) for 3 days
at 37.degree. C. Each well was pulsed with 1 uCi 3H-thymidine on
day 2 and plates were harvested and counted 16 hours later. Unlike
our results with mouse T cells, our preliminary data suggests that
human zalpha11 Ligand co-stimulates CD4+, but not CD8+, human T
cells in a dose-dependent fashion.
Example 43
[0571] Real Time PCR Shows Zalpha11 Ligand Expression in Human CD4+
Cells
[0572] A. Purified Human T Cells as a Primary Source Used to Assess
Human zalpha11 Ligand Expression
[0573] Whole blood (150 ml) was collected from a healthy human
donor and mixed 1:1 with PBS in 50 ml conical tubes. Thirty ml of
diluted blood was then underlayed with 15 ml of Ficoll Paque Plus
(Amersham Pharmacia Biotech, Uppsala, Sweden). These gradients were
centrifuged 30 min at 500 g and allowed to stop without braking.
The RBC-depleted cells at the interface (PBMC) were collected and
washed 3 times with PBS. The isolated human PBMC yield was
200.times.10.sup.6 prior to selection described below.
[0574] The PBMCs were suspended in 1.5 ml MACS buffer (PBS, 0.5%
EDTA, 2 mM EDTA) and 3.times.10.sup.6 cells were set aside for
control RNA and for flow cytometric analysis. We next added 0.25 ml
anti-human CD8 microbeads (Miltenyi Biotec) and the mixture was
incubated for 15 min at 4.degree. C. These cells labeled with CD8
beads were washed with 30 ml MACS buffer, and then resuspended in 2
ml MACS buffer.
[0575] A VS+ column (Miltenyi) was prepared according to the
manufacturer's instructions. The VS+ column was then placed in a
VarioMACS magnetic field (Miltenyi). The column was equilibrated
with 5 ml MACS buffer. The isolated primary mouse cells were then
applied to the column. The CD8 negative cells were allowed to pass
through. The column was rinsed with 9 ml (3.times.3 ml) MACS
buffer. The column was then removed from the magnet and placed over
a 15 ml falcon tube. CD8+ cells were eluted by adding 5 ml MACS
buffer to the column and bound cells flushed out using the plunger
provided by the manufacturer. The yield of CD8+ selected human
peripheral T cells was about 51.times.106 total cells. The
CD8-negative flow through cells were collected, counted, stained
with anti-human CD4 coated beads, then incubated and passed over a
new VS+column at the same concentrations as described above. The
yield of CD4+ selected human peripheral T cells was
42.times.10.sup.6 total cells.
[0576] A sample of each of the CD8+ and CD4+ selected human T cells
was removed for staining and sorting on a fluorescence activated
cell sorter (FACS) to assess their purity. A PE-conjugated
anti-human CD4 antibody, an anti-human CD8-FITC Ab, and an
anti-human CD19-CyChrome Ab (all from PharMingen) were used for
staining the CD8+ and CD4+ selected cells. The CD8-selected cells
in this first experiment were 80% CD8+, and the CD4-selected cells
were 85% CD4+. In 2 subsequent experiments (Example 43B), the CD8+
purified cells were 84% and 81% pure, and the CD4+ cells were 85%
and 97% pure, respectively. In one experiment, we stained the
non-binding (flow-through) cells with anti-human CD19-coated beads
(Miltenyi) and ran them over a third magnetic bead column to
isolate CD19+B cells (these were 92% pure).
[0577] The human CD8+, CD4+ and CD19+ selected cells were activated
by incubating 0.5.times.106 cells/ml in RPMI+5% human ultraserum
(Gemini Bioproducts, Calabasas, Calif.)+PMA 10 ng/ml and Ionomycin
0.5 .mu.g/ml (Calbiochem) for about 4, 16, or 24 hours at
37.degree. C. The T-cells (2.5.times.10.sup.6/well) were
alternately stimulated in 24-well plates pre-coated overnight with
0.5 .mu.g/ml plate-bound anti-CD3 mAb UCHT1 (PharMingen) with or
without soluble anti-CD28 mAb (PharMingen) at 5 .mu.g/ml. At each
timepoint, the cells were harvested, pelleted, washed once with
PBS, and pelleted again. The supernatant was removed and the
pellets were snap-frozen in a dry ice/ethanol bath, then stored at
-80.degree. C. for RNA preparation at a later date.
[0578] Real Time-PCR was performed on these human CD8+, CD4+ and
CD19+ selected cells as described in Example 43B and 43C below for
assessing human zalpha11 Ligand and receptor expression.
[0579] B. Primers and Probes for Quantitative RT-PCR for Human
zalpha11 Ligand Expression
[0580] Real-time quantitative RT-PCR using the ABI PRISM 7700
Sequence Detection System (PE Applied Biosystems, Inc., Foster
City, Calif.) has been previously described (see, Heid, C A et al.,
Genome Research 6:986-994, 1996; Gibson, U E M et al., Genome
Research 6: 995-1001, 1996; and Sundaresan, S et al., Endocrinology
139:4756-4764, 1998). This method incorporates use of a gene
specific probe containing both reporter and quencher dyes. When the
probe is intact the reporter dye emission is negated due to the
proximity of the quencher dye. During PCR extension using
additional gene-specific forward and reverse primers, the probe is
cleaved by 5' nuclease activity of Taq polymerase which releases
the reporter dye resulting in an increase in fluorescent
emission.
[0581] The primers and probes used for real-time quantitative
RT-PCR analyses were designed using the primer design software
Primer Express.TM. (PE Applied Biosystems). Primers for human
zalpha11 Ligand were designed spanning an intron-exon junction to
eliminate amplification of genomic DNA. The forward primer,
ZC22,281 (SEQ ID NO:90) and the reverse primer, ZC22,279 (SEQ ID
NO:91) were both used at 300 nM concentration to synthesize an 80
bp product. The corresponding zalpha11 Ligand TaqMan probe, ZG32
(SEQ ID NO:92) was synthesized by PE Applied Biosystems. The probe
was labeled with a reporter fluorescent dye (6-carboxy-fluorescein)
(FAM) (PE Applied Biosystems) at the 5' end and a quencher
fluorescent dye (6-carboxy-tetramethyl-rhodamine) (TAMRA) (PE
Applied Biosystems) at the 3' end. In order to test the integrity
or quality of all the RNA samples, they were screened for rRNA
using the primer and probe set ordered from PE Applied Biosystems
(cat No. 4304483). The reporter fluorescent dye for this probe is
VIC (PE Applied Biosystems). The rRNA results will allow for the
normalization of the zalpha11 Ligand results.
[0582] RNA was prepared from pellets provided in Example 43A, using
RNeasy Miniprep* Kit (Qiagen, Valencia, Calif.) per the
manufacturer's instructions. Control RNA was prepared from about 10
million BHK cells expressing human zalpha11 Ligand.
[0583] C. Primers and Probes for Quantitative RT-PCR for Human
zalpha11 Receptor Expression
[0584] Real time PCR was performed to assess the expression of
zalpha11 receptor as per Example 43B and Example 43D, using the
cells prepared under the conditions detailed in 43A, and probes
specific for the zalpha11 receptor. The forward primer, ZC22,277
(SEQ ID NO:93) and the reverse primer, ZC22,276 (SEQ ID NO:94) were
used in a PCR reaction (above) at about 300 nM concentration to
synthesize a 143 bp product. The corresponding zalpha11 TaqMan.RTM.
probe, designated ZG31 (SEQ ID NO:95) was synthesized and labeled
by PE Applied Biosystems. RNA from BaF3 cells expressing human
zalpha11 receptor was used to generate appropriate control for
standard curves for the real-time PCR described in Example 43D
below.
[0585] D. Real-Time Quantitative RT-PCR
[0586] Relative levels of zalpha11 Ligand RNA were determined by
analysis of total RNA samples using the One-Step RT-PCR method (PE
Applied Biosystems). RNA from BHK cells expressing human zalpha11
Ligand was used to generate a standard curve. The curve consisted
of serial dilutions ranging from 2.5-2.5.times.10-4 ng for the rRNA
screen and 25-0.0025 ng for the zalpha11 Ligand screen with each
point analyzed in triplicate. The total RNA samples were also
analyzed in triplicate for human zalpha11 Ligand transcript levels
and for levels of rRNA as an endogenous control. Each One-step
RT-PCR reaction consisted of 25 ng of total RNA in buffer A (50 mM
KCL, 10 mM Tris-HCL, and the internal standard dye, ROX (PE Applied
Biosystems)), appropriate primers (50 nM for rRNA samples, 300 nM
for zalpha11 Ligand samples) and probe (50 nM for rRNA, 100 nM for
zalpha11 Ligand), 5.5 mM MgCl2, 300 .mu.M each d-CTP, d-ATP, and
d-GTP and 600 .mu.M of d-UTP, reverse transcriptase (0.25 U/.mu.l),
AmpliTaq DNA polymerase (0.025 U/.mu.l) and RNase Inhibitor (0.4
U/.mu.l) in a total volume of 25 .mu.l. Thermal cycling conditions
consisted of an initial RT step at 48.degree. C. for 30 minutes, an
AmpliTaq Gold activation step of 95.degree. C. for 10 minutes,
followed by 40 cycles of amplification for 15 seconds at 95.degree.
C. and 1 minute at 60.degree. C. Relative zalpha11 Ligand RNA
levels were determined by the Standard Curve Method as described in
User Bulletin No. 2 (PE Biosystems; User Bulletin #2: ABI Prism
7700 Sequence Detection System, Relative Quantitation of Gene
Expression, Dec. 11, 1997) using the rRNA measurements to normalize
the zalpha11 Ligand levels. Samples were compared relative to the
calibrator within each experiment. The calibrator was arbitrarily
chosen based on good quality RNA and an expression level to which
other samples could significantly be compared. Results of the
experiments analyzing the expression of the zalpha11 Ligand and
zalpha receptor in stimulated and unstimulated cells (Example 43A)
are as described in Example 43E below.
[0587] E. Expression of Human zalpha11 Receptor and Ligand in CD4+,
CD8+ and CD19+Cells
[0588] The first experiment used RT-PCR, described above, to assess
zalpha11 receptor expression in unstimulated and anti-CD3
stimulated CD4+ and CD8+ samples at timepoints of Oh (unstimulated
("resting") cells), and at 4 h, 15.5 h and 24 h, after stimulatoin.
The resting CD4+ sample was arbitrarily chosen as the calibrator
and given a value of 1.00. There was approximately a 4-fold
increase in receptor expression in unstimulated CD4+ cells from 4 h
to 24 h of culture and about an 8-fold increase over the same time
period in anti-CD3 stimulated CD4+ cells. The CD8+ cells showed a
7-fold increase in zalpha11 receptor expression that peaked at 4
hrs and decreased over time. With anti-CD3 stimulation, the CD8+
cells had a constant 8-fold increase in receptor expression.
[0589] This first experiment also used RT-PCR to assess zalpha11
Ligand expression in the same anti-CD3 stimulated and unstimulated
CD4+ and CD8+ samples. The 4 hr anti-CD3 stimulated CD8+ sample was
arbitrarily chosen as the calibrator and given a value of 1.00. The
results showed that unstimulated CD4+ and CD8+ cells do not express
zalpha11 Ligand. We observed a significant elevation of expression
in the anti-CD3 stimulated CD4+ cells at 4 h, with about a 300-fold
increase in signal observed at 15.5 h. The CD8+ cells expressed a
small amount of ligand upon anti-CD3 stimulation, however this is
probably due to contamination of the CD8+ population with a small
number of CD4+ cells.
[0590] The second experiment used RT-PCR to assess zalpha11
receptor expression in anti-CD3-stimulated,
PMA+Ionomycin-stimulated and unstimulated CD4+ and CD8+ samples at
timepoints of 0 h, and at 3.5 h, 16 h and 24 h after activation.
The resting CD8+ sample was arbitrarily chosen as the calibrator
and given a value of 1.00. The resting CD4+ and CD8+ cells did not
have significant amounts of receptor expression. The expression was
about 3 fold higher in the PMA+Ionomycin-stimulated CD4+ samples at
3.5 h, 16 h and 24 h after stimulation. The expression in anti-CD3
activated CD4+ cells peaked at 10-fold above background levels at
3.5 h after stimulation, then fell back to levels 4-fold above
background at 16 h after stimulation. The CD8+ cells showed a
4-fold expression increase at 3.5 h after PMA+Ionomycin
stimulation, with expression decreasing at subsequent timepoints.
As in the first experiment, the anti-CD3 stimulated CD8+ cells
again exhibited an 8-fold above background induction of receptor
expression.
[0591] These samples from the second experiment were also used to
assess zalpha11 Ligand expression. The 24 hr PMA+Ionomycin
stimulated CD4+ sample was arbitrarily chosen as the calibrator and
given a value of 1.00. The results showed that again none of the
unstimulated cells expressed zalpha11 Ligand. There was about a
30-fold induction of ligand expression in the CD4+ cells stimulated
with anti-CD3 at 3.5 h, as seen in the previous experiment (at 4
h). However, there was only about a 5-fold induction with
PMA+Ionomycin stimulation at 3.5 h that went down at subsequent
timepoints. Again, the CD8+ cells expressed a very small amount of
Ligand that was probably attributed to contaminating CD4+
cells.
[0592] The final experiment used RT-PCR to assess zalpha11 receptor
expression in anti-CD3- and anti-CD3/anti-CD28-stimulated and
unstimulated CD4+ and CD8+ samples at timepoints of 0 h, and at 2
h, 4 h, and 16 h after stimulation. CD19+ cells activated with
PMA+Ionomycin were also screened for receptor expression at the
same time intervals. The resting CD4+ sample was arbitrarily chosen
as the calibrator and given a value of 1.00. The 2 h anti-CD3
stimulated CD4+ cells only had a 4-fold induction of receptor,
compared to the 10-fold induction seen at 3.5 h in the previous
experiment. The combination of anti-CD3 and anti-CD28 increased
zalpha11 receptor expression to 8-fold above background. The 16 h
anti-CD3/anti-CD28 stimulated CD8+ cells had very low zalpha11
receptor expression levels, as seen in the CD8+ cells in previous
experiments (above). The CD19+ cells stimulated with PMA+Ionomycin
had the most significant zalpha11receptor expression with a 19-fold
increase at 2 h, but the expression levels decreased back to those
of resting cells by 16 h.
[0593] These samples from the final experiment were also used to
assess zalpha11 Ligand by RT-PCR. The 16 h anti-CD3/anti-CD28
stimulated CD8+ sample was arbitrarily chosen as the calibrator and
given a value of 1.00. The results showed that at 2 h the CD4+
cells had about a 2-fold induction of zalpha11 Ligand expression
with anti-CD3 stimulation and a 5-fold induction with anti-CD3 plus
anti-CD28 stimulation. These stimulation conditions induced Ligand
expression over time, with the 16 h stimulated CD4+ cells
exhibiting Ligand expression levels 70-fold above background. CD8+
and CD19+ cells showed no zalpha11 Ligand expression.
[0594] A certain amount of variation was expected between blood
draws (i.e. multiple samples at different times from the same
patient and between multiple patients). Therefore, data trends were
analyzed within each study or from a single blood sample and the
three experiments above were compared for an overall conclusion.
The trend from the Real Time PCR experiments described above is
that of all the cell types tested, CD19+B cells activated with PMA+
ionomycin expressed the highest levels of zalpha11 receptor RNA.
CD4+ and CD8+ cells can also be stimulated to express receptor, but
at lower levels than in B cells. Zalpha11 Ligand was expressed
almost exclusively in stimulated CD4+ T cells (and not by CD8+ T
cells or CD19+B cells). Although stimulation with PMA+Ionomycin
induced a good zalpha11 Ligand signal in this assay, a
significantly higher signal was obtained from CD4+ T cells
stimulated with anti-CD3 mAb or a combination of anti-CD3 and
anti-CD28 mAbs, conditions that better mimic an antigen encounter
in vivo.
Example 44
[0595] Zalpha11 Ligand-Dependent Proliferation of B-Cell Cells
Stimulated Anti-CD40 or Anti-IgM
[0596] A. Purification of Human B Cells
[0597] A vial containing 1.times.10.sup.8 frozen, apheresed human
peripheral blood mononuclear cells (PBMCs) was quickly thawed in a
37.degree. C. water bath and resuspended in 25 ml B cell medium
(RPMI Medium 1640 (JRH Biosciences. Lenexa, Kans.), 10% Heat
inactivated fetal bovine serum, 5% L-glutamine, 5% Pen/Strep)
(Gibco BRL)) in a 50 ml tube (Falcon VWR, Seattle, Wash.). Cells
were tested for viability using Trypan Blue (Gibco BRL). Ten
milliliters of Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) was layered under the cell suspension and
spun for 30 minutes at 1800 rpm and allowed to stop with the brake
off. The interface was then removed and transferred to a fresh 50
ml Falcon tube, brought up to a final volume of 40 ml with PBS and
spun for 10 minutes at 1200 rpm with the brake on. The viability of
the isolated cells was again tested using Trypan Blue. Alternately
fresh drawn human blood was diluted 1:1 with PBS (Gibco BRL) and
layered over Ficoll/Hypaque Plus (Pharmacia), spun and washed as
above. Cells isolated from either fresh or frozen sources gave
equivalent results.
[0598] B cells were purified from the Ficoll floated peripheral
blood cells of normal human donors (above) with anti-CD19 magnetic
beads (Miltenyi Biotec, Auburn, Calif.) following the
manufacturer's instructions. The purity of the resulting
preparations was monitored by flow cytometric analysis with
anti-CD22 FITC Ab (Pharmingen, San Diego, Calif.). B cell
preparations were typically >90% pure.
[0599] B. Purification of Murine B Cells
[0600] A suspension of murine splenocytes was prepared by teasing
adult C57B1/6 mouse (Charles River Laboratories, Wilmington, Mass.)
spleens apart with bent needles in B cell medium. RBCs were removed
by hypotonic lysis. CD43 positive cells were removed with CD43
magnetic beads (Miltenyi Biotec) following the manufacturer's
instructions. The purity of the resulting preparations was
monitored by flow cytometric analysis with anti-CD45R FITC Ab
(Pharmingen). B cell preparations were typically >90% pure.
[0601] C. Proliferation of Anti-CD40-Stimulated B-Cells in the
Presence of Human or Murine zalpha11 Ligand
[0602] The B cells from either the human or mouse source were
resuspended at a final concentration of 1.times.10.sup.6 cells/ml
in B cell medium and plated at 100 .mu.l/well in a 96 well U bottom
plate (Falcon, VWR) containing various stimulation conditions to
bring the final volume to 200 .mu.l/well. For anti-CD40 stimulation
human cultures were supplemented with 1 .mu.g/ml anti-human CD40
(Genzyme, Cambridge, Mass.) and mouse cultures were supplemented
with 1 .mu.g/ml anti-murine CD40 (Serotec, UK). Human or murine
zalpha11 Ligand was added at dilutions ranging from 1 pg/ml-100
ng/ml. The specificity of the effect of zalpha11 Ligand was
confirmed by inhibition of zalpha11 Ligand with 25 mg/ml soluble
human zalpha11CEE (Example 10A). All treatments were performed in
triplicate. The cells were then incubated at 37.degree. C. in a
humidified incubator for 120 hours (human) or 72 hours (mouse).
Sixteen hours prior to harvesting, 1 .mu.Ci 3H-thymidine (Amersham,
Piscataway, N.J.) was added to all wells to assess whether the
B-cells had proliferated. The cells were harvested into a 96 well
filter plate (UniFilter GF/C, Packard, Meriden, Conn.) using a cell
harvester (Packard) and collected according to manufacturer's
instructions. The plates were dried at 55.degree. C. for 20-30
minutes and the bottom of the wells were sealed with an opaque
plate sealer. To each well was added 0.25 ml of scintillation fluid
(Microscint-O, Packard) and the plate was read using a TopCount
Microplate Scintillation Counter (Packard).
[0603] Incubation with Zalpha11 Ligand at concentrations of 3 ng/ml
or more enhanced the proliferation induced by soluble anti-CD40 in
a dose dependent manner in both murine and human B cells by as much
as 30 fold. The murine and human B cells responded equally as well
to their respective zalpha11 Ligand. In both species, the
stimulation was specific to zalpha11 Ligand, as it was reversed by
the presence of soluble zalpha11 receptor in the culture.
[0604] D. Proliferation of Anti-IgM-stimulated B-Cells in the
Presence of Human or Murine zalpha11 Ligand
[0605] The B cells from either human or mouse source as described
above (Example 44A and Example 44B) were plated as described above
(Example 44C). For anti-IgM stimulation of human cells the plates
were pre-coated overnight with 10 mg/ml F(ab')2 anti-human IgM Abs
(Southern Biotech Associates, Birmingham, Ala.) and washed with
sterile media just prior to use. The cultures were supplemented
with 0-10 ng/ml hu rIL-4 (R&D Systems, Minneapolis, Minn.). For
anti-IgM stimulation of murine cells soluble anti-IgM (Biosource,
Camarillo, Calif.) was added to the cultures at 10 mg/ml. To each
of the preceding anti-IgM/IL-4 conditions, human or murine Zalpha11
ligand was added at dilutions ranging from 1 pg/ml-100 ng/ml as
described above. The specificity of the effect of zalpha11 Ligand
was confirmed by inhibition with soluble human zalpha11 receptor as
described above (Example 44C). All treatments were performed in
triplicate. The cells were incubated, labeled with 3H-thymidine,
harvested, and analyzed as described in Example 44C.
[0606] Incubation with Zalpha11 ligand at concentrations of 0.3
ng/ml or more inhibited the proliferation induced by insoluble
anti-IgM (mouse) or anti-IgM and IL-4 (human) in a dose-dependent
manner. This inhibition was specific to zalpha11 Ligand, as it was
reversed by the presence of soluble zalpha11 receptor in the
culture.
Example 45
[0607] Expression of Human zalpha11 Soluble Receptor in E. coli
[0608] A. Construction of Expression Vector pCZR225 that Expresses
huzalpha11/MBP-6H Fusion Polypeptide
[0609] An expression plasmid containing a polynucleotide encoding a
human zalpha11 soluble receptor fused C-terminally to maltose
binding protein (MBP) was constructed via homologous recombination.
A fragment of human zalpha11 cDNA (SEQ ID NO:7) was isolated using
PCR. The polynucleotide sequence for the MBP-zalpha11 soluble
receptor fusion polypeptide is shown in SEQ ID NO:96. Two primers
were used in the production of the human zalpha11 fragment in a PCR
reaction: (1) Primer ZC20,187 (SEQ ID NO:98), containing 40 bp of
the vector flanking sequence and 25 bp corresponding to the amino
terminus of the human zalpha11, and (2) primer ZC20,185 (SEQ ID
NO:99), containing 40 bp of the 3' end corresponding to the
flanking vector sequence and 25 bp corresponding to the carboxyl
terminus of the human zalpha11. The PCR Reaction conditions were as
follows: 25 cycles of 94.degree. C. for 30 seconds, 50.degree. C.
for 30 seconds, and 72.degree. C. for 1 minute; followed by
4.degree. C. soak, run in duplicate. Two .mu.l of the 100 .mu.l PCR
reaction was run on a 1.0% agarose gel with 1.times.TBE buffer for
analysis, and the expected approximately 660 bp fragment was seen.
The remaining 90 .mu.l of PCR reaction was combined with the second
PCR tube precipitated with 400 .mu.l of absolute ethanol. The
precipitated DNA used for recombining into the Sma1 cut recipient
vector pTAP98 (Example 31) to produce the construct encoding the
MBP-zalpha11 fusion. Clones were transformed, identified and grown
up as described in Example 31. The positive clones were designated
pCZR225 and subjected to sequence analysis. The polynucleotide
sequence for the MBP-zalpha11 soluble receptor fusion polypeptide
is shown in SEQ ID NO:96, and corresponding polypeptide sequence is
shown in SEQ ID NO:97. The positive clones were used to grow up in
E. coli as described in Example 31 for protein purification of the
huzalpha11/MBP-6H fusion protein (Example 46, below).
Example 46
[0610] Purification of huzalpha11/MBP-6H Soluble Receptor from E.
coli Fermentation
[0611] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying
huzalpha11/MBP-6H soluble receptor polypeptide. E. coli cells
containing the pCZR225 construct and expressing huzalpha11/MBP-6H
soluble receptor (Example 45) were grown up in SuperBroth 11 (12
g/L Casein, 24 g/L Yeast Extract, 11.4 g/L di-potassium phosphate,
1.7 g/L Mono-potassium phosphate; Becton Dickenson, Cockeysville,
Md.), and frozen in 0.5% glycerol. Twenty grams of the frozen cells
in SuperBroth II+Glycerol were used to purify the protein. The
frozen cells were thawed and diluted 1:10 in a protease inhibitor
solution (Extraction buffer) prior to lysing the cells and
releasing the huzalpha11/MBP-6H soluble receptor protein. The
diluted cells contained final concentrations of 20 mM Tris (JT
Baker, Philipsburg, N.J.) 100 mM Sodium Chloride (NaCl,
Mallinkrodt, Paris, Ky.), 0.5 mM pheynlmethylsulfonyl fluoride
(PMSF, Sigma Chemical Co., St. Louis, Mo.), 2 .mu.g/ml Leupeptin
(Fluka, Switzerland), and 2 .mu.g/ml Aprotinin (Sigma). A French
Press cell breaking system (Constant Systems Ltd., Warwick, UK)
with temperature of -7 to -10.degree. C. and 30K PSI was used to
lyse the cells. The diluted cells were checked for breakage by A600
readings before and after the French Press. The lysed cells were
centrifuged at 18,000G for 45 minutes to remove the broken cell
debris, and the supernatant used to purify the protein. Total
target protein concentrations of the supernatant was determined via
BCA Protein Assay (Pierce, Rockford, Ill.), according to
manufacturer's instructions.
[0612] A 25 ml column of Talon Metal Affinity resin (Clontech, Palo
Alto, Calif.) (prepared as described below) was poured in a
Bio-Rad, 2.5 cm D.times.10 cm H glass column. The column was packed
and equilibrated by gravity with 10 column volumes (CVs) of Talon
Equilibration buffer (20 mM Tris, 100 mM NaCl, pH 8.0). The
supernatant was batch loaded to Talon metal affinity resin and was
rocked overnight. The resin was poured back into the column and was
washed with 10 CV's of Talon Equilibration buffer by gravity, then
gravity eluted with 140 ml of Elution buffer (Talon Equilibration
buffer+200 mM Imidazole-Fluka Chemical). The talon column was
cleaned with 5 CVs of 20 mM 2-(N-Morhpholino) ethanesulfonic acid
pH 5.0 (MES, Sigma), 5 CVs of distilled H.sub.2O, then stored in
20% Ethanol/0.1% Sodium Azide. Fourteen ml fractions were collected
over the entire elution chromatography and the fractions were read
with absorbance at 280 and 320 nM and BCA protein assay; the pass
through and wash pools were also saved and analyzed. The protein
elution fractions of interest were pooled and loaded straight to
Amylose resin (New England Biolabs, Beverly, Mass.).
[0613] To obtain more pure huzalpha11/MBP-6H polypeptide, the talon
affinity elution pooled fractions were subjected to Amylose resin
(22 mls) at pH 7.4. A 2.5 cm D.times.10 cm H Bio-Rad column was
poured, packed and equilibrated in 10 CVs of Amylose equilibration
buffer-20 mM Tris (JT Baker), 100 mM NaCl (Mallinkrodt), 1 mM PMSF
(Sigma), 10 mM beta-Mercaptoethanol (BME, ICN Biomedicals Inc.,
Aurora, Ohio) pH 7.4. The sample was loaded by gravity flow rate of
0.5 ml/min. The column was washed for 10 CVs with Amylose
equilibration buffer, then eluted with about 2 CV of Amylose
equilibration buffer+10 mM Maltose (Fluka Biochemical, Switzerland)
by gravity. 5 ml fractions were collected over the entire
chromatography and absorbance at 280 and 320 nM were read. The
Amylose column was regenerated with 1 CV of distilled H.sub.2O, 5
CVs of 0.1% (w/v) SDS (Sigma), 5 CVs of distilled H.sub.2O, and
then 5 CVs of Amylose equilibration buffer.
[0614] Fractions of interest were pooled and dialyzed in a
Slide-A-Lyzer (Pierce) with 4.times.4L PBS pH 7.4 (Sigma) to remove
low molecular weight contaminants, buffer exchange and desalt.
After the changes of PBS, the material harvested represented the
purified huzalpha11/MBP-6H polypeptide. The purified
huzalpha11/MBP-6H polypeptide was analyzed via SDS-PAGE Coomassie
staining and Western blot analysis with the anti-rabbit HRP
conjugated antibody (Rockland, Gilbertsville, Pa.). The
concentration of the huzalpha11/MBP-6H polypeptide was 1.92 mg/ml
as determined by BCA analysis.
[0615] Purified huzalpha11/MBP-6H polypeptide was prepared for
injection into rabbits and sent to R & R Research and
Development (Stanwood, Wash.) for antibody production. Rabbits were
injected to produce anti anti-huzalpha11/MBP-6H serum (Example 47,
below).
Example 47
[0616] Zalpha11 Receptor Polyclonal Antibodies
[0617] Polyclonal antibodies were prepared by immunizing two female
New Zealand white rabbits with the purified huzalpha11/MBP-6H
polypeptide (Example 46), or the purified recombinant zalpha11CEE
soluble receptor (Example 110A). Corresponding polyclonal
antibodies were designated rabbit anti-huzalpha11/MBP-6H and rabbit
anti-huzalpha11-CEE-BHK respectively. The rabbits were each given
an initial intraperitoneal (IP) injection of 200 mg of purified
protein in Complete Freund's Adjuvant (Pierce, Rockford, Ill.)
followed by booster IP injections of 100 mg purified protein in
Incomplete Freund's Adjuvant every three weeks. Seven to ten days
after the administration of the third booster injection, the
animals were bled and the serum was collected. The rabbits were
then boosted and bled every three weeks.
[0618] The zalpha11-specific polyclonal antibodies were affinity
purified from the rabbit serum using an CNBr-SEPHAROSE 4B protein
column (Pharmacia LKB) that was prepared using 10 mg of the
purified huzalpha11/MBP-6H polypeptide (Example 32) per gram
CNBr-SEPHAROSE, followed by 20.times. dialysis in PBS overnight.
Zalpha11-specific antibodies were characterized by an ELISA titer
check using 1 mg/ml of the appropriate protein antigen as an
antibody target. The lower limit of detection (LLD) of the rabbit
anti-huzalpha11/MBP-6H affinity purified antibody is a dilution of
500 pg/ml. The LLD of the rabbit anti-huzalpha11-CEE-BHK affinity
purified antibody is a dilution of 50 pg/ml.
Example 48
[0619] Zalpha11 Receptor Distribution
[0620] To assess zalpha11 receptor distribution on various cells
types, we generated both rabbit polyclonal and mouse monoclonal
antibodies (mAbs) directed against the human receptor (Example 35
and Example 47) and conjugated these antibodies to biotin for use
in flow cytometry. We initially used the polyclonal antibodies,
which were of relatively low affinity, to stain a panel of cell
lines: IL-3 dependent murine pre-B cell line wild-type BaF3 cells
(Palacios and Steinmetz, ibid.; Mathey-Prevot et al., ibid.); BaF3
cells transfected with human zalpha11 (Example 4); human Burkitt's
lymphoma cell lines Raji (ATCC No. CCL-86), Ramos (ATCC No.
CRL-1596), RPMI 8226 (ATCC No. CCL-155), and Daudi (ATCC No.
CCL-213); human T cell leukemia cell line Jurkat (ATCC No.
TIB-152); human myelomonocytic leukemia cell lines Thp-1 (ATCC No.
TIB-202) and UT937 (ATCC No. CRL-1593.2); human pro-myelomonocytic
cells HLT60 (ATCC No. CCL-240); murine B cell lymphoma cell line
A20 (ATCC No TIB-208); and murine thymoma cell line EL4 (ATCC No.
TIB-39).
[0621] The cells were harvested, washed once with FACS wash buffer
with serum (WBS). WBS consisted of Hank's balanced salt solution
(Gibco/BRL)+10 mM HEPES (Gibco/BRL)+1% BSA (Sigma)+10% normal goat
serum (Gemini Bioproducts, Woodland, Calif.)+10% normal rabbit
serum (Sigma). Wash buffer (WB) was identical to WBS except that it
was serum free. After washing, the cells were resuspended in 100
.mu.l WB containing 10 .mu.g/ml rabbit anti-zalpha11 polyclonal
antibodies (Example 47). The cells were kept on ice with Ab for 20
min, then washed with WB and resuspended in WB containing goat
anti-rabbit-FITC (BioSource, International), incubated another 20
min on ice, then washed and resuspended in 400 .mu.l WB for
analysis on a FACSCalibur flow cytometer (Becton Dickinson).
Control samples were stained with the secondary goat
anti-rabbit-FITC Ab only. Positive staining was defined as a shift
above the staining with secondary alone. Although the polyclonal
antibodies were of low affinity, we confidently detected zalpha11
expression on the BaF3/zalpha11 transfectant, on all four human
Burkitt's lymphomas (Raji, Ramos, Daudi, and RPMI 8226), and on
Jurkat T cells. Resting (undifferentiated) HL-60 cells did not bind
the anti-zalpha11 antibodies, but we did detect a positive signal
on HL-60 cells activated for 24 hours with PMA (Calbiochem, La
Jolla, Calif.) which induces HL-60 cell differentiation into a
monocyte-like cell. We also saw a positive signal on UT937 and
Thp-1 cells, although this signal may have been due to non-specific
binding. The polyclonal antibodies weakly cross-reacted on the
mouse B cell line A20, but we saw no staining of the EL4 murine
thymoma.
[0622] The four anti-zalpha11 monoclonal antibodies (Example 35)
were conjugated to biotin, and a subset of the cells described
above were screened for zalpha11 receptor expression (BaF3,
BaF3/zalpha11, Raji, Jurkat, and resting HL-60). Cells were
harvested, washed, then resuspended in 100 .mu.l WB containing 15
.mu.g/ml of one of each of the 4 biotinylated mAbs. The cells were
incubated with mAb for 20 min on ice, then washed with 1.5 ml WB
and pelleted in a centrifuge. The supernatant was removed by
aspiration and the pellets were resuspended in 100 .mu.l of
CyChrome-conjugated streptavidin (CyC-SA; PharMingen), then
incubated on ice for another 20 min and washed and pelleted as
before. Control tubes contained cells stained only with CyC-SA.
Pellets were resuspended in 400 .mu.l WB and flow cytometry
performed as above. Positive staining was defined as a signal
exceeding the background level of staining with CyC-SA alone. Using
the BaF3/zalpha11 transfectant as a control, we were able to rank
the 4 mAbs in terms of their respective mean fluorescence
intensities (MFI), which can reflect antibody affinity and/or the
extent of biotinylation of the mAbs. The mAbs were ranked as
follows, from highest to lowest MFI: 249.28.2.1.2.2,
247.10.2.15.4.6, 249.19.2.2.3.5, and 249.15.2.4.2.7. The Raji cells
stained positive with the zalpha11 monoclonal antibodies. The
Jurkats cells positively stained with the zalpha11 monoclonal
antibodies, but not as strongly as that on B cells (Raji). Thus
the, zalpha11 receptor was expressed on these B and T cell lines.
The staining patterns on non-activated HL60 cells were identical
for all the mAbs, and the signal was very weak. We believe that
this signal does not reflect actual expression of zalpha11 by HL-60
cells, but rather may be due to non-specific binding of the mouse
mAbs to the human cells, probably via Fc-receptors.
Example 49
[0623] Human zalpha11 Ligand Effect on B-Cells and Zalpha11 Ligand
Toxic Saporin Fusion
[0624] The effects of human zalpha11 Ligand were tested on the
following human B-cell lines: and human Burkitt's lymphoma cell
lines Raji (ATCC No. CCL-86), and Ramos (ATCC No. CRL-1596); human
EBV B-cell lymphoma cell line RPMI 1788 (ATCC No. CRL-156); human
myeloma/plasmacytoma cell line IM-9 (ATCC No. CRL159); and human
EBV transformed B-cell line DAKIKI (ATCC No. TIB-206), and HS
Sultan cells (ATCC No. CRL-1484). Following about 2-5 days
treatment with zalpha11 Ligand, changes in surface marker
expression were found in IM-9, Raji, Ramos, and RPM11788 cell
lines, showing that these cells can respond to zalpha11 Ligand.
Human B-cell lines treated with zalpha11 Ligand grew much more
slowly than untreated cells when re-plated in cell culture dishes.
These cells also had an increased expression of FAS ligand, as
assessed by flow cytometry (Example 49D and Example 49E), and
moderately increased sensitivity to an activating FAS antibody
(Example 49A). This results indicate that zalpha11 Ligand could
control some types of B-cell neoplasms by inducing them to
differentiate to a less proliferative and or more FAS ligand
sensitive state. Moreover, zalpha11 receptor is expressed on the
surface of several of these cell lines (See Example 48). Thus,
zalpha11 Ligand and the human zalpha11 Ligand-saporin immunotoxin
conjugate (Example 49B, below), or other zalpha11 Ligand-toxin
fusion could be therapeutically used in B-cell leukemias and
lymphomas.
[0625] A. The Effect of Human zalpha11 Ligand on B-Cell Lines.
[0626] IM-9 cells were seeded at about 50,000 cells per ml +/-50
.mu.g/ml purified human zalpha11 Ligand (Example 29). After 3 days
growth the cells were harvested, washed and counted then re-plated
at about 2500 cells/ml in 96 well plates in to wells with 0, 0.033,
0.1 or 0.33 .mu.g/ml anti-FAS antibody (R&D Systems,
Minneapolis). After 2 days an Alamar blue fluorescence assay was
performed (Example 2B) to assess proliferation of the cells.
[0627] Zalpha11 Ligand-treated IM-9 cells grew to only 27% the
density of the untreated cells in the absence of anti-FAS antibody.
In the presence of 0.33 .mu.g/ml anti-FAS antibody, the zalpha11
Ligand-treated cells were inhibited an additional 52% while the
untreated cells were inhibited by only 30%. The overall inhibition
of cell growth with both zalpha11 Ligand and 0.33 .mu.g/ml anti-FAS
antibody treatment was 86%.
[0628] When the IM-9 cells were pretreated for three days with or
without zalpha11 Ligand and then re-plated at 100 cells per well
and grown with or without anti-FAS antibody for 6 days, the growth
of untreated cells assessed by Alamar Blue assay (Example 2B) was
inhibited only 25% by anti-FAS antibody while the growth of
zalpha11 Ligand-treated cells was inhibited 95% relative to the
growth of untreated cells in zero anti-FAS antibody.
[0629] B. The Effect of Human zalpha11 Ligand-Saporin Immunotoxin
on B-Cell Lines.
[0630] The human zalpha11 Ligand-saporin immunotoxin conjugate
(zalpha11L-sap) construction and purification is described in
Example 50. The human zalpha11L-sap was far more potent than the
saporin alone in inhibiting cell growth. When the treated cell are
re-plated after a three or four day treatment the human
zalpha11L-sap treated cells grew very poorly.
[0631] IM-9, Ramos and K562 (ATCC No. CCL-243) cells were seeded at
about 2500 cells/well in 96 well plates with zero to 250 ng/ml
human zalpha11L-sap conjugate or 0-250 ng/ml saporin (Stirpe et
al., Biotechnology 10,:405-412, 1992) only as a control. The plates
were incubated 4 days then an Alamar Blue proliferation assay was
performed (Example 5B). At the maximal concentration of human
zalpha11-sap conjugate, the growth of IM-9 cells and RAMOS cells
was inhibited by 79% and 65% respectively. K562 cells which are
low/negative by flow for expression of the zalpha11 receptor were
not affected by the zalpha11-sap, thus showing the specificity of
the conjugate's effect.
[0632] IM-9 cells were seeded a 50,000 cells/ml into 6 well plates
at zero and 50 ng/ml human zalpha11L-sap conjugate. After 3 days
the cells were harvested and counted then re-plated from 100 to 0.8
cells per well in 2 fold serial dilutions, and 12 wells per cell
dilution without the human zalpha11 Ligand-saporin immunotoxin.
After 6 days the number of wells with growth at each cell dilution
was scored according to the results of an Alamar blue proliferation
assay (Example 2B).
[0633] When cell number was assessed, by Alamar blue assay (Example
2B), after 6 days of growth control cells seeded at about 12.5 and
6.25 cells per well had equivalent growth to zalpha11-sap treated
cells seeded at 100 and 50 cells/well respectively. Thus, the
growth of the surviving treated IM-9 cells was markedly impaired
even after the removal, by re-plating, of the zalpha11-sap
immunotoxin.
[0634] The limited tissue distribution of the human zalpha11
receptor (Example 48), and the specificity of action of the
zalpha11-sap to receptor-expressing cell lines suggest that this
conjugate may be tolerated in vivo.
[0635] C. The effect of Human zalpha11 Ligand-Saporin Immunotoxin
on B-Cell Line Viability.
[0636] HS Sultan cells (ATCC No. CRL-1484) were seeded at about
40,000 cells per ml into 12 well plates and grown for five days
with either no added cytokines or 4 0 ng/ml purified human zalpha11
Ligand (Example 29) or 25 ng/ml human zalpha11L-sap conjugate
(Example 50, below) or with 20 ng/ml IFN-alpha (RDI) or zalpha11
Ligand and IFN-alpha. Zalpha11 ligand inhibited the outgrowth of Hs
Sultan cells by 63%. IFN-alpha inhibited the growth by 38%.
Zalpha11 ligand plus IFN-alpha inhibited growth 78%, indicating
that the growth inhibitory effects of human zalpha11 Ligand and
IFN-alpha may be additive. The human zalpha11L-sap inhibited growth
of the HS Sultans by 92%.
[0637] The results above support the possible use of zalpha11
Ligand or human zalpha11L-sap in the treatment of malignancies or
other diseases that express the zalpha11 receptor, particularly
those of B-cell origin. The combination of zalpha11 Ligand with
IFN-alpha is specifically suggested by their additive effect in the
inhibition of HS Sultan cells. Some other types of lymphoid
malignancies and diseases may also express the zalpha11 receptor,
as activated T-cells also express the receptor mRNA (Example 48),
and some of these diseases may also be responsive to zalpha11
Ligand of zalpha11 Ligand-toxic fusion therapy.
[0638] D. FAS (CD95) Expression on Human B-Cell Lines is Increased
by Human zalpha11 Ligand Stimulation
[0639] Human B-cell lines HS Sultan (ATCC No. CRL-1484), IM-9 (ATCC
No. CRL159), RPMI 8226 (ATCC No. CCL-155), RAMOS (ATCC No.
CRL-1596), DAKIKI (ATCC No. TIB-206), and RPMI 1788 (ATCC No.
CRL-156), were all treated with or without purified 10 to 50 ng/ml
human zalpha11 Ligand (Example 29) for 2 to 8 days. The cells were
then stained with anti-CD95 PE-conjugated antibody (PharMingen, San
Diego, Calif.), per manufacturer's protocol, and analyzed on a
FACScalibur (Becton Dickinson, San Jose, Calif.). In all cell
lines, anti-CD95 (FAS or APO-1) staining was increased, in some
cases more than two fold, upon treatment with human zalpha11
Ligand.
[0640] E. FAS (CD95) Expression on Primary Mouse Spleen B-cells is
Increased by Human zalpha11 Ligand Stimulation
[0641] Primary mouse splenocytes were obtained by chopping up
spleens from 8 to 12 week old C57/BL6 mice. Erythrocytes were lysed
by treating the preparation for 5 seconds with water then put
through a 70 micron sieve. The remaining splenocytes were washed
and plated in RPMI (JRH Bioscience) plus 10% HIA-FBS (Hyclone,
Logan, Utah). Interleukin 2 (IL-2) (R and D Systems) with or
without human zalpha11 Ligand, as described above. They were then
incubated at 37.degree. C., in 5% CO2 for 5 days. The splenocytes
were harvested and stained with anti-CD95 PE conjugated antibody
(PharMingen) and anti-CD19 FITC conjugated antibody (PharMingen)
per manufacturer's protocol. The cells were analyzed by flow
cytometry on a FACScalibur (Becton Dickinson). Upon gating on the
CD19+ mouse B-cells, it was found that anti-CD95 staining was
increased on B-cells treated with IL-2 plus human zalpha11 Ligand
compared to those in IL-2 alone. The anti-CD95 staining was 37
relative fluorescent units (RFU) on the B-cells in IL-2 alone and
55 RFU on the B-cells cultured in IL-2 and human zalpha11
Ligand.
Example 50
[0642] Construction and Purification of Zalpha11 Ligand Toxic
Fusion
[0643] Under a supply contract, 10 mg human zalpha11 Ligand
(Example 29) was sent to Advanced Targeting Systems (ATS, San
Diego, Calif.) for conjugation to the plant toxin saporin (Stirpe
et al., Biotechnology 10:405-412, 1992). ZymoGenetics received from
ATS 1.3 mg of a protein conjugate comprised of 1.1 molecules
saporin per molecule of human zalpha11 Ligand, formulated at a
concentration of 1.14 mg/ml in 20 nM Sodium phosphate, 300 nM
sodium chloride, pH 7.2.
Example 51
[0644] Zalpha11 Ligand Toxic Fusion In Vivo
[0645] A. Testing zalpha11-Saporin Conjugate in Mice
[0646] Zalpha11-saporin conjugate (Example 49) was administered to
C57BL6 mice (female, 12 weeks of age, purchased from Taconic) at
two different dosages: 0.5 and 0.05 mg/kg. Injections were given
i.v. in vehicle consisting of 0.1% BSA (ICN, Costa Mesa, Calif.).
Three injections were given over a period of one week (day 0, 2,
and 7). Blood samples were taken from the mice on day 0
(pre-injection) and on days 2 and 8 (post-injection). Blood was
collected into heparinized tubes (Bectin Dickenson, Franklin Lakes,
N.J.), and cell counts were determined using an automated
hematology analyzer (Abbot Cell-Dyn model No. CD-3500CS, Abbot
Park, Ill.). Animals were euthanized and necropsied on day 8
following blood collection. Spleen, thymus, liver, kidney and bone
marrow were collected for histopathology. Spleen and thymus were
weighed, and additional blood sample was collected in serum
separator tubes. Serum was sent to Phoenix Central Labs, Everett,
Wash., for testing in a standard chemistry panel. Samples were also
collected for flow cytometric analysis as described herein.
[0647] Circulating blood cell counts and serum chemistry
measurements did not differ significantly between zalpha11
conjugate treated mice and mice treated with an equivalent dose of
unconjugated toxin (saporin). Histological analysis of tissues in
zalpha11-saporin treated mice showed no significant changes
relative to mice treated with an equivalent dose of unconjugated
toxin. These results indicated that the saporin conjugate was not
toxic in vivo.
[0648] B. Testing Zalpha11 Ligand Toxic Saporin Fusion on B-Cell
Derived Tumors In Vivo
[0649] The effects of human zalpha11 Ligand and the human zalpha11
Ligand toxic saporin fusion (Example 50) on human tumor cells are
tested in vivo using a mouse tumor xenograft model described
herein. The xenograft models are initially tested using cell lines
selected on the basis of in vitro experiments, such as those
described in Example 49. These cell lines include, but are not
limited to: human Burkitt's lymphoma cell lines Raji (ATCC No.
CCL-86), and Ramos (ATCC No. CRL-1596); human cell line RPMI 1788
(ATCC No. CRL-156); human myeloma/plasmacytoma cell line IM-9 (ATCC
No. CRL159); human cell line DAKIKI (ATCC No. TIB-206), and HS
Sultan cells (ATCC No. CRL-1484). Cells derived directly from human
tumors can also be used in this type of model. In this way,
screening of patient samples for sensitivity to treatment with
zalpha11 Ligand or with a zalpha11 Ligand toxic saporin fusion can
be used to select optimal indications for use of zalpha11 in
anti-tumor therapy.
[0650] After selection of the appropriate zenograft in vivo model,
described above, zalpha11 Ligand-induced activity of natural killer
cells and/or zalpha11 Ligand effects on B-cell derived tumors are
assessed in vivo. Human zalpha11 Ligand is tested for its ability
to generate cytotoxic effector cells (e.g. NK cells) with activity
against B-cell derived tumors using mouse tumor xenograft models
described herein. Moreover, direct affects of human zalpha11 Ligand
on tumors can be assessed. The xenograft models to be carried out
are selected as described above. A protocol using zalpha11 Ligand
stimulated human cells is developed and tested for efficacy in
depleting tumor cells and promoting survival in mice innoculated
with cell lines or primary tumors.
Example 52
[0651] Identification of P1 Artificial Chromosome clones Containing
Genomic Human zalpha11 Ligand DNA
[0652] The human zalpha11 Ligand cDNA insert was amplified by PCR
using vector-based primers. The PCR product was .sup.32P-labeled
and hybridized to high-density filters representing a PAC (P1
Artificial Chromosome) library. Filters and frozen library stocks
were obtained from Roswell Park Cancer Institute, Buffalo, N.Y.;
the library segment was RPCI6, with a four-fold depth of coverage.
Filters were hybridized overnight at 65.degree. C. in ExpressHyb
(Clontech) and were washed according to manufacturer's suggestions.
Decoding the positive signals resulted in identification of four
PAC clones, designated 23H17, 34A9, 105G9, and 236H14. PCR analysis
using primers specific for the 5' end (ZC22,452 (SEQ ID NO:100) and
ZC 22,451 (SEQ ID NO:101)) and 3' end (ZC 22,450 (SEQ ID NO:102)
and ZC 22,449 (SEQ ID NO:103)) of the coding region showed that
PACs 34A9 and 105G9 contained both ends, while PACs 23H17 and
236H14 contained the 5' end only. PAC 23H17 was digested with Eco
RI and Not I, and a 9 kb fragment was identified which hybridized
with the zalpha11 Ligand cDNA probe. This fragment was isolated and
subcloned, using methods described herein, into pBluescript II SK
(+) (Stratagene) previously digested with Eco RI and Not I.
Sequencing revealed that this fragment contained about 380 base
pairs of the promoter region, exons 1, 2, and 3, all of introns 1
and 2, and ended within intron 3.
[0653] The 3' end of the human zalpha11 Ligand gene was obtained by
PCR using DNA from PAC 34A9 as template, with primers ZC23,771 (SEQ
ID NO:104) and ZC22,449. (SEQ ID NO:103). Taq DNA polymerase was
used, with buffer provided, with the addition of 4% DMSO. Reaction
conditions were as follows: 94.degree. C., 5 min.; followed by 35
cycles of 94.degree. C. for 30 sec., 52.degree. C. for 1 min.,
72.degree. C. for 3 min.; then 72.degree. C. for 7 min. This
generated a 2.9 kb fragment which contained part of exon 3, all of
introns 3 and 4, all of exon 4, and the coding portion of exon
5.
[0654] The genomic structure of the human zalpha11 Ligand gene is
as follows from 5' to 3': SEQ ID NO:105, containing about 240 bp of
the promoter, exon 1 (nucleotide number 240-455 of SEQ ID NO:105),
intron 1 (nucleotide number 456-562 of SEQ ID NO:105), exon 2
(nucleotide number 563-598 of SEQ ID NO:105), and part of intron 2
containing the 5' 748 base pairs (nucleotide number 599-1347 of SEQ
ID NO:105); a gap of approximately 3 kb; SEQ ID NO:106, containing
the 3' 718 bp of intron 2, exon 3 (nucleotide number 719-874 of SEQ
ID NO:106), and part of the 5' end of intron 3 (nucleotide number
875-1656 of SEQ ID NO:106); a gap of less than about 800 bp; SEQ ID
NO:107, containing 644 bp of intron 3; a gap of less than about 800
bp; and SEQ ID NO:108, containing the 3' 435 bp of intron 3, exon 4
(nucleotide number 436-513 of SEQ ID NO:108), intron 4 (nucleotide
number 514-603 of SEQ ID NO: 108), and part of the 5' end of exon 5
(nucleotide number 604-645 of SEQ ID NO: 108).
Example 53
[0655] .sup.125I-Labeled Human zalpha11 Ligand Binding Study in
Cell Lines
[0656] 25 micrograms of purified human zalpha11 Ligand (Example 29)
was labeled with 2 mCI .sup.125I using iodobeads (Pierce, Rockford
Ill.), according to manufacturer's instructions. This labeled
protein was used to assess human zalpha11 Ligand binding to human
Raji cells (ATCC No. CCL-86), using binding to wild-type murine
BaF3 cells, and BaF3 cells transfected with zalpha11 receptor
(BaF3/hzalpha11 cells) as controls. Zalpha11 Ligand binding to
BaF3/hzalpha11 cells was expected (positive control), while no
binding to wild-type BaF3 cells was expected (negative control),
based on proliferation assay results (Example 5). About
5.times.10.sup.5 Raji cells/well, 1.times.10.sup.6 BaF3/hzalpha11
and 1.times.10.sup.6 BaF3 cells cells/well, were each plated in
96-well plates. Ten ng/ml of labeled human zalpha11 Ligand was
added in duplicate to wells, with a dilution series of unlabeled
human zalpha11 Ligand competitor added from 250 fold molar excess
in 1:4 dilutions down to 0.061 fold molar excess. Each point was
run in duplicate. After the labeled human zalpha11 Ligand was added
to wells, it was allowed to incubate at 4.degree. C. for 2 h to
allow for binding of Ligand to the cells. The cells were then
washed 3.times. in binding buffer (RPMI-1710 (JRH Bioscience) with
1% BSA (Sigma)), and counted on the COBRA II AUTO-GAMMA gamma
counter (Packard Instrument Company, Meriden, Conn.).
[0657] Binding of the labeled zalpha11 Ligand to cells was evident
in the Raji and the BaF3/hzalpha11 cells. In addition, for Raji
cells, an average 250 fold molar excess of unlabeled zalpha11
Ligand decreased binding 3 fold in the presence of a non-specific
unlabeled competitor (Interferon Gamma from R&D Systems,
Minneapolis, Minn.), and 3.7 fold relative to no competitor.
Competition was observed in a dose dependent fashion for the
specific unlabeled competitor, human zalpha11 Ligand. Thus, the
zalpha11 Ligand binding to Raji cells was specific. Similarly, for
positive control BaF3/zalpha11 cells, the 250 fold molar excess of
unlabeled zalpha11 Ligand decreased binding 2 fold relative to the
non-specific competitor and 3.06 fold relative to no competitor.
Thus, the zalpha11 Ligand binding to BaF3/zalpha11 cells also was
specific. No compeatable binding was observed with the wild-type
BaF3 cells. Thus, the zalpha11 Ligand was shown to bind
specifically to Raji cells, and to BaB/hzalpha11 cells, but not to
the negative control Baf3 cells.
Example 54
[0658] Zalpha11 Receptor Expression On Human Blood Cells
[0659] A. Preparation and Culture of Human Peripheral Blood
Cells
[0660] Fresh drawn human blood was diluted 1:1 with PBS (GIBCO BRL)
and layered over Ficoll/Hypaque Plus (Pharmacia LKB Biotechnology
Inc., Piscataway, N.J.) and spun for 30 minutes at 1800 rpm and
allowed to stop with the brake off. The interface layer was removed
and transferred to a fresh 50 ml Falcon tube (Falcon, VWR, Seattle,
Wash.), brought up to a final volume of 40 ml with PBS and spun for
10 minutes at 1200 rpm with the brake on. The viability of the
isolated cells was tested using Trypan Blue (GIBCO BRL) and the
cells were resuspended at a final concentration of 1.times.106
cells/ml cell medium (RPMI Medium 1640, 10% Heat inactivated fetal
bovine serum, 5% L-glutamine, 5% Pen/Strep) (GIBCO BRL).
[0661] Cells were cultured in 6 well plates (Falcon, VWR) for 0, 4
or 24 hours with a variety of different stimuli described below.
Anti-IgM, anti-CD40 and anti-CD3 stimulation were done as in
Example 44 and Example 42. Phorbol myristate acetate (PMA) and
ionomycin (Sigma, St. Louis, Mo.) (Example 5C) were added to
appropriate wells at 10 ng/ml and 0.5 mg/ml respectively. The cells
were incubated at 37.degree. C. in a humidified incubator for
various times.
[0662] B. Antibody Staining and Analysis
[0663] Cells were collected out of the plates, washed and
resuspended in ice cold staining media (HBSS, 1% fetal bovine
serum, 0.1% sodium azide) at a concentration of about ten million
cells per milliliter. Blocking of Fc receptor and non-specific
binding of antibodies to the cells was achieved by adding 10%
normal goat serum (Gemini Bioproducts, Woodland, Calif.) and 10%
normal human serum (Ultraserum, Gemini) to the cell suspension.
Aliquots of the cell suspensions were mixed with a FITC labeled
monoclonal antibody against one of the lineage markers CD3, CD19 or
CD14 (PharMingen, La Jolla, Calif.) and a biotinylated monoclonal
antibody against the human zalpha11 receptor (hu-zalpha11) (Example
35). Staining specificity was determined by competition using
zalpha11CEE soluble receptor (Example 10A) at a ten fold mass
excess. After incubation on ice for 60 minutes the cells were
washed twice with ice cold staining media and resuspended in 50 ml
staining media containing streptavidin-PE (Caltag, Burlingame,
Calif.). After a 30 minute incubation on ice, the cells were washed
twice with ice cold wash buffer (PBS, 1% fetal bovine serum, 0.1%
sodium azide) and resuspended in wash buffer containing 1 mg/ml
7-AAD (Molecular Probes, Eugene, Oreg.) as a viability marker. Flow
data was acquired on living cells using a FACSCalibur flow
cytometer (BD Immunocytometry Systems, San Jose, Calif.). Both
acquisition and analysis were performed using CellQuest software
(BD Immunocytometry Systems).
[0664] Results of staining by anti-zalpha11 antibody showed that
the human zalpha11 receptor is expressed on human peripheral blood
cells expressing either CD3, CD19 or CD14. Staining on CD3 and CD19
cells was specific, as evidenced by absolute competion with the
zalpha11 soluble receptor. Staining on CD14 cells showed some
specificity for the Ligand, as evidenced by partial competion with
the soluble receptor. Activation of either T cells with anti-CD3 or
B cells with anti-CD40 resulted in an increased level of cell
surface zalpha11 at 24 hours. No increase in the level of
expression of zalpha11 was seen at 4 hours with any stimulus on
either cell population. Treatment of the cells with zalpha11 ligand
resulted in a decrease of zalpha11 staining on CD3 positive and
CD19 positive cells but not CD14 positive cells at both 4 and 24
hours.
Example 55
[0665] Preliminary Evaluation of the Aqueous Stability of Human
zalpha11 Ligand
[0666] Preliminary studies were conducted to evaluate the aqueous
stability characteristics of human zalpha11 Ligand in support of
bioprocessing, formulation, and in vivo administration. The
objectives were to: 1) verify the stability and recovery from Alzet
Minipumps & general storage and handling, 2) determine the
stability-indicating nature of several analytical methods including
cation-exchange HPLC (CX-HPLC), reverse-phase HPLC (RP-HPLC), size
exclusion HPLC (SEC-HPLC), & bioassay (BaF3/zalpha11R
proliferation (e.g., Example 2 and Example 4), and 3) determine the
stability-limiting degradation pathways and their kinetic
dependencies.
[0667] Aliquots of purified human zalpha11 Ligand (Example 29) were
prepared by dilution to 2 mg/mL in PBS (pH 7.4) and stored in low
density polyethylene (LDPE) cryovials (Nalgene, 1.8 mL) at
-80.degree. C. (control), 5.degree. C., 30.degree. C., and
37.degree. C. Samples were assayed intermittently over 29 days by
CX-, RP-, SEC-HPLC, and bioassay. Aliquots were also stored at
-80.degree. C. and subjected to freeze-thaw (f/t) cycling
(-80.degree. C./RT; 5.times. f/t, 10.times. f/t). Recovery of human
zalpha11 Ligand was determined relative to the -80.degree. C.
control (1 f/t) in all assays.
[0668] The remaining human zalpha11 Ligand solution from the
-80.degree. C. control samples were refrozen (80.degree. C.) after
analysis. This aliquot (2 f/t) was used to evaluate the thermal and
conformational stability of human zalpha11 Ligand as a function of
pH using circular dichroism (CD). The 2 mg/mL solution was diluted
to 100 .mu.g/mL in PBS buffers ranging from pH 3.3-8.8. The far-UV
CD spectra was monitored over the temperature range 5-90.degree. C.
in 5.degree. C. intervals (n=3/pH). The CD spectropolarimeter used
was a Jasco 715 (Jasco, Easton, Md.). The thermal unfolding was
monitored by changes in ellipticity at 222 nm as a function of
temperature. Estimates of the Tm were estimated assuming a
two-state unfolding model. The data was fit (sigmoidal) using
SlideWrite Plus for Windows v4.1 (Advanced Graphics Software;
Encinitas, Calif.).
[0669] Recovery and stability from Alzet Minipumps (Model No.
1007D; ALZA Corporation, Mountain View, Calif.) was assessed by
filling pumps with 100 .mu.L of the 2 mg/mL human zalpha11 Ligand
solution, placing the pumps in 1.8 mL LDPE containing 1 mL of PBS
(pH 7.4), and storing them at 37.degree. C. The release/recovery of
human zalpha11 Ligand from the minipumps was assessed by CX-, RP-,
and SEC-HPLC on days 2, 4, and 7. The activity was assessed by
bioassay on day 7. The study was designed to evaluate the release
from 3 pumps per sampling time.
[0670] The chromatographic data suggested that the CX- &
SEC-HPLC methods were stability-indicating, whereas the RP-HPLC
method was not. At least 3 additional peaks indicating apparent
degradation products were observed by CX-HPLC. The SEC-HPLC method
resolved an apparent human zalpha11 Ligand aggregate, eluting prior
to human zalpha11 Ligand. However, no significant additional peaks
were observed eluting after the human zalpha11 Ligand peak. This
suggests that the degradation products observed by CX-HPLC most
probably result from amino acid modifications such as deamidation,
rather than hydrolysis/proteolysis processes leading to clipped
variants. A small degree of fronting/tailing was observed by
RP-HPLC (relative to control) in samples which had been shown to
have undergone significant degradation by SEC- & CX-HPLC.
However, apparent degradation products were not resolved by
RP-HPLC. The degradation observed by CX-HPLC increased as a
function of time-temperature, and followed apparent first-order
kinetics. The % human zalpha11 Ligand recovered by CX-HPLC after 29
days at 37.degree. C., 30.degree. C., and 5.degree. C. was 39%,
63%, and 98%, respectively. Aggregation also increased in a
time-temperature dependent fashion. The % aggregate found in
preparations stored for 29 days at 37.degree. C., 30.degree. C.,
and 5.degree. C. was 7.4, 3.4, and below detectable limits (BDL),
respectively. No significant differences were observed by bioassay
in any sample, suggesting the degradation products have equivalent
activity to intact human zalpha11 Ligand. No degradation was
observed by any assay in samples subjected to up to 10 f/t
cycles.
[0671] The release of human zalpha11 Ligand from Alzet Minipumps
was consistent with the theoretical expected volume release. This
suggests that significant surface adsorption would not impair the
delivery of human zalpha11 Ligand using the Alzet Minipumps with a
2 mg/mL fill concentration. The degradation consistent with that
previously noted was observed. The % purity determined by CX-HPLC
of human zalpha11 Ligand released after 2, 4, and 7 days was 96%,
90%, and 79%, receptively. It should be recognized that degradation
also occurs after human zalpha11 Ligand is released into or diluted
with release medium. Therefore, the % purity within the minipump
may be somewhat different than that determined to be in the release
medium. The bioactivity of each sample was consistent with the
expected amount of human zalpha11 Ligand released from the
minipumps.
[0672] The human zalpha11 Ligand far-UW CD spectra, as expected,
was consistent with interleukins, such as IL-3 (J. Biochem,
23:352-360, 1991), IL-4 (Biochemistry, 30:1259-1264, 1991), and
IL-6 mutants (Biochemistry, 35:11503-11511, 1996). Gross changes in
the far-uv CD spectra as a function of pH were not observed.
Results showed that the pH of maximum thermal/conformational
stability was .about.pH 7.4. Analysis of the unfolding curves were
based on a two-state unfolding mechanism to allow comparison of the
thermal/conformational stability as a function of pH/composition.
However, one or more intermediates may exist during the unfolding
process since the cooperatively was relatively low, based on the
shallowness of the unfolding curve. Although studies were not
specifically designed to determine whether human zalpha11 Ligand
refolds following thermal unfolding to 90.degree. C., preliminary
data suggests that at least partial refolding occurs after the
temperature of the sample is cooled back to 20.degree. C.
[0673] These studies allow an analytical paradigm to be identified
to evaluate the purity and verify the stability of human zalpha11
Ligand. For instance, SEC-HPLC can be used to characterize the
extent and rate of aggregation in aqueous solution. Likewise,
CX-HPLC can be used to characterize the extent and rate of
degradation of human zalpha11 Ligand by mechanisms other than
aggregation. The bioassay can be used to verify activity of human
zalpha11 Ligand and it's aqueous degradation products. For
instance, the human zalpha11 Ligand variants generated in aqueous
solution & resolved by CX-HPLC may themselves be useful as
therapeutic agents, since they have equivalent bioactivity. Also,
the fact that human zalpha11 Ligand degrades by several different
processes (aggregation, amino acid modifications) suggests a
preferred or unique formulation which minimizes the rate of each
degradation process may be necessary for long-term stability of a
solution product.
[0674] Identification of the nature of the aqueous degradation
products and determination of their kinetic dependencies (pH,
concentration, excipients) is underway. Human zalpha11 Ligand
stability in serum/plasma is determined to support the design and
interpretation of in vivo studies.
[0675] 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
115 1 642 DNA Homo sapiens CDS (47)...(532) 1 gctgaagtga aaacgagacc
aaggtctagc tctactgttg gtactt atg aga tcc 55 Met Arg Ser 1 agt cct
ggc aac atg gag agg att gtc atc tgt ctg atg gtc atc ttc 103 Ser Pro
Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe 5 10 15 ttg
ggg aca ctg gtc cac aaa tca agc tcc caa ggt caa gat cgc cac 151 Leu
Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His 20 25
30 35 atg att aga atg cgt caa ctt ata gat att gtt gat cag ctg aaa
aat 199 Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys
Asn 40 45 50 tat gtg aat gac ttg gtc cct gaa ttt ctg cca gct cca
gaa gat gta 247 Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro
Glu Asp Val 55 60 65 gag aca aac tgt gag tgg tca gct ttt tcc tgt
ttt cag aag gcc caa 295 Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys
Phe Gln Lys Ala Gln 70 75 80 cta aag tca gca aat aca gga aac aat
gaa agg ata atc aat gta tca 343 Leu Lys Ser Ala Asn Thr Gly Asn Asn
Glu Arg Ile Ile Asn Val Ser 85 90 95 att aaa aag ctg aag agg aaa
cca cct tcc aca aat gca ggg aga aga 391 Ile Lys Lys Leu Lys Arg Lys
Pro Pro Ser Thr Asn Ala Gly Arg Arg 100 105 110 115 cag aaa cac aga
cta aca tgc cct tca tgt gat tct tat gag aaa aaa 439 Gln Lys His Arg
Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys 120 125 130 cca ccc
aaa gaa ttc cta gaa aga ttc aaa tca ctt ctc caa aag atg 487 Pro Pro
Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met 135 140 145
att cat cag cat ctg tcc tct aga aca cac gga agt gaa gat tcc 532 Ile
His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser 150 155 160
tgaggatcta acttgcagtt ggacactatg ttacatactc taatatagta gtgaaagtca
592 tttctttgta ttccaagtgg aggagcccta ttaaattata taaagaaata 642 2
162 PRT Homo sapiens 2 Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile
Val Ile Cys Leu Met 1 5 10 15 Val Ile Phe Leu Gly Thr Leu Val His
Lys Ser Ser Ser Gln Gly Gln 20 25 30 Asp Arg His Met Ile Arg Met
Arg Gln Leu Ile Asp Ile Val Asp Gln 35 40 45 Leu Lys Asn Tyr Val
Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro 50 55 60 Glu Asp Val
Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln 65 70 75 80 Lys
Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile 85 90
95 Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala
100 105 110 Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp
Ser Tyr 115 120 125 Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe
Lys Ser Leu Leu 130 135 140 Gln Lys Met Ile His Gln His Leu Ser Ser
Arg Thr His Gly Ser Glu 145 150 155 160 Asp Ser 3 486 DNA
Artificial Sequence Degenerate polynucleotide sequence for human
zalpha11 ligand misc_feature (1)...(486) n = A,T,C or G 3
atgmgnwsnw snccnggnaa yatggarmgn athgtnatht gyytnatggt nathttyytn
60 ggnacnytng tncayaarws nwsnwsncar ggncargaym gncayatgat
hmgnatgmgn 120 carytnathg ayathgtnga ycarytnaar aaytaygtna
aygayytngt nccngartty 180 ytnccngcnc cngargaygt ngaracnaay
tgygartggw sngcnttyws ntgyttycar 240 aargcncary tnaarwsngc
naayacnggn aayaaygarm gnathathaa ygtnwsnath 300 aaraarytna
armgnaarcc nccnwsnacn aaygcnggnm gnmgncaraa rcaymgnytn 360
acntgyccnw sntgygayws ntaygaraar aarccnccna argarttyyt ngarmgntty
420 aarwsnytny tncaraarat gathcaycar cayytnwsnw snmgnacnca
yggnwsngar 480 gaywsn 486 4 535 DNA Mus musculus source (0)...(0)
EST1483966 ; GenBank Acc #AA764063 4 taaacatgta tcatataagg
atatgtcata ataaggatta atattatata attataaata 60 atttataata
cttataatat cattgtttgg ttcactaata aatctatgga tacatggtca 120
aaatggaaat gaatattttg ccaattatta atccccaaag tcattgaaaa taagcataac
180 cattctactg acttgttaga ctctaaacta acataaaata cattttcaga
aataaattca 240 accgatctta cctttacatc ttgtggagct gatagaagtt
caggatccta agaaaattaa 300 ccaaagagta ttagttctga gttggtgata
caagtcaaaa ggctcctttt gcattaatta 360 aaaaaatatt atttaaattg
cattgtgaca aacatggcct taccaagtca ttttcataga 420 ttttcagctg
ttcaacaatg tcaataaggt gacgaagtct aatcaggagg cgatctggcc 480
cttgggggct tgatttatgg gccactgtcc ccaagaagat gactaccaga cagac 535 5
33 DNA Artificial Sequence Oligonucleotide primer ZC17212 5
ggggaattcg aagccatgcc ctcttgggcc ctc 33 6 30 DNA Artificial
Sequence Oligonucleotide primer ZC19914 6 caatggatgg gtctttagca
gcagtaggcc 30 7 1614 DNA Homo sapiens 7 atgccgcgtg gctgggccgc
ccccttgctc ctgctgctgc tccagggagg ctggggctgc 60 cccgacctcg
tctgctacac cgattacctc cagacggtca tctgcatcct ggaaatgtgg 120
aacctccacc ccagcacgct cacccttacc tggcaagacc agtatgaaga gctgaaggac
180 gaggccacct cctgcagcct ccacaggtcg gcccacaatg ccacgcatgc
cacctacacc 240 tgccacatgg atgtattcca cttcatggcc gacgacattt
tcagtgtcaa catcacagac 300 cagtctggca actactccca ggagtgtggc
agctttctcc tggctgagag catcaagccg 360 gctccccctt tcaacgtgac
tgtgaccttc tcaggacagt ataatatctc ctggcgctca 420 gattacgaag
accctgcctt ctacatgctg aagggcaagc ttcagtatga gctgcagtac 480
aggaaccggg gagacccctg ggctgtgagt ccgaggagaa agctgatctc agtggactca
540 agaagtgtct ccctcctccc cctggagttc cgcaaagact cgagctatga
gctgcaggtg 600 cgggcagggc ccatgcctgg ctcctcctac caggggacct
ggagtgaatg gagtgacccg 660 gtcatctttc agacccagtc agaggagtta
aaggaaggct ggaaccctca cctgctgctt 720 ctcctcctgc ttgtcatagt
cttcattcct gccttctgga gcctgaagac ccatccattg 780 tggaggctat
ggaagaagat atgggccgtc cccagccctg agcggttctt catgcccctg 840
tacaagggct gcagcggaga cttcaagaaa tgggtgggtg cacccttcac tggctccagc
900 ctggagctgg gaccctggag cccagaggtg ccctccaccc tggaggtgta
cagctgccac 960 ccaccacgga gcccggccaa gaggctgcag ctcacggagc
tacaagaacc agcagagctg 1020 gtggagtctg acggtgtgcc caagcccagc
ttctggccga cagcccagaa ctcggggggc 1080 tcagcttaca gtgaggagag
ggatcggcca tacggcctgg tgtccattga cacagtgact 1140 gtgctagatg
cagaggggcc atgcacctgg ccctgcagct gtgaggatga cggctaccca 1200
gccctggacc tggatgctgg cctggagccc agcccaggcc tagaggaccc actcttggat
1260 gcagggacca cagtcctgtc ctgtggctgt gtctcagctg gcagccctgg
gctaggaggg 1320 cccctgggaa gcctcctgga cagactaaag ccaccccttg
cagatgggga ggactgggct 1380 gggggactgc cctggggtgg ccggtcacct
ggaggggtct cagagagtga ggcgggctca 1440 cccctggccg gcctggatat
ggacacgttt gacagtggct ttgtgggctc tgactgcagc 1500 agccctgtgg
agtgtgactt caccagcccc ggggacgaag gacccccccg gagctacctc 1560
cgccagtggg tggtcattcc tccgccactt tcgagccctg gaccccaggc cagc 1614 8
30 DNA Artificial Sequence Oligonucleotide primer ZC19913 8
ggcctactgc tgctaaagac ccatccattg 30 9 33 DNA Artificial Sequence
Oligonucleotide primer ZC20097 9 acatctagat tagctggcct ggggtccagg
cgt 33 10 21 DNA Artificial Sequence Oligonucleotide primer ZC12700
10 ggaggtctat ataagcagag c 21 11 21 DNA Artificial Sequence
Oligonucleotide primer ZC5020 11 cactggagtg gcaacttcca g 21 12 20
DNA Artificial Sequence Oligonucleotide primer ZC6675 12 gtggatgccg
aacccagtcc 20 13 21 DNA Artificial Sequence Oligonucleotide primer
ZC7727 13 tgttcacagc tacctgggct c 21 14 26 DNA Artificial Sequence
Oligonucleotide primer ZC8290 14 ccaccgagac tgcttggatc accttg 26 15
20 DNA Artificial Sequence Oligonucleotide primer ZC19572 15
gtcctgtggc tgtgtctcag 20 16 21 DNA Artificial Sequence
Oligonucleotide primer ZC6622 16 ctgggctgga aactggcaca c 21 17 18
DNA Artificial Sequence Oligonucleotide primer ZC7736 17 cactgtcaga
aatggagc 18 18 24 DNA Artificial Sequence Oligonucleotide primer
ZC9273 18 ggtccctccc cgggcaccga gaga 24 19 36 DNA Artificial
Sequence Oligonucleotide primer ZC19905 19 acaggatccg tcagcatgcc
gcgtggctgg gccgcc 36 20 33 DNA Artificial Sequence Oligonucleotide
primer ZC19906 20 acagaattct tagctggcct ggggtccagg cgt 33 21 22 DNA
Artificial Sequence Oligonucleotide primer ZC20114 21 cctgccttct
acatgctgaa gg 22 22 21 DNA Artificial Sequence Oligonucleotide
primer ZC19459 22 ctcctcctgc ttgtcatagt c 21 23 18 DNA Artificial
Sequence Oligonucleotide primer ZC19954 23 actgggctgg gggactgc 18
24 22 DNA Artificial Sequence Oligonucleotide primer ZC20116 24
agcacagtca ctgtgtcaat gg 22 25 40 DNA Artificial Sequence
Oligonucleotide primer ZC13946 25 ccctgcagtg atcaacatgg ccaagttgac
cagtgccgtt 40 26 45 DNA Artificial Sequence Oligonucleotide primer
ZC13945 26 gcccatggac tagtttcgaa aggtcgagtg tcagtcctgc tcctc 45 27
34 DNA Artificial Sequence Oligonucleotide primer ZC18698 27
tttttttctc gagacttttt tttttttttt tttt 34 28 25 DNA Artificial
Sequence Oligonucleotide primer ZC14063 28 caccagacat aatagctgac
agact 25 29 6 PRT Artificial Sequence Glu-Glu (CEE) Tag amino acid
sequence 29 Glu Tyr Met Pro Met Glu 1 5 30 36 DNA Artificial
Sequence Oligonucleotide primer ZC19931 30 ggttggtacc gcaagatgcc
gcgtggctgg gccgcc 36 31 29 DNA Artificial Sequence Oligonucleotide
primer ZC19932 31 cggaggatcc gtgagggttc cagccttcc 29 32 66 DNA
Artificial Sequence Oligonucleotide primer spanning vector flanking
region and the 5' end of the zalpha11 extracellular domain 32
tccactttgc ctttctctcc acaggtgtcc agggaattca tcgataatgc cgcgtggctg
60 ggccgc 66 33 699 DNA Homo sapiens 33 gagcccagat cttcagacaa
aactcacaca tgcccaccgt gcccagcacc tgaagccgag 60 ggggcaccgt
cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 120
acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc
180 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg
ggaggagcag 240 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc
tgcaccagga ctggctgaat 300 ggcaaggagt acaagtgcaa ggtctccaac
aaagccctcc catcctccat cgagaaaacc 360 atctccaaag ccaaagggca
gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 420 gatgagctga
ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 480
gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
540 cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt
ggacaagagc 600 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc
atgaggctct gcacaaccac 660 tacacgcaga agagcctctc cctgtctccg
ggtaaataa 699 34 62 DNA Artificial Sequence First Oligonucleotide
primer spanning 3' end of the zalpha11 extracellular domain and the
5' end of Fc4 34 gcacggtggg catgtgtgag ttttgtctga agatctgggc
tcgtgagggt tccagccttc 60 ct 62 35 61 DNA Artificial Sequence Second
Oligonucleotide primer spanning 3' end of the zalpha11
extracellular domain and the 5' end of Fc4 35 agacccagtc agaggagtta
aaggaaggct ggaaccctca cgagcccaga tcttcagaca 60 a 61 36 67 DNA
Artificial Sequence Oligonucleotide primer spanning the 3' end of
Fc4 and the vector flanking region 36 gtgggcctct ggggtgggta
caaccccaga gctgttttaa tctagattat ttacccggag 60 acaggga 67 37 8 PRT
Artificial Sequence C-terminal FLAG amino acid sequence 37 Asp Tyr
Lys Asp Asp Asp Asp Lys 1 5 38 26 DNA Artificial Sequence
Oligonucleotide primer ZC7764a 38 tttttttttt tttttttttt ttttta 26
39 26 DNA Artificial Sequence Oligonucleotide primer ZC7764b 39
tttttttttt tttttttttt tttttc 26 40 19 DNA Artificial Sequence
Oligonucleotide primer ZC22034 40 ttcaaatcac ttctccaaa 19 41 20 DNA
Artificial Sequence Oligonucleotide primer ZC22035 41 ttttggagaa
gtgatttgaa 20 42 16 DNA Artificial Sequence Oligonucleotide primer
ZC22050 42 gaatgcgtca acttat 16 43 16 DNA Artificial Sequence
Oligonucleotide primer ZC22051 43 ggaccaagtc attcac 16 44 25 DNA
Artificial Sequence Oligonucleotide primer ZC22056 44 gtctgtctgg
tagtcatctt cttgg 25 45 25 DNA Artificial Sequence Oligonucleotide
primer ZC22057 45 cttgtggagc tgatagaagt tcagg 25 46 26 DNA
Artificial Sequence Oligonucleotide primer ZC22205 46 agctgttcaa
caatgtcaat aaggtg 26 47 24 DNA Artificial Sequence Oligonucleotide
primer ZC22206 47 cctcctgatt agacttcgtc acct 24 48 27 DNA
Artificial Sequence Oligonucleotide primer ZC9739 48 ccatcctaat
acgactcact atagggc 27 49 23 DNA Artificial Sequence Oligonucleotide
primer ZC9719 49 actcactata gggctcgagc ggc 23 50 25 DNA Artificial
Sequence Oligonucleotide primer ZC14063 50 caccagacat aatagctgac
agact 25 51 21 DNA Artificial Sequence Oligonucleotide primer
ZC5020 51 cactggagtg gcaacttcca g 21 52 20 DNA Artificial Sequence
Oligonucleotide primer ZC22421 52 ctaaaatggc tccttcaaaa 20 53 40
DNA Artificial Sequence Oligonucleotide primer ZC22604 53
cacacaggcc ggccaccatg ggcttccagc ctccggccgc 40 54 20 DNA Artificial
Sequence Oligonucleotide primer ZC22641 54 atgcgttggt tctgattgtg 20
55 3072 DNA mus musculus CDS (54)...(491) 55 gagaaccaga ccaaggccct
gtcatcagct cctggagact cagttctggt ggc atg 56 Met 1 gag agg acc ctt
gtc tgt ctg gta gtc atc ttc ttg ggg aca gtg gcc 104 Glu Arg Thr Leu
Val Cys Leu Val Val Ile Phe Leu Gly Thr Val Ala 5 10 15 cat aaa tca
agc ccc caa ggg cca gat cgc ctc ctg att aga ctt cgt 152 His Lys Ser
Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu Arg 20 25 30 cac
ctt att gac att gtt gaa cag ctg aaa atc tat gaa aat gac ttg 200 His
Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp Leu 35 40
45 gat cct gaa ctt cta tca gct cca caa gat gta aag ggg cac tgt gag
248 Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys Glu
50 55 60 65 cat gca gct ttt gcc tgt ttt cag aag gcc aaa ctc aag cca
tca aac 296 His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro
Ser Asn 70 75 80 cct gga aac aat aag aca ttc atc att gac ctc gtg
gcc cag ctc agg 344 Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val
Ala Gln Leu Arg 85 90 95 agg agg ctg cct gcc agg agg gga gga aag
aaa cag aag cac ata gct 392 Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys
Lys Gln Lys His Ile Ala 100 105 110 aaa tgc cct tcc tgt gat tcg tat
gag aaa agg aca ccc aaa gaa ttc 440 Lys Cys Pro Ser Cys Asp Ser Tyr
Glu Lys Arg Thr Pro Lys Glu Phe 115 120 125 cta gaa aga cta aaa tgg
ctc ctt caa aag atg att cat cag cat ctc 488 Leu Glu Arg Leu Lys Trp
Leu Leu Gln Lys Met Ile His Gln His Leu 130 135 140 145 tcc
tagaacacat aggacccgaa gattcctgag gatccgagaa gattcccgag 541 Ser
gactgaggag acgccggaca ctatagacgc tcacgaatgc aggagtacat cttgcctctt
601 gggattgcaa gtggagaagt acgatacgtt atgataagaa caactcagaa
aagctatagg 661 ttaagatcct ttcgcccatt aactaagcag acattgtggt
tccctgcaca gactccatgc 721 tgtcaacatg gaaaatctca actcaacaag
agcccagctt cccgtgtcag ggatttctgg 781 tgcttctcaa gctgtggctt
catcttattg cccaactgtg acattctttg attggaaggg 841 gaaaactaaa
gcttttagca aaaatacagc tagggaattt gtcgatctgc gagagtaaga 901
cctcttatga tcctaacgga atgatgtaag ctggaaataa taagcataag atgaaattga
961 aaattgaagt ctttattctt taagaaaaac tttgtacttg aaagcatgtc
tgaagagttt 1021 actcattacc acaaacatct agcatattga taactaacat
ctttatactc tacaagagag 1081 gctttccaga taggtacagt ttttcttctc
tattaggtct atcaaaattt aacctattat 1141 gagggtcacc cctggctttc
actgtttttc taaagaggca agggtgtagt aagaagcagg 1201 cttaagttgc
cttcctccca atgtcaagtt cctttataag ctaatagttt aatcttgtga 1261
agatggcaat gaaagcctgt ggaagtgcaa acctcactat cttctggagc caagtagaat
1321 tttcaagttt gtagctctca cctcaagtgg ttatgggtgt cctgtgatga
atctgctagc 1381 tccagcctca gtctcctctc ccacatcctt tcctttcttt
cctctttgaa acttctaaga 1441 aaaagcaatc caaacaagtt cagcacttaa
gacacattgc atgcacactt ttgataagtt 1501 aaatccaacc atctatttaa
aatcaaaatc aggagatgag ccaagagacc agaggttctg 1561 ttccagtttt
aaacagactt ttactgaaca tcccaatctt ttaaccacag aggctaaatt 1621
gagcaaatag ttttgccatt tgatataatt tccaacagta tgtttcaatg tcaagttaaa
1681 aagtctacaa agctattttc cctggagtgg tatcatcgct ttgagaattt
cttatggtta 1741 aaatggatct gagatccaag catggcctgg gggatggttt
tgatctaagg aaaaaggtgt 1801 ctgtacctca cagtgccttt aaaacaagca
gagatcccgt gtaccgccct aagatagcac 1861 agactagtgt taactgattc
ccagaaaagt gtcacaatca gaaccaacgc attctcttaa 1921 actttaaaaa
tatgtattgc aaagaacttg tgtaactgta aatgtgtgac tgttgatgac 1981
attatacaca catagcccac gtaagtgtcc aatggtgcta gcattggttg ctgagtttgc
2041 tgctcgaaag ctgaagcaga gatgcagtcc ttcacaaagc aatgatggac
agagagggga 2101 gtctccatgt tttattcttt tgttgtttct ggctgtgtaa
ctgttgactt cttgacattg 2161 tgatttttat atttaagaca atgtatttat
tttggtgtgt ttattgttct agccttttaa 2221 atcactgaca atttctaatc
aagaagtaca aataattcaa tgcagcacag gctaagagct 2281 tgtatcgttt
ggaaaagcca gtgaaggctt ctccactagc catgggaaag ctacgcttta 2341
gagtaaacta gacaaaattg cacagcagtc ttgaacctct ctgtgctcaa gactcagcca
2401 gtcctttgac attattgttc actgtgggtg ggaacacatt ggacctgaca
cactgttgtg 2461 tgtccatgaa ggttgccact ggtgtaagct ttttttggtt
ttcattctct tatctgtaga 2521 acaagaatgt ggggctttcc taagtctatt
ctgtatttta ttctgaactt cgtatgtctg 2581 agttttaatg ttttgagtac
tcttacagga acacctgacc acacttttga gttaaatttt 2641 atcccaagtg
tgatatttag ttgttcaaaa agggaaggga tatacataca tacatacata 2701
catacataca tatatatata tatatataca tatatatata tatatatatg tatatatata
2761 tatatataga gagagagaga gagagagaga gagaaagaga gagaggttgt
tgtaggtcat 2821 aggagttcag aggaaatcag ttatggccgt taatactgta
gctgaaagtg ttttctttgt 2881 gaataaattc atagcattat tgatctatgt
tattgctctg ttttatttac agtcacacct 2941 gagaatttag ttttaatatg
aatgatgtac tttataactt aatgattatt tattatgtat 3001 ttggttttga
atgtttgtgt tcatggcttc ttatttaaga cctgatcata ttaaatgcta 3061
cccagtccgg a 3072 56 146 PRT mus musculus 56 Met Glu Arg Thr Leu
Val Cys Leu Val Val Ile Phe Leu Gly Thr Val 1 5 10 15 Ala His Lys
Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu 20 25 30 Arg
His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp 35 40
45 Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys
50 55 60 Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys
Pro Ser 65 70 75 80 Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu
Val Ala Gln Leu 85 90 95 Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly
Lys Lys Gln Lys His Ile 100 105 110 Ala Lys Cys Pro Ser Cys Asp Ser
Tyr Glu Lys Arg Thr Pro Lys Glu 115 120 125 Phe Leu Glu Arg Leu Lys
Trp Leu Leu Gln Lys Met Ile His Gln His 130 135 140 Leu Ser 145 57
34 DNA Artificial Sequence Oligonucleotide primer ZC22283 57
cgctcgagac catggagagg acccttgtct gtct 34 58 31 DNA Artificial
Sequence Oligonucleotide primer ZC22284 58 gctctagaat cttctcggat
cctcaggaat c 31 59 100 DNA Artificial Sequence Oligonucleotide
ZC12749 59 gtaccttccc gtaaatccct ccccttcccg gaattacaca cgcgtatttc
ccagaaaagg 60 aactgtagat ttctaggaat tcaatccttg gccacgcgtc 100 60
100 DNA Artificial Sequence Oligonucleotide ZC12748 60 tcgagacgcg
tggccaagga ttgaattcct agaaatctac agttcctttt ctgggaaata 60
cgcgtgtgta attccgggaa ggggagggat ttacgggaag 100 61 32 DNA
Artificial Sequence Oligonucleotide primer ZC22143 61 cgtatcggcc
ggccaccatg agatccagtc ct 32 62 32 DNA Artificial Sequence
Oligonucleotide primer ZC22144 62 cgtacgggcg cgcctcagga atcttcactt
cc 32 63 483 DNA homo sapiens 63 tccagtcctg gcaacatgga gaggattgtc
atctgtctga tggtcatctt cttggggaca 60 ctggtccaca aatcaagctc
ccaaggtcaa gatcgccaca tgattagaat gcgtcaactt 120 atagatattg
ttgatcagct gaaaaattat gtgaatgact tggtccctga atttctgcca 180
gctccagaag atgtagagac aaactgtgag tggtcagctt tttcctgttt tcagaaggcc
240 caactaaagt cagcaaatac aggaaacaat gaaaggataa tcaatgtatc
aattaaaaag 300 ctgaagagga aaccaccttc cacaaatgca gggagaagac
agaaacacag actaacatgc 360 ccttcatgtg attcttatga gaaaaaacca
cccaaagaat tcctagaaag attcaaatca 420 cttctccaaa agatgattca
tcagcatctg tcctctagaa cacacggaag tgaagattcc 480 tga 483 64 57 DNA
Artificial Sequence Oligonucleotide primer ZC22052 64 tcatataggc
cggccatatg cccgggcgcc accatggatt ccagtcctgg caacatg 57 65 57 DNA
Artificial Sequence Oligonucleotide primer ZC22053 65 gtacaacccc
agagctgttt taaggcgcgc ctctagatca ggaatcttca cttccgt 57 66 32 DNA
Artificial Sequence Oligonucleotide primer ZC23115 66 gtatacggcc
ggccaccatg gagaggaccc tt 32 67 32 DNA Artificial Sequence
Oligonucleotide primer ZC23116 67 cgtatcggcg cgccctagga gagatgctga
tg 32 68 35 DNA Artificial Sequence Oligonucleotide primer ZC20892
68 gtatacgttt aaacgccacc atgccgcgtg gctgg 35 69 32 DNA Artificial
Sequence Oligonucleotide primer ZC20893 69 cgtatcggcg cgccttacaa
tggatgggtc tt 32 70 39 DNA Artificial Sequence Oligonucleotide
primer ZC22054 70 cccggggtcg acaccatgga ttccagtcct ggcaacatg 39 71
32 DNA Artificial Sequence Oligonucleotide primer ZC22055 71
tgcagtttaa actcaggaat cttcacttcc gt 32 72 40 PRT Artificial
Sequence Huzalpha11L-1 peptide 72 Gln Asp Arg His Met Ile Arg Met
Arg Gln Leu Ile Asp Ile Val Asp 1 5 10 15 Gln Leu Lys Asn Tyr Val
Asn Asp Leu Val Pro Glu Phe Leu Pro Ala 20 25 30 Pro Glu Asp Val
Glu Thr Asn Cys 35 40 73 32 PRT Artificial Sequence Huzalpha11L-3
peptide 73 Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu
Phe Leu 1 5 10 15 Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His
Gln His Leu Ser 20 25 30 74 29 DNA Artificial Sequence
Oligonucleotide primer ZC23444 74 gcccgggcgg atccatggat tccagtcct
29 75 32 DNA Artificial Sequence Oligonucleotide primer ZC23445 75
cgcgccctcg agtcaggaat cttcacttcc gt 32 76 17 DNA Artificial
Sequence Oligonucleotide primer ZC447 76 taacaatttc acacagg 17 77
18 DNA Artificial Sequence Oligonucleotide primer ZC976 77
cgttgtaaaa cgacggcc 18 78 66 DNA Artificial Sequence
Oligonucleotide primer ZC22128 78 tcaccacgcg aattcggtac cgctggttcc
gcgtggatcc caagatcgcc acatgattag 60 aatgcg 66 79 68 DNA Artificial
Sequence Oligonucleotide primer ZC22127 79 tctgtatcag gctgaaaatc
ttatctcatc cgccaaaaca tcaggaatct tcacttccgt 60 gtgttcta 68 80 40
DNA Artificial Sequence Oligonucleotide primer ZC19372 80
tgtcgatgaa gccctgaaag acgcgcagac taattcgagc 40 81 60 DNA Artificial
Sequence Oligonucleotide primer ZC19351 81 acgcgcagac taattcgagc
tcccaccatc accatcacca cgcgaattcg gtaccgctgg 60 82 60 DNA Artificial
Sequence Oligonucleotide primer ZC19352 82 actcactata gggcgaattg
cccgggggat ccacgcggaa ccagcggtac cgaattcgcg 60 83 42 DNA Artificial
Sequence Oligonucleotide primer ZC19371 83 acggccagtg aattgtaata
cgactcacta tagggcgaat tg 42 84 1560 DNA Artificial Sequence CDS
(1)...(1560) MBP-human zalpha11 Ligand fusion polynucleotide 84 atg
aaa act gaa gaa ggt aaa ctg gta atc tgg att aac ggc gat aaa 48 Met
Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5 10
15 ggc tat aac ggt ctc gct gaa gtc ggt aag aaa ttc gag aaa gat acc
96 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr
20 25 30 gga att aaa gtc acc gtt gag cat ccg gat aaa ctg gaa gag
aaa ttc 144 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu
Lys Phe 35 40 45 cca cag gtt gcg gca act ggc gat ggc cct gac att
atc ttc tgg gca 192 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile
Ile Phe Trp Ala 50 55 60 cac gac cgc ttt ggt ggc tac gct caa tct
ggc ctg ttg gct gaa atc 240 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser
Gly Leu Leu Ala Glu Ile 65 70 75 80 acc ccg gac aaa gcg ttc cag gac
aag ctg tat ccg ttt acc tgg gat 288 Thr Pro Asp Lys Ala Phe Gln Asp
Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 gcc gta cgt tac aac ggc
aag ctg att gct tac ccg atc gct gtt gaa 336 Ala Val Arg Tyr Asn Gly
Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 gcg tta tcg ctg
att tat aac aaa gat ctg ctg ccg aac ccg cca aaa 384 Ala Leu Ser Leu
Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 acc tgg
gaa gag atc ccg gcg ctg gat aaa gaa ctg aaa gcg aaa ggt 432 Thr Trp
Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140
aag agc gcg ctg atg ttc aac ctg caa gaa ccg tac ttc acc tgg ccg 480
Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145
150 155 160 ctg att gct gct gac ggg ggt tat gcg ttc aag tat gaa aac
ggc aag 528 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn
Gly Lys 165 170 175 tac gac att aaa gac gtg ggc gtg gat aac gct ggc
gcg aaa gcg ggt 576 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly
Ala Lys Ala Gly 180 185 190 ctg acc ttc ctg gtt gac ctg att aaa aac
aaa cac atg aat gca gac 624 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn
Lys His Met Asn Ala Asp 195 200 205 acc gat tac tcc atc gca gaa gct
gcc ttt aat aaa ggc gaa aca gcg 672 Thr Asp Tyr Ser Ile Ala Glu Ala
Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 atg acc atc aac ggc ccg
tgg gca tgg tcc aac atc gac acc agc aaa 720 Met Thr Ile Asn Gly Pro
Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 gtg aat tat
ggt gta acg gta ctg ccg acc ttc aag ggt caa cca tcc 768 Val Asn Tyr
Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255 aaa
ccg ttc gtt ggc gtg ctg agc gca ggt att aac gcc gcc agt ccg 816 Lys
Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265
270 aac aaa gag ctg gca aaa gag ttc ctc gaa aac tat ctg ctg act gat
864 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp
275 280 285 gaa ggt ctg gaa gcg gtt aat aaa gac aaa ccg ctg ggt gcc
gta gcg 912 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala
Val Ala 290 295 300 ctg aag tct tac gag gaa gag ttg gcg aaa gat cca
cgt att gcc gcc 960 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro
Arg Ile Ala Ala 305 310 315 320 acc atg gaa aac gcc cag aaa ggt gaa
atc atg ccg aac atc ccg cag 1008 Thr Met Glu Asn Ala Gln Lys Gly
Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 atg tcc gct ttc tgg tat
gcc gtg cgt act gcg gtg atc aac gcc gcc 1056 Met Ser Ala Phe Trp
Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 agc ggt cgt
cag act gtc gat gaa gcc ctg aaa gac gcg cag act aat 1104 Ser Gly
Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365
tcg agc tcc cac cat cac cat cac cac gcg aat tcg gta ccg ctg gtt
1152 Ser Ser Ser His His His His His His Ala Asn Ser Val Pro Leu
Val 370 375 380 ccg cgt gga tcc caa gat cgc cac atg att aga atg cgt
caa ctt ata 1200 Pro Arg Gly Ser Gln Asp Arg His Met Ile Arg Met
Arg Gln Leu Ile 385 390 395 400 gat att gtt gat cag ctg aaa aat tat
gtg aat gac ttg gtc cct gaa 1248 Asp Ile Val Asp Gln Leu Lys Asn
Tyr Val Asn Asp Leu Val Pro Glu 405 410 415 ttt ctg cca gct cca gaa
gat gta gag aca aac tgt gag tgg tca gct 1296 Phe Leu Pro Ala Pro
Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala 420 425 430 ttt tcc tgt
ttt cag aag gcc caa cta aag tca gca aat aca gga aac 1344 Phe Ser
Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn 435 440 445
aat gaa agg ata atc aat gta tca att aaa aag ctg aag agg aaa cca
1392 Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys
Pro 450 455 460 cct tcc aca aat gca ggg aga aga cag aaa cac aga cta
aca tgc cct 1440 Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg
Leu Thr Cys Pro 465 470 475 480 tca tgt gat tct tat gag aaa aaa cca
ccc aaa gaa ttc cta gaa aga 1488 Ser Cys Asp Ser Tyr Glu Lys Lys
Pro Pro Lys Glu Phe Leu Glu Arg 485 490 495 ttc aaa tca ctt ctc caa
aag atg att cat cag cat ctg tcc tct aga 1536 Phe Lys Ser Leu Leu
Gln Lys Met Ile His Gln His Leu Ser Ser Arg 500 505 510 aca cac gga
agt gaa gat tcc tga 1560 Thr His Gly Ser Glu Asp Ser 515 85 519 PRT
Artificial Sequence MBP-human zalpha11 Ligand fusion polypeptide 85
Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5
10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu
Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp
Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln
Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln
Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn
Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser
Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135
140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu
Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala
Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys
Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu
Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly
Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn
Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260
265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly
Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp
Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly
Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr
Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln
Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser
Ser His His His His His His Ala Asn Ser Val Pro Leu Val 370 375 380
Pro Arg Gly Ser Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile 385
390 395 400 Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val
Pro Glu 405 410 415 Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys
Glu Trp Ser Ala 420 425 430 Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys
Ser Ala Asn Thr
Gly Asn 435 440 445 Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu
Lys Arg Lys Pro 450 455 460 Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys
His Arg Leu Thr Cys Pro 465 470 475 480 Ser Cys Asp Ser Tyr Glu Lys
Lys Pro Pro Lys Glu Phe Leu Glu Arg 485 490 495 Phe Lys Ser Leu Leu
Gln Lys Met Ile His Gln His Leu Ser Ser Arg 500 505 510 Thr His Gly
Ser Glu Asp Ser 515 86 64 DNA Artificial Sequence Oligonucleotide
primer ZC22849 86 tcaccacgcg aattcggtac cgctggttcc gcgtggatcc
ccagatcgcc tcctgattag 60 actt 64 87 64 DNA Artificial Sequence
Oligonucleotide primer ZC22850 87 tctgtatcag gctgaaaatc ttatctcatc
cgccaaaaca ctaggagaga tgctgatgaa 60 tcat 64 88 1533 DNA Artificial
Sequence MBP-mouse zalpha11 Ligand fusion polynucleotide CDS
(1)...(1533) 88 atg aaa act gaa gaa ggt aaa ctg gta atc tgg att aac
ggc gat aaa 48 Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn
Gly Asp Lys 1 5 10 15 ggc tat aac ggt ctc gct gaa gtc ggt aag aaa
ttc gag aaa gat acc 96 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys
Phe Glu Lys Asp Thr 20 25 30 gga att aaa gtc acc gtt gag cat ccg
gat aaa ctg gaa gag aaa ttc 144 Gly Ile Lys Val Thr Val Glu His Pro
Asp Lys Leu Glu Glu Lys Phe 35 40 45 cca cag gtt gcg gca act ggc
gat ggc cct gac att atc ttc tgg gca 192 Pro Gln Val Ala Ala Thr Gly
Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 cac gac cgc ttt ggt
ggc tac gct caa tct ggc ctg ttg gct gaa atc 240 His Asp Arg Phe Gly
Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 acc ccg gac
aaa gcg ttc cag gac aag ctg tat ccg ttt acc tgg gat 288 Thr Pro Asp
Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 gcc
gta cgt tac aac ggc aag ctg att gct tac ccg atc gct gtt gaa 336 Ala
Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105
110 gcg tta tcg ctg att tat aac aaa gat ctg ctg ccg aac ccg cca aaa
384 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys
115 120 125 acc tgg gaa gag atc ccg gcg ctg gat aaa gaa ctg aaa gcg
aaa ggt 432 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala
Lys Gly 130 135 140 aag agc gcg ctg atg ttc aac ctg caa gaa ccg tac
ttc acc tgg ccg 480 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr
Phe Thr Trp Pro 145 150 155 160 ctg att gct gct gac ggg ggt tat gcg
ttc aag tat gaa aac ggc aag 528 Leu Ile Ala Ala Asp Gly Gly Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys 165 170 175 tac gac att aaa gac gtg ggc
gtg gat aac gct ggc gcg aaa gcg ggt 576 Tyr Asp Ile Lys Asp Val Gly
Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 ctg acc ttc ctg gtt
gac ctg att aaa aac aaa cac atg aat gca gac 624 Leu Thr Phe Leu Val
Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 acc gat tac
tcc atc gca gaa gct gcc ttt aat aaa ggc gaa aca gcg 672 Thr Asp Tyr
Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 atg
acc atc aac ggc ccg tgg gca tgg tcc aac atc gac acc agc aaa 720 Met
Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230
235 240 gtg aat tat ggt gta acg gta ctg ccg acc ttc aag ggt caa cca
tcc 768 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro
Ser 245 250 255 aaa ccg ttc gtt ggc gtg ctg agc gca ggt att aac gcc
gcc agt ccg 816 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala
Ala Ser Pro 260 265 270 aac aaa gag ctg gca aaa gag ttc ctc gaa aac
tat ctg ctg act gat 864 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn
Tyr Leu Leu Thr Asp 275 280 285 gaa ggt ctg gaa gcg gtt aat aaa gac
aaa ccg ctg ggt gcc gta gcg 912 Glu Gly Leu Glu Ala Val Asn Lys Asp
Lys Pro Leu Gly Ala Val Ala 290 295 300 ctg aag tct tac gag gaa gag
ttg gcg aaa gat cca cgt att gcc gcc 960 Leu Lys Ser Tyr Glu Glu Glu
Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 acc atg gaa aac
gcc cag aaa ggt gaa atc atg ccg aac atc ccg cag 1008 Thr Met Glu
Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 atg
tcc gct ttc tgg tat gcc gtg cgt act gcg gtg atc aac gcc gcc 1056
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340
345 350 agc ggt cgt cag act gtc gat gaa gcc ctg aaa gac gcg cag act
aat 1104 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln
Thr Asn 355 360 365 tcg agc tcc cac cat cac cat cac cac gcg aat tcg
gta ccg ctg gtt 1152 Ser Ser Ser His His His His His His Ala Asn
Ser Val Pro Leu Val 370 375 380 ccg cgt gga tcc cca gat cgc ctc ctg
att aga ctt cgt cac ctt att 1200 Pro Arg Gly Ser Pro Asp Arg Leu
Leu Ile Arg Leu Arg His Leu Ile 385 390 395 400 gac att gtt gaa cag
ctg aaa atc tat gaa aat gac ttg gat cct gaa 1248 Asp Ile Val Glu
Gln Leu Lys Ile Tyr Glu Asn Asp Leu Asp Pro Glu 405 410 415 ctt cta
tca gct cca caa gat gta aag ggg cac tgt gag cat gca gct 1296 Leu
Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys Glu His Ala Ala 420 425
430 ttt gcc tgt ttt cag aag gcc aaa ctc aag cca tca aac cct gga aac
1344 Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser Asn Pro Gly
Asn 435 440 445 aat aag aca ttc atc att gac ctc gtg gcc cag ctc agg
agg agg ctg 1392 Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu
Arg Arg Arg Leu 450 455 460 cct gcc agg agg gga gga aag aaa cag aag
cac ata gct aaa tgc cct 1440 Pro Ala Arg Arg Gly Gly Lys Lys Gln
Lys His Ile Ala Lys Cys Pro 465 470 475 480 tcc tgt gat tcg tat gag
aaa agg aca ccc aaa gaa ttc cta gaa aga 1488 Ser Cys Asp Ser Tyr
Glu Lys Arg Thr Pro Lys Glu Phe Leu Glu Arg 485 490 495 cta aaa tgg
ctc ctt caa aag atg att cat cag cat ctc tcc tga 1533 Leu Lys Trp
Leu Leu Gln Lys Met Ile His Gln His Leu Ser 500 505 510 89 510 PRT
Artificial Sequence MBP-mouse zalpha11 Ligand fusion polypeptide 89
Met Lys Thr Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys 1 5
10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp
Thr 20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu
Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp
Ile Ile Phe Trp Ala 50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln
Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr Pro Asp Lys Ala Phe Gln
Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90 95 Ala Val Arg Tyr Asn
Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu 100 105 110 Ala Leu Ser
Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys 115 120 125 Thr
Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly 130 135
140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro
145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu
Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala
Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys
Asn Lys His Met Asn Ala Asp 195 200 205 Thr Asp Tyr Ser Ile Ala Glu
Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215 220 Met Thr Ile Asn Gly
Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn
Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser 245 250 255
Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro 260
265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr
Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly
Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp
Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly
Glu Ile Met Pro Asn Ile Pro Gln 325 330 335 Met Ser Ala Phe Trp Tyr
Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340 345 350 Ser Gly Arg Gln
Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 Ser Ser
Ser His His His His His His Ala Asn Ser Val Pro Leu Val 370 375 380
Pro Arg Gly Ser Pro Asp Arg Leu Leu Ile Arg Leu Arg His Leu Ile 385
390 395 400 Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp Leu Asp
Pro Glu 405 410 415 Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys
Glu His Ala Ala 420 425 430 Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys
Pro Ser Asn Pro Gly Asn 435 440 445 Asn Lys Thr Phe Ile Ile Asp Leu
Val Ala Gln Leu Arg Arg Arg Leu 450 455 460 Pro Ala Arg Arg Gly Gly
Lys Lys Gln Lys His Ile Ala Lys Cys Pro 465 470 475 480 Ser Cys Asp
Ser Tyr Glu Lys Arg Thr Pro Lys Glu Phe Leu Glu Arg 485 490 495 Leu
Lys Trp Leu Leu Gln Lys Met Ile His Gln His Leu Ser 500 505 510 90
22 DNA Artificial Sequence Oligonucleotide primer ZC22281 90
tgtgaatgac ttggtccctg aa 22 91 23 DNA Artificial Sequence
Oligonucleotide primer ZC22279 91 aacaggaaaa agctgaccac tca 23 92
31 DNA Artificial Sequence Human zalpha11 Ligand TaqMan probe, ZG32
92 tctgccagct ccagaagatg tagagacaaa c 31 93 20 DNA Artificial
Sequence Oligonucleotide primer ZC22277 93 ccaggagtgt ggcagctttc 20
94 21 DNA Artificial Sequence Oligonucleotide primer ZC22276 94
gcttgccctt cagcatgtag a 21 95 23 DNA Artificial Sequence Human
zalpha11 TaqMan probe, ZG31 95 cggctccccc tttcaacgtg act 23 96 1821
DNA Artificial Sequence CDS (1)...(1821) MBP-zalpha11 soluble
receptor polynucleotide sequence 96 atg aaa atc gaa gaa ggt aaa ctg
gta atc tgg att aac ggc gat aaa 48 Met Lys Ile Glu Glu Gly Lys Leu
Val Ile Trp Ile Asn Gly Asp Lys 1 5 10 15 ggc tat aac ggt ctc gct
gaa gtc ggt aag aaa ttc gag aaa gat acc 96 Gly Tyr Asn Gly Leu Ala
Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30 gga att aaa gtc
acc gtt gag cat ccg gat aaa ctg gaa gag aaa ttc 144 Gly Ile Lys Val
Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 cca cag
gtt gcg gca act ggc gat ggc cct gac att atc ttc tgg gca 192 Pro Gln
Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60
cac gac cgc ttt ggt ggc tac gct caa tct ggc ctg ttg gct gaa atc 240
His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65
70 75 80 acc ccg gac aaa gcg ttc cag gac aag ctg tat ccg ttt acc
tgg gat 288 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr
Trp Asp 85 90 95 gcc gta cgt tac aac ggc aag ctg att gct tac ccg
atc gct gtt gaa 336 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro
Ile Ala Val Glu 100 105 110 gcg tta tcg ctg att tat aac aaa gat ctg
ctg ccg aac ccg cca aaa 384 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu
Leu Pro Asn Pro Pro Lys 115 120 125 acc tgg gaa gag atc ccg gcg ctg
gat aaa gaa ctg aaa gcg aaa ggt 432 Thr Trp Glu Glu Ile Pro Ala Leu
Asp Lys Glu Leu Lys Ala Lys Gly 130 135 140 aag agc gcg ctg atg ttc
aac ctg caa gaa ccg tac ttc acc tgg ccg 480 Lys Ser Ala Leu Met Phe
Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 ctg att gct
gct gac ggg ggt tat gcg ttc aag tat gaa aac ggc aag 528 Leu Ile Ala
Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 tac
gac att aaa gac gtg ggc gtg gat aac gct ggc gcg aaa gcg ggt 576 Tyr
Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185
190 ctg acc ttc ctg gtt gac ctg att aaa aac aaa cac atg aat gca gac
624 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp
195 200 205 acc gat tac tcc atc gca gaa gct gcc ttt aat aaa ggc gaa
aca gcg 672 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu
Thr Ala 210 215 220 atg acc atc aac ggc ccg tgg gca tgg tcc aac atc
gac acc agc aaa 720 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile
Asp Thr Ser Lys 225 230 235 240 gtg aat tat ggt gta acg gta ctg ccg
acc ttc aag ggt caa cca tcc 768 Val Asn Tyr Gly Val Thr Val Leu Pro
Thr Phe Lys Gly Gln Pro Ser 245 250 255 aaa ccg ttc gtt ggc gtg ctg
agc gca ggt att aac gcc gcc agt ccg 816 Lys Pro Phe Val Gly Val Leu
Ser Ala Gly Ile Asn Ala Ala Ser Pro 260 265 270 aac aaa gag ctg gca
aaa gag ttc ctc gaa aac tat ctg ctg act gat 864 Asn Lys Glu Leu Ala
Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285 gaa ggt ctg
gaa gcg gtt aat aaa gac aaa ccg ctg ggt gcc gta gcg 912 Glu Gly Leu
Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 ctg
aag tct tac gag gaa gag ttg gcg aaa gat cca cgt att gcc gcc 960 Leu
Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310
315 320 acc atg gaa aac gcc cag aaa ggt gaa atc atg ccg aac atc ccg
cag 1008 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile
Pro Gln 325 330 335 atg tcc gct ttc tgg tat gcc gtg cgt act gcg gtg
atc aac gcc gcc 1056 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala
Val Ile Asn Ala Ala 340 345 350 agc ggt cgt cag act gtc gat gaa gcc
ctg aaa gac gcg cag act aat 1104 Ser Gly Arg Gln Thr Val Asp Glu
Ala Leu Lys Asp Ala Gln Thr Asn 355 360 365 tcg agc tcc cac cat cac
cat cac cac gcg aat tcg gta ccg ctg gtt 1152 Ser Ser Ser His His
His His His His Ala Asn Ser Val Pro Leu Val 370 375 380 ccg cgt gga
tcc tgc ccc gac ctc gtc tgc tac acc gat tac ctc cag 1200 Pro Arg
Gly Ser Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln 385 390 395
400 acg gtc atc tgc atc ctg gaa atg tgg aac ctc cac ccc agc acg ctc
1248 Thr Val Ile Cys Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr
Leu 405 410 415 acc ctt acc tgg caa gac cag tat gaa gag ctg aag gac
gag gcc acc 1296 Thr Leu Thr Trp Gln Asp Gln Tyr Glu Glu Leu Lys
Asp Glu Ala Thr 420 425 430 tcc tgc agc ctc cac agg tcg gcc cac aat
gcc acg cat gcc acc tac 1344 Ser Cys Ser Leu His Arg Ser Ala His
Asn Ala Thr His Ala Thr Tyr 435 440 445 acc tgc cac atg gat gta ttc
cac ttc atg gcc gac gac att ttc agt 1392 Thr Cys His Met Asp Val
Phe His Phe Met Ala Asp Asp Ile Phe Ser 450 455 460 gtc aac atc aca
gac cag tct ggc aac tac tcc cag gag tgt ggc agc 1440 Val Asn Ile
Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser 465 470 475 480
ttt ctc ctg gct gag agc atc aag ccg gct ccc cct ttc aac gtg act
1488 Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val
Thr 485 490 495 gtg acc ttc tca gga cag tat aat atc tcc tgg cgc tca
gat tac gaa 1536 Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser Asp Tyr Glu 500 505
510 gac cct gcc ttc tac atg ctg aag ggc aag ctt cag tat gag ctg cag
1584 Asp Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu
Gln 515 520 525 tac agg aac cgg gga gac ccc tgg gct gtg agt ccg agg
aga aag ctg 1632 Tyr Arg Asn Arg Gly Asp Pro Trp Ala Val Ser Pro
Arg Arg Lys Leu 530 535 540 atc tca gtg gac tca aga agt gtc tcc ctc
ctc ccc ctg gag ttc cgc 1680 Ile Ser Val Asp Ser Arg Ser Val Ser
Leu Leu Pro Leu Glu Phe Arg 545 550 555 560 aaa gac tcg agc tat gag
ctg cag gtg cgg gca ggg ccc atg cct ggc 1728 Lys Asp Ser Ser Tyr
Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly 565 570 575 tcc tcc tac
cag ggg acc tgg agt gaa tgg agt gac ccg gtc atc ttt 1776 Ser Ser
Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe 580 585 590
cag acc cag tca gag gag tta aag gaa ggc tgg aac cct cac tag 1821
Gln Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His 595 600 605
97 606 PRT Artificial Sequence MBP-zalpha11 soluble receptor
polypeptide sequence 97 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp
Ile Asn Gly Asp Lys 1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly
Lys Lys Phe Glu Lys Asp Thr 20 25 30 Gly Ile Lys Val Thr Val Glu
His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45 Pro Gln Val Ala Ala
Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60 His Asp Arg
Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile 65 70 75 80 Thr
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90
95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro
Pro Lys 115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu
Lys Ala Lys Gly 130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu
Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175 Tyr Asp Ile Lys Asp
Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190 Leu Thr Phe
Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205 Thr
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215
220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys
225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly
Gln Pro Ser 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile
Asn Ala Ala Ser Pro 260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu
Glu Asn Tyr Leu Leu Thr Asp 275 280 285 Glu Gly Leu Glu Ala Val Asn
Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300 Leu Lys Ser Tyr Glu
Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met
Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330 335
Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala 340
345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr
Asn 355 360 365 Ser Ser Ser His His His His His His Ala Asn Ser Val
Pro Leu Val 370 375 380 Pro Arg Gly Ser Cys Pro Asp Leu Val Cys Tyr
Thr Asp Tyr Leu Gln 385 390 395 400 Thr Val Ile Cys Ile Leu Glu Met
Trp Asn Leu His Pro Ser Thr Leu 405 410 415 Thr Leu Thr Trp Gln Asp
Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr 420 425 430 Ser Cys Ser Leu
His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr 435 440 445 Thr Cys
His Met Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser 450 455 460
Val Asn Ile Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser 465
470 475 480 Phe Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn
Val Thr 485 490 495 Val Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg
Ser Asp Tyr Glu 500 505 510 Asp Pro Ala Phe Tyr Met Leu Lys Gly Lys
Leu Gln Tyr Glu Leu Gln 515 520 525 Tyr Arg Asn Arg Gly Asp Pro Trp
Ala Val Ser Pro Arg Arg Lys Leu 530 535 540 Ile Ser Val Asp Ser Arg
Ser Val Ser Leu Leu Pro Leu Glu Phe Arg 545 550 555 560 Lys Asp Ser
Ser Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly 565 570 575 Ser
Ser Tyr Gln Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe 580 585
590 Gln Thr Gln Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His 595 600
605 98 65 DNA Artificial Sequence Oligonucleotide primer ZC20187 98
tcaccacgcg aattcggtac cgctggttcc gcgtggatcc tgccccgacc tcgtctgcta
60 caccg 65 99 68 DNA Artificial Sequence Oligonucleotide primer
ZC20185 99 tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ctagtgaggg
ttccagcctt 60 cctttaac 68 100 21 DNA Artificial Sequence
Oligonucleotide primer ZC22452 100 tcttcttggg gacactggtc c 21 101
21 DNA Artificial Sequence Oligonucleotide primer ZC22451 101
aatcatgtgg cgatcttgac c 21 102 21 DNA Artificial Sequence
Oligonucleotide primer ZC22450 102 cagactaaca tgcccttcat g 21 103
23 DNA Artificial Sequence Oligonucleotide primer ZC22449 103
ttcacttccg tgtgttctag agg 23 104 18 DNA Artificial Sequence
Oligonucleotide primer ZC23771 104 accaccttcc acaaatgc 18 105 1347
DNA Homo sapiens 105 gaattcaccc attctctctt tttcctgtca aagatgcaga
tggggcacat ttcgttgact 60 ccatcaatcc ctgcccccac acattagcac
atgcacacgt atacctagcc agtggaaaag 120 aaaaaagagt tactcacatt
catccatttt acaaagattt ccaggctgca atgggagggc 180 tttacctctc
cctgaaggat gaataaatag gtagcttaac tgacaacctg ttctcagtca 240
agctgaagtg aaaacgagac caaggtctag ctctactgtt ggtacttatg agatccagtc
300 ctggcaacat ggagaggatt gtcatctgtc tgatggtcat cttcttgggg
acactggtcc 360 acaaatcaag ctcccaaggt caagatcgcc acatgattag
aatgcgtcaa cttatagata 420 ttgttgatca gctgaaaaat tatgtgaatg
acttggtaag actatatttg tcacaacaaa 480 atctaaatca tacttttcaa
ttaatataaa aggagggttt ggcttataaa aataactcag 540 aacaaatttt
cttttgctct aggtccctga atttctgcca gctccagaag atgtagaggt 600
aagaccagtt gaatttattt ctgaaaatac attggacata agtttttaaa tccaataaga
660 aagacattag catgattata taggagtata ctgaatttta atgaacttag
cggtctaata 720 attgatgaaa tatttattta tattttggtt aaattcattg
atttaccaaa aaccaactaa 780 aaaatgctat attatattcc tcataaacta
tgtttatctt caagaatctc taagagtact 840 cctaagtagt attgctgaga
cagaataaca aaactagaaa cgaaatctat actctgatca 900 gtttctgaac
aatgcacagc tagttactct ttaagagccc ttgggcatga aagcttttga 960
gccttctttg ttatcctacc gaagaaacat agatacatac agtaggaagc agaattaacc
1020 ttttaataac aaacttaaaa aagaaagaaa gaaagaatta gattacaggg
acagcatgga 1080 gaaatggtgg tgtggaaatc aaagctgtcc tttagaatat
aattcacata taccttggcc 1140 tcagtgagtc ttgtctttgg ccttccgtga
ggtcttttga aagaaccatt ttcaacaatt 1200 catcccgtct cttaagccat
ttaaatccat tagagttcca ggaagaagag gcctggcatg 1260 agttcagagt
gctgtcccgc tgatcttttt ctcagtaact tctacgatct gatcttctgg 1320
tctggtaccc tgaggtataa atgcaat 1347 106 1656 DNA Homo sapiens 106
cctcaactgc ttgattcagg cagaatccta accctaaact gagctgggag tatgaaaagg
60 gttttagaaa agtcatggtg tgatctatgg caagtatatt gattcttaga
tgtaaaatat 120 gctatcagag ggaggtaccc acttcctttc tccaaaggag
gggctttaat tcattttctt 180 catctgttaa ctttacaaat atatgttgat
cattaactgg caagacacta tgcctggcgc 240 tgtacagaat aaaatgctgc
tcaagacatg tcatgataga tacattaaca gaaaccacaa 300 acaaatgaaa
aatgttcttc atcagactat aacataattt acccaaagct gccactagtc 360
acagtgtaag ttttagagcc tcataactca gcaaatgtgt cctaaaccga actaactctc
420 ctttataaaa cacaaaggtc ttgtccacca cccagacatc aaaatggtcc
tctgtgtagc 480 atcaggaata aagcattgtg aagaagtgag gctcctttct
ctcttatctg cgaagcaggg 540 gattgtccct ttttcccatc ccaaagatta
agtaggaggt gaaatcatac ctcactcatc 600 tgttgaaacg atgtaatgca
cgacattgca gaagagatag aaatagagga ttgggaaagc 660 tatcttttac
tttctgaata atgtttgtta acatatatac aaattgttta tctttcagac 720
aaactgtgag tggtcagctt tttcctgttt tcagaaggcc caactaaagt cagcaaatac
780 aggaaacaat gaaaggataa taaatgtatc aattaaaaag ctgaagagga
aaccaccttc 840 cacaaatgca gggagaagac agaaacacag actagtaaga
ttgtcatttg tcatctctct 900 tatttgtact tataaactat atatcttgca
ttacataaac atacacacac acctgtagcc 960 agggctgctg gtgtcttcct
tacctatagt tatgccttat tatacatggt gctttttttt 1020 tttaagacag
agtctcactc tgtcacccag gctggagtgc agtggcgtga tctctgctca 1080
ccgcaagatc cacctcccgg tttcacgcca ttctcctcct acctcagcct cctgagtacc
1140 tgggactaca ggtgcccgcc accatgcccg gctaattttg tttttgtatt
tttagtaaag 1200 acagggtttc accatgttag ccaggatggt ctcgatctcc
tgaccccgtg atccgcccgc 1260 cttggcctcc caaagtgtgg ggattacagg
catgagccac cgcacccggc ctatacgtgg 1320 tgcattttaa gaagtagggt
cactctttta agcccacaga cttgaaagta ttcaaaaacc 1380 caattataat
ttcctagtag tccttggcag ctggaatatg ttaatatagc ttctcaaggt 1440
gaggaagtca ttaggcagag aatccaactg tgattttgga gttaagaact atttcctctc
1500 atatggtcac agataacttg tattcttatt aacaggagct agatcctagc
tttctaacaa 1560 gaaaagagcc tacaagaaga ctagggcaaa tcttaaactt
tgcctcctct ctaaatcata 1620 ttactatctg tacatcagca gagtcagtat tgaatt
1656 107 644 DNA Homo sapiens 107 agctaaactt agaactctcc agttaagcat
gttcatctta tagatgagga aaagtgagat 60 ctacaaagga gttaagtcac
tagccccaag ttccataaat agtgtcagaa tgagaattag 120 aacgtatatc
tactatcttt tagtgaaatg ctctcactac aacatcacac tggcattgag 180
atgctaacta ccaagcaatg gcttggtgtt tggatctaaa tagggataaa gacaaagagc
240 ataaactaag aaagcttttt aaaaatctaa gtgagcaatc catatatgaa
aaactgttca 300 atctccctag taatcacata aatgcgagtt aaaacaagga
aatcctgttt tttccaatta 360 aacattttaa acaataccct ataataataa
gaatgctcca agtgaaaaga ggtaaaaccc 420 tttataatgt atatcaaagc
cttaaaattt ttatcccttt aatttagtaa ttctacttct 480 aggaatatat
caaataagca aagatatata tgaaaaatta tttacagaga tgttctttgg 540
agtaatgtag acaaaaataa aaagttagat acagctgggt gtggtggctc atgcctgtat
600 tcccagcact ttgggaggcc gaggcaggcg gatcacctga gatc 644 108 645
DNA Homo sapiens misc_feature (1)...(645) n = A,T,C or G 108
aaaaaaaaaa aaaaaaaaaa gttagatgca ccnttgggtc caaaaatagt aagagtgcat
60 cctatgtgga aacagaccaa ccactacatg tcatattttt gaagattatt
taacacttag 120 gaaatcctgt gatatgttaa gtgtgaaaaa aaaaaagcaa
atcaccaact ggtataaata 180 atgtaaatgc acaataataa ttaaaaatac
ccaaaacaca gagagaatat acattaaaac 240 attgcagtgg gattcctatc
tctgggaatg ggattacaag gactttttcc attgttactt 300 tccaaacagt
tttatgtact tctcgaatgt ttttcagtga acataattta tgtttttaat 360
gaaaaaaaat tttaagaaac attttattac gaaaaaaatt ttaaagaaga ctgttacttt
420 ttcattgatt tctagacatg cccttcatgt gattcttatg agaaaaaacc
acccaaagaa 480 ttcctagaaa gattcaaatc acttctccaa aaggtatcta
ccttaagttt catttgattt 540 tctgctttat ctttacctat ccagatttgc
ttcttagtta ctcacggtat actatttcca 600 cagatgattc atcagcatct
gtcctctaga acacacggaa gtgaa 645 109 36 DNA Artificial Sequence
Oligonucleotide primer ZC25970 109 atgcattcta gactaggaga gatgctgatg
aatcat 36 110 36 DNA Artificial Sequence Oligonucleotide primer
ZC25969 110 atgcattccg gacataaatc aagcccccaa gggcca 36 111 153 PRT
Homo sapiens 111 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu
Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser
Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp
Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro
Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys
Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95
Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100
105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr
Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp
Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150
112 153 PRT Homo sapiens 112 Met Gly Leu Thr Ser Gln Leu Leu Pro
Pro Leu Phe Phe Leu Leu Ala 1 5 10 15 Cys Ala Gly Asn Phe Val His
Gly His Lys Cys Asp Ile Thr Leu Gln 20 25 30 Glu Ile Ile Lys Thr
Leu Asn Ser Leu Thr Glu Gln Lys Thr Leu Cys 35 40 45 Thr Glu Leu
Thr Val Thr Asp Ile Phe Ala Ala Ser Lys Asn Thr Thr 50 55 60 Glu
Lys Glu Thr Phe Cys Arg Ala Ala Thr Val Leu Arg Gln Phe Tyr 65 70
75 80 Ser His His Glu Lys Asp Thr Arg Cys Leu Gly Ala Thr Ala Gln
Gln 85 90 95 Phe His Arg His Lys Gln Leu Ile Arg Phe Leu Lys Arg
Leu Asp Arg 100 105 110 Asn Leu Trp Gly Leu Ala Gly Leu Asn Ser Cys
Pro Val Lys Glu Ala 115 120 125 Asn Gln Ser Thr Leu Glu Asn Phe Leu
Glu Arg Leu Lys Thr Ile Met 130 135 140 Arg Glu Lys Tyr Ser Lys Cys
Ser Ser 145 150 113 162 PRT Homo sapiens 113 Met Arg Ile Ser Lys
Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu
Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val
Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40
45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp
Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu
Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser
Ile His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn
Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys
Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu
Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr
Ser 114 144 PRT Homo sapiens 114 Met Trp Leu Gln Ser Leu Leu Leu
Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser
Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile
Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala
Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60
Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65
70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu
Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr
Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser
Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe
Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 115 538 PRT Homo
sapiens 115 Met Pro Arg Gly Trp Ala Ala Pro Leu Leu Leu Leu Leu Leu
Gln Gly 1 5 10 15 Gly Trp Gly Cys Pro Asp Leu Val Cys Tyr Thr Asp
Tyr Leu Gln Thr 20 25 30 Val Ile Cys Ile Leu Glu Met Trp Asn Leu
His Pro Ser Thr Leu Thr 35 40 45 Leu Thr Trp Gln Asp Gln Tyr Glu
Glu Leu Lys Asp Glu Ala Thr Ser 50 55 60 Cys Ser Leu His Arg Ser
Ala His Asn Ala Thr His Ala Thr Tyr Thr 65 70 75 80 Cys His Met Asp
Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val 85 90 95 Asn Ile
Thr Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe 100 105 110
Leu Leu Ala Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val
115
120 125 Thr Phe Ser Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu
Asp 130 135 140 Pro Ala Phe Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu
Leu Gln Tyr 145 150 155 160 Arg Asn Arg Gly Asp Pro Trp Ala Val Ser
Pro Arg Arg Lys Leu Ile 165 170 175 Ser Val Asp Ser Arg Ser Val Ser
Leu Leu Pro Leu Glu Phe Arg Lys 180 185 190 Asp Ser Ser Tyr Glu Leu
Gln Val Arg Ala Gly Pro Met Pro Gly Ser 195 200 205 Ser Tyr Gln Gly
Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln 210 215 220 Thr Gln
Ser Glu Glu Leu Lys Glu Gly Trp Asn Pro His Leu Leu Leu 225 230 235
240 Leu Leu Leu Leu Val Ile Val Phe Ile Pro Ala Phe Trp Ser Leu Lys
245 250 255 Thr His Pro Leu Trp Arg Leu Trp Lys Lys Ile Trp Ala Val
Pro Ser 260 265 270 Pro Glu Arg Phe Phe Met Pro Leu Tyr Lys Gly Cys
Ser Gly Asp Phe 275 280 285 Lys Lys Trp Val Gly Ala Pro Phe Thr Gly
Ser Ser Leu Glu Leu Gly 290 295 300 Pro Trp Ser Pro Glu Val Pro Ser
Thr Leu Glu Val Tyr Ser Cys His 305 310 315 320 Pro Pro Arg Ser Pro
Ala Lys Arg Leu Gln Leu Thr Glu Leu Gln Glu 325 330 335 Pro Ala Glu
Leu Val Glu Ser Asp Gly Val Pro Lys Pro Ser Phe Trp 340 345 350 Pro
Thr Ala Gln Asn Ser Gly Gly Ser Ala Tyr Ser Glu Glu Arg Asp 355 360
365 Arg Pro Tyr Gly Leu Val Ser Ile Asp Thr Val Thr Val Leu Asp Ala
370 375 380 Glu Gly Pro Cys Thr Trp Pro Cys Ser Cys Glu Asp Asp Gly
Tyr Pro 385 390 395 400 Ala Leu Asp Leu Asp Ala Gly Leu Glu Pro Ser
Pro Gly Leu Glu Asp 405 410 415 Pro Leu Leu Asp Ala Gly Thr Thr Val
Leu Ser Cys Gly Cys Val Ser 420 425 430 Ala Gly Ser Pro Gly Leu Gly
Gly Pro Leu Gly Ser Leu Leu Asp Arg 435 440 445 Leu Lys Pro Pro Leu
Ala Asp Gly Glu Asp Trp Ala Gly Gly Leu Pro 450 455 460 Trp Gly Gly
Arg Ser Pro Gly Gly Val Ser Glu Ser Glu Ala Gly Ser 465 470 475 480
Pro Leu Ala Gly Leu Asp Met Asp Thr Phe Asp Ser Gly Phe Val Gly 485
490 495 Ser Asp Cys Ser Ser Pro Val Glu Cys Asp Phe Thr Ser Pro Gly
Asp 500 505 510 Glu Gly Pro Pro Arg Ser Tyr Leu Arg Gln Trp Val Val
Ile Pro Pro 515 520 525 Pro Leu Ser Ser Pro Gly Pro Gln Ala Ser 530
535
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References