U.S. patent application number 12/506060 was filed with the patent office on 2010-02-18 for human tumor necrosis factor tr21 and methods based thereon.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to David Hilbert, Steven M. Ruben.
Application Number | 20100040624 12/506060 |
Document ID | / |
Family ID | 37018867 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100040624 |
Kind Code |
A1 |
Ruben; Steven M. ; et
al. |
February 18, 2010 |
Human Tumor Necrosis Factor TR21 and Methods Based Thereon
Abstract
The present invention relates to TR21 polypeptides. In
particular, isolated nucleic acid molecules are provided encoding
human TR21 protein. TR21 polypeptides are also provided as are
vectors, host cells and recombinant methods for producing the same.
The invention further relates to screening methods for identifying
agonists and antagonists of TR21 activity.
Inventors: |
Ruben; Steven M.;
(Brookeville, MD) ; Hilbert; David; (Bethesda,
MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
37018867 |
Appl. No.: |
12/506060 |
Filed: |
July 20, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11517438 |
Sep 8, 2006 |
|
|
|
12506060 |
|
|
|
|
10229352 |
Aug 28, 2002 |
7112410 |
|
|
11517438 |
|
|
|
|
60315357 |
Aug 29, 2001 |
|
|
|
Current U.S.
Class: |
424/139.1 ;
424/133.1; 424/183.1; 435/320.1; 435/7.1; 514/1.1; 530/324;
530/387.9; 536/23.1 |
Current CPC
Class: |
A61P 13/12 20180101;
A61P 29/00 20180101; A61P 25/00 20180101; G01N 33/6863 20130101;
A61P 3/10 20180101; A61P 37/00 20180101; G01N 2800/101 20130101;
G01N 2333/715 20130101; A61P 35/02 20180101; G01N 2500/02 20130101;
A61P 37/06 20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/139.1 ;
530/324; 536/23.1; 435/320.1; 530/387.9; 514/12; 435/7.1;
424/183.1; 424/133.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/00 20060101 C07K014/00; C07H 21/00 20060101
C07H021/00; C12N 15/63 20060101 C12N015/63; C07K 16/00 20060101
C07K016/00; A61K 38/16 20060101 A61K038/16; G01N 33/53 20060101
G01N033/53; A61P 37/00 20060101 A61P037/00 |
Claims
1. An isolated polypeptide comprising an amino acid sequence at
least 90% identical to a sequence selected from the group
consisting of: (a) amino acids from 2 to 81 in SEQ ID NO:2 (FIG.
1); (b) amino acids from 2 to 38 in SEQ ID NO:2 (FIG. 1); (c) amino
acids from 2 to 46 in SEQ ID NO:2 (FIG. 1); (d) amino acids from 2
to 62 in SEQ ID NO:2 (FIG. 1); and optionally, a heterologous
polypeptide sequence
2. An isolated nucleic acid molecule encoding the polypeptide of
claim 1.
3. A recombinant vector or a recombinant host cell comprising
nucleic acid molecule of claim 2.
4. An isolated antibody that binds specifically to a polypeptide
consisting of amino acid residues selected from the group
consisting of: (a) amino acids 2 to 81 in SEQ ID NO:2; (b) amino
acids 2 to 38 in SEQ ID NO:2; (c) amino acids 2 to 46 in SEQ ID
NO:2; (d) amino acids 2 to 62 in SEQ ID NO:2; (e) amino acids 3 to
15 in SEQ ID NO:2; (f) amino acids 39 to 45 in SEQ ID NO:2; (g)
amino acids 51 to 53 in SEQ ID NO:2; (h) amino acids 59 to 61 in
SEQ ID NO:2; (i) amino acids 104 to 112 in SEQ ID NO:2; (j) amino
acids 115 to 122 in SEQ ID NO:2; (k) amino acids 136 to 138 in SEQ
ID NO:2; (l) amino acids 147 to 153 in SEQ ID NO:2; and (m) amino
acids 178 to 182 in SEQ ID NO:2.
5. The antibody of claim 4 which binds a polypeptide consisting of
amino acids 2 to 38 of SEQ ID NO:2 and a polypeptide consisting of
amino acids 2 to 81 of SEQ ID NO:2.
6. A method of treating an autoimmune disease or condition
associated with an autoimmune disease, comprising administering an
effective amount of the polypeptide of claim 1, to a patient in
need thereof; wherein said autoimmune disease is selected from the
group consisting of: (a) rheumatoid arthritis; (b) systemic lupus
erythmatosus; (c) multiple sclerosis; (d) Sjogren's syndrome; (e)
IgA nephropathy; (f) glomerulonephritis; (g) diabetes mellitus; and
(h) myasthenia gravis.
7. A method of treating an autoimmune disease or condition
associated with an autoimmune disease comprising, administering an
effective amount of the antibody of claim 4, to a patient in need
thereof; wherein said autoimmune disease is selected from the group
consisting of: (a) rheumatoid arthritis; (b) systemic lupus
erythmatosus; (c) multiple sclerosis; (d) Sjogren's syndrome; (e)
IgA nephropathy; (f) glomerulonephritis; (g) diabetes mellitus; and
(h) myasthenia gravis.
8. A method of treating an immunodeficiency or condition associated
with an immunodeficiency, comprising administering an effective
amount of the antibody of claim 4, to a patient in need thereof;
wherein said immunodeficiency is selected from the group consisting
of: (a) common variable immunodeficiency (CVID); (b) acquired
immunodeficiency syndrome (AIDS); (c) severe combined
immunodeficiency (SCID); (d) selective IgA deficiency; (e)
hypogammaglobulinemia; and (f) Wiskott-Aldrich syndrome.
9. A method of diagnosing an immunodeficiency or an autoimmune
disease, comprising contacting the polypeptide of claim 1 with
cells or bodily fluids from an individual, and assaying for binding
to said polypeptide.
10. A method of diagnosing an immunodeficiency or an autoimmune
disease, comprising contacting the antibody of claim 4 with cells
or bodily fluids from an individual, and assaying for binding to
said antibody.
11. A method of increasing B cell proliferation or immunoglobulin
production, comprising administering an effective amount of the
antibody of claim 4, to a patient in need thereof.
12. A method of inhibiting B cell proliferation or immunoglobulin
production, comprising administering an effective amount of the
polypeptide of claim 1 to a patient in need thereof.
13. A method of inhibiting B cell proliferation or immunoglobulin
production, comprising administering an effective amount of the
antibody of claim 4 to a patient in need thereof.
14. A method of killing a cell that expresses TR21 polypeptide on
its cell surface, comprising contacting said cell with an antibody
or portion thereof that specifically binds a polypeptide consisting
of amino acid residues 1-184 of SEQ ID NO:2; wherein said antibody
or portion thereof is conjugated to a toxin.
15. The method of claim 14 performed in vivo.
16. The method of claim 14 wherein said antibody or portion thereof
specifically binds to a polypeptide consisting of amino acid
residues 1-81 of SEQ ID NO:2.
17. The method of claim 14 wherein the antibody or portion thereof
is monoclonal, chimeric or human.
18. The method of claim 14 wherein the antibody or portion thereof
is a single chain antibody or a Fab fragment.
19. The method of claim 14 wherein said cell is a lymphocyte.
20. The method of claim 19 wherein said lymphocyte is leukemic.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a continuation of U.S. application Ser.
No. 11/517,438, filed Sep. 8, 2006, which is a divisional of U.S.
application Ser. No. 10/229,352, filed Aug. 28, 2002, which claims
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application No. 60/315,357 filed Aug. 29, 2001, each of which is
hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING AS TEXT FILE
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text file. The text file contains a
document entitled "PF562D1C1_SequenceListing.txt" (4,724 bytes,
created Jul. 17, 2009), which is incorporated by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to TR21, a member of the tumor
necrosis factor family of receptors. More specifically, isolated
nucleic acid molecules are provided encoding TR21 and variants
thereof. TR21 polypeptides are also provided, as are vectors, host
cells, and recombinant and synthetic methods for producing the
same. The invention also relates to diagnostic and therapeutic
methods using TR21 nucleic acid molecules, polypeptides and/or TR21
agonists or antagonists, such as for example agonistic anti-TR21
antibodies, and antagonistic anti-TR21 antibodies. The invention
further relates to screening methods for identifying agonists and
antagonists of TR21 activity.
[0005] 2. Related Art
[0006] Many biological actions, for instance, response to certain
stimuli and natural biological processes, are controlled by
factors, such as cytokines. Many cytokines act through receptors by
engaging the receptor and producing an intra-cellular response.
[0007] For example, tumor necrosis factors (TNF) alpha and beta are
cytokines, which act through TNF receptors to regulate numerous
biological processes, including protection against infection and
induction of shock and inflammatory disease. The TNF molecules
belong to the "TNF-ligand" superfamily, and act together with their
receptors or counter-ligands, the "TNF-receptor" superfamily. So
far, more than ten members of the TNF ligand superfamily have been
identified and more than ten members of the TNF-receptor
superfamily have been characterized.
[0008] Among the ligands there are included TNF-alpha,
lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta
(found in complex heterotrimer LT-2-beta), FasL, CD40L, CD27L,
CD30L, 4-1BBL, OX40L and nerve growth factor (NGF). The superfamily
of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF
receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30,
4-1BB, OX40, low affinity p75 and NGF-receptor (A. Meager,
Biologicals 22:291-295 (1994)).
[0009] Many members of the TNF-ligand superfamily are expressed by
activated T-cells, implying that they are necessary for T-cell
interactions with other cell types which underlie cell ontogeny and
functions. (A. Meager, supra).
[0010] Considerable insight into the essential functions of several
members of the TNF receptor family has been gained from the
identification and creation of mutants that abolish the expression
of these proteins. For example, naturally occurring mutations in
the FAS antigen and its ligand cause lymphoproliferative disease
(R. Watanabe-Fukunaga et al., Nature 356:314 (1992)), perhaps
reflecting a failure of programmed cell death. Mutations of the
CD40 ligand cause an X-linked immunodeficiency state characterized
by high levels of immunoglobulin M and low levels of immunoglobulin
G in plasma, indicating faulty T-cell-dependent B-cell activation
(R. C. Allen et al., Science 259:990 (1993)). Targeted mutations of
the low affinity nerve growth factor receptor cause a disorder
characterized by faulty sensory innervation of peripheral
structures (K. F. Lee et al., Cell 69:737 (1992)).
[0011] TNF alpha and LT-alpha are capable of binding to two TNF
receptors (the 55- and 75-kd TNF receptors). A large number of the
biological effects elicited by TNF alpha and LT-alpha are mediated
through their receptors, include hemorrhagic necrosis of
transplanted tumors, cytotoxicity, a role in endotoxic shock,
inflammation, immunoregulation, proliferation and anti-viral
responses, as well as protection against the deleterious effects of
ionizing radiation. TNF alpha and LT-alpha are involved in the
pathogenesis of a wide range of diseases, including endotoxic
shock, cerebral malaria, tumors, autoimmune disease, AIDS and
graft-host rejection (B. Beutler and C. Von Huffel, Science
264:667-668 (1994)). Mutations in the p55 receptor cause increased
susceptibility to microbial infection.
[0012] Moreover, a domain of about 80 amino acids near the
C-terminus of TNFR1 (p55) and Fas was reported as the "death
domain," which is responsible for transducing signals for
programmed cell death (Tartaglia et al., Cell 74:845 (1993)).
[0013] Apoptosis, or programmed cell death, is a physiologic
process essential to the normal development and homeostasis of
multicellular organisms (H. Steller, Science 267:1445-1449 (1995)).
Derangements of apoptosis contribute to the pathogenesis of several
human diseases including cancer, neurodegenerative disorders, and
acquired immune deficiency syndrome (C. B. Thompson, Science
267:1456-1462 (1995)). Recently, much attention has focused on the
signal transduction and biological function of two cell surface
death receptors, Fas/APO-1 and TNFR-1 (J. L. Cleveland et al., Cell
81:479-482 (1995); A. Fraser et al., Cell 85:781-784 (1996); S.
Nagata et al. Science 267:1449-56 (1995)). Both are members of the
TNF receptor family, which also include TNFR-2, low affinity NGFR,
CD40, and CD30, among others (C. A. Smith et al., Science 248:
1019-23 (1990); M. Tewari et al., in Modular Texts in Molecular and
Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall,
London, 1995). While family members are defined by the presence of
cysteine-rich repeats in their extracellular domains, Fas/APO-1 and
TNFR-1 also share a region of intracellular homology, appropriately
designated the "death domain," which is distantly related to the
Drosophila suicide gene, reaper (P. Golstein et al., Cell 81:185-6
(1995); K. White et al., Science 264:677-83 (1994)). This shared
death domain suggests that both receptors interact with a related
set of signal transducing molecules that, until recently, remained
unidentified. Activation of Fas/APO-1 recruits the death
domain-containing adapter molecule FADD/MORT1 (A. M. Chinnaiyan et
al., Cell 81:505-512 (1995); M. P. Boldin et al., J. Biol. Chem.
270:7795-8 (1995); F. C. Kischkel et al., EMBO 14:5579-5588
(1995)), which in turn binds and presumably activates FLICE/MACH1,
a member of the ICE/CED-3 family of pro-apoptotic proteases (M.
Muzio et al., Cell 85: 817-827 (1996); M. P. Boldin et al., Cell
85:803-815 (1996)). While the central role of Fas/APO-1 is to
trigger cell death, TNFR-1 can signal an array of diverse
biological activities-many of which stem from its ability to
activate NF-kB (L. A. Tartaglia et al., Immunol Today 13:151-153
(1992)). Accordingly, TNFR-1 recruits the multivalent adapter
molecule TRADD, which like FADD, also contains a death domain (H.
Hsu et al., Cell 81:495-504 (1995); H. Hsu et al., Cell 84:299-308
(1996)). Through its associations with a number of signaling
molecules including FADD, TRAF2, and RIP, TRADD can signal both
apoptosis and NF-kB activation (H. Hsu et al., Cell 84:299-308
(1996); H. Hsu et al., Immunity 4:387-396 (1996)).
[0014] Recently, Human Genome Sciences has demonstrated that the
TNF ligand family member Neutrokine-alpha (International
publication number WO 98/18921) induces both in vitro and in vivo B
cell proliferation. B lymphocytes are responsible for the
production of immunoglobulins, the major effector molecules of the
humoral immune system. Immune system related disorders associated
with B cells include, for example, immunodeficiencies and
autoimmune disease.
[0015] Accordingly, there is a need to provide cytokines similar to
TNF that are involved in pathological conditions. Such novel
cytokines may be used to make novel antibodies or other antagonists
that bind these TNF-like cytokines for diagnosis and therapy of
disorders related to TNF-like cytokines. More particularly, there
is a need to provide Neutrokine-alpha binding proteins that may be
involved in pathological conditions. Such novel Neutrokine-alpha
binding proteins may be used, for example, as therapeutics to treat
or prevent diseases, disorders or conditions associated with
aberrant Neutrokine-alpha mediated activity.
SUMMARY OF THE INVENTION
[0016] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding at least a portion
of TR21. Thus, the present invention provides, for example,
isolated nucleic acid molecules comprising a polynucleotide
encoding the TR21 receptor having the amino acid sequence shown in
FIG. 1 (SEQ ID NO:2).
[0017] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells. The
invention further provides for the use of such recombinant vectors
in the production of TR21 polypeptides by recombinant
techniques.
[0018] The invention further provides an isolated TR21 polypeptide
having an amino acid sequence encoded by a polynucleotide described
herein.
[0019] The present invention also provides diagnostic assays such
as quantitative and diagnostic assays for detecting levels of TR21
protein. Thus, for instance, a diagnostic assay in accordance with
the invention for detecting over-expression of TR21, or soluble
form thereof, compared to normal control tissue samples may be used
to detect the presence of tumors.
[0020] Tumor Necrosis Factor (TNF) family ligands are known to be
among the most pleiotropic cytokines, inducing a large number of
cellular responses, including cell proliferation, cytotoxicity,
anti-viral activity, immunoregulatory activities, hematopoiesis,
and the transcriptional regulation of several genes. Cellular
responses to TNF-family ligands include not only normal
physiological responses, but such responses may lead to diseases
associated with dysregulation of these physiological responses,
such as, for example, diseases associated with increased apoptosis
or the inhibition of apoptosis. Apoptosis-programmed cell death is
a physiological mechanism involved in the deletion of peripheral T
lymphocytes of the immune system, and its dysregulation can lead to
a number of different pathogenic processes. Diseases associated
with increased cell survival, unregulated cell proliferation, or
the inhibition of apoptosis, include cancers, autoimmune disorders,
viral infections, inflammation, graft vs. host disease, acute graft
rejection, and chronic graft rejection. Diseases associated with
increased apoptosis include AIDS, neurodegenerative disorders,
myelodysplastic syndromes, ischemic injury, toxin-induced liver
disease, septic shock, cachexia, and anorexia.
[0021] Thus, the invention further provides a method comprising
contacting cells which express the TR21 polypeptide with a
candidate compound and a TNF-family ligand (e.g. Neutrokine-alpha
(International Publication Number WO 97/33902)), and assaying for
the inhibition of TR21 mediated signaling, and/or activation of
transcription factors, such as, for example, AP-1 and/or NF-kappaB,
induced by, for example, a TNF-family ligand (e.g.,
Neutrokine-alpha) which involves administering to a cell which
expresses the TR21 polypeptide (e.g., a B cell) an effective amount
of a TR21 antagonist capable of decreasing TR21 mediated
signaling.
[0022] The present invention is also directed to methods for
enhancing TR21 mediated signaling induced by a TNF-family ligand
(e.g., Neutrokine-alpha) which involves administering to a cell
which expresses the TR21 polypeptide (e.g., a B cell) an effective
amount of a TR21 agonist capable of increasing TR21 mediated
signaling.
[0023] Whether any candidate "agonist" or "antagonist" of the
present invention can enhance or inhibit TR21 mediated signaling
can be determined using or routinely modifying TNF-family
ligand/receptor cellular response assays known in the art,
including, for example, those described in von Bulow et al.
(Science 278:138-141 (1997)) and herein (see, e.g., Examples 17 and
18). Thus, in a further embodiment, a screening method is provided
for determining whether a candidate agonist or antagonist is
capable of enhancing or decreasing TR21 mediated cellular response
to TNF-ligand (e.g., Neutrokine-alpha). The method involves
contacting cells expressing TR21 with the candidate compound (i.e.,
candidate agonist or antagonist compound) and TNF-ligand (e.g.,
Neutrokine-alpha), and measuring the TR21 mediated cellular
response (e.g., activation of transcription factors such as, for
example, NF-AT, AP-1, and/or NF-kappaB), and comparing the cellular
response to a standard cellular response. The standard cellular
response being measured when contact is made with the TNF-family
ligand (e.g., Neutrokine-alpha) in absence of the candidate
compound. An increased cellular response over the standard
indicates that the candidate compound is an agonist of the ligand
(e.g., Neutrokine-alpha)/TR21 signaling pathway and a decreased
cellular response compared to the standard indicates that the
candidate compound is an antagonist of the ligand (e.g.,
Neutrokine-alpha)/TR21 signaling pathway. By the invention, a cell
expressing the TR21 polypeptide can be contacted with either an
endogenous or exogenously administered TNF-family ligand (e.g.,
Neutrokine-alpha.).
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino
acid sequence (SEQ ID NO:2) of TR21. Predicted amino acids from
about 1 to about 81 constitute the extracellular domain (SEQ ID
NO:2); amino acids from about 82 to about 101 constitute the
transmembrane domain (SEQ ID NO:2); and amino acids from about 102
to about 184 constitute the intracellular domain (SEQ ID NO:2).
[0025] FIG. 2 shows an analysis of the TR21 amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity; amphipathic
regions; flexible regions; antigenic index and surface probability
are shown. The regions were determined by analyzing the amino acid
sequence of FIG. 1 (SEQ ID NO:2) using the default parameters of
the recited computer programs. In the "Antigenic
Index--Jameson-Wolf" graph, amino acid residues 3 to 15, 39 to 45,
51 to 53, 59 to 61, 104 to 112, 115 to 122, 136 to 138, 147 to 153
and 178 to 182 in FIG. 1 (SEQ ID NO:2) correspond to highly
antigenic regions of the TR21 protein.
[0026] A tabular representation of the data summarized graphically
in FIG. 2 can be found in Table I. In Table I, the columns are
labeled with the headings "Res," "Position," and Roman numerals
I-XIV. The column headings refer to the Following Features of the
amino acid sequence presented in FIG. 2 and Table I: "Res": amino
acid residue of SEQ ID NO:2 and FIG. 1; "Position": position of the
corresponding residue within SEQ ID NO:2 and FIG. 1; "I": Alpha
Regions-Garnier-Robson; "II": Alpha Regions-Chou-Fasman; "III":
Beta Regions-Garnier-Robson; "IV": Beta Regions-Chou-Fasman; "V":
Turn Regions-Garnier-Robson; "VI": Turn Regions-Chou-Fasman; "VII":
Coil Regions-Garnier-Robson; "VIII": Hydrophilicity
Plot-Kyte-Doolittle; "IX": Hydrophobicity Plot-Hopp-Woods; "X":
Alpha Amphipathic Regions-Eisenberg; "XI": Beta Amphipathic
Regions-Eisenberg; "XII": Flexible Regions-Karplus-Schulz; "XIII":
Antigenic Index-Jameson-Wolf; "XIV": Surface Probability
Plot-Emini.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding TR21, such as, for
example, polynucleotides having the nucleotide sequence shown in
FIG. 1 (SEQ ID NO:1). The present invention provides isolated
nucleic acid molecules comprising a polynucleotide encoding a TR21
polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID
NO:2).
TR21 Nucleic Acid Molecules
[0028] The determined nucleotide sequence of TR21 (FIG. 1; SEQ ID
NO:1) contains an open reading frame encoding a protein of about
184 amino acid residues, with a deduced molecular weight of about
18.9 kDa. The amino acid sequence of the predicted mature TR21
receptor is shown in SEQ ID NO:2 from amino acid residue about 1 to
residue about 184.
[0029] As indicated, the present invention also provides the mature
form(s) of the TR21 receptors of the present invention. According
to the signal hypothesis, proteins secreted by mammalian cells have
a signal or secretory leader sequence which is cleaved from the
mature protein once export of the growing protein chain across the
rough endoplasmic reticulum has been initiated. Most mammalian
cells and even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species on the protein. Further, it has long been known that the
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide.
[0030] The present invention provides a nucleotide sequence
encoding the mature TR21 polypeptide having the amino acid sequence
shown in FIG. 1. By the mature TR21 protein having the amino acid
sequence shown in FIG. 1 is meant the mature form(s) of the TR21
receptor predicted by computer analysis or produced by expression
of the coding sequence shown in FIG. 1 in a mammalian cell (e.g.,
COS cells, as described below). As indicated below, the mature TR21
receptor having the amino acid sequence encoded by the coding
sequence shown in FIG. 1 may or may not differ from the predicted
mature TR21 protein shown in FIG. 1 (amino acids from about 1 to
about 184) depending on the accuracy of the predicted cleavage site
based on computer analysis.
[0031] Methods for predicting whether a protein has a secretory
leader as well as the cleavage point for that leader sequence are
available. For instance, the method of McGeoch (Virus Res.
3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690
(1986)) can be used. The accuracy of predicting the cleavage points
of known mammalian secretory proteins for each of these methods is
in the range of 75-80% (von Heinje, supra). However, the two
methods do not always produce the same predicted cleavage point(s)
for a given protein.
[0032] The polypeptide sequence of the TR21 depicted in FIG. 1 can
routinely be examined by computer programs. For example, the mature
form, intracellular form, extracellular form, and transmembrane
domains of the TR21 polypeptides of the invention can routinely be
predicted via analysis using the "PSORT" computer program (K. Nakai
and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert
system for predicting the cellular location of a protein based on
the amino acid sequence. As part of this computational prediction
of localization, the methods of McGeoch and von Heinje are
incorporated into the PSORT program.
[0033] The predicted TR21 polypeptide comprises about 184 amino
acids. However, as one of ordinary skill in the art would
appreciate, the actual TR21 polypeptide may be anywhere in the
range of 174-194 amino acids due to the possibilities of sequencing
errors as well as the variability of cleavage sites for leaders in
different known proteins. It will further be appreciated that, the
domains described herein have been predicted by computer analysis,
and accordingly, that depending on the analytical criteria used for
identifying various functional domains, the exact "address" of, for
example, the extracellular domain, intracellular domain,
cysteine-rich motif, and transmembrane domain of TR21 may differ
slightly from the predicted locations. For example, the exact
location of the TR21 extracellular domain in FIG. 1 (SEQ ID NO:2)
may vary slightly (e.g., the address may "shift" by about 1 to
about 20 residues, more likely about 1 to about 5 residues)
depending on the criteria used to define the domain. In any event,
as discussed in more detail below, the invention further provides
polypeptides having various residues deleted from the N-terminus
and/or C-terminus of the complete TR21 polypeptide, including
polypeptides lacking one or more amino acids from the N-termini of
the TR21 extracellular domains described herein, which constitute
soluble forms of the extracellular domain of the TR21 polypeptides
respectively.
[0034] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA may be the coding strand,
also known as the sense strand, or it may be the non-coding strand,
also referred to as the anti-sense strand.
[0035] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced naturally, recombinantly or synthetically.
However, a nucleic acid molecule contained in a clone that is a
member of a mixed clone library (e.g., a genomic or cDNA library)
and that has not been isolated from other clones of the library
(e.g., in the form of a homogeneous solution containing the clone
without other members of the library) or a chromosome isolated or
removed from a cell or a cell lysate (e.g., a "chromosome spread",
as in a karyotype), or a preparation of randomly sheared or genomic
DNA cut with one or more restriction enzymes, is not "isolated" for
the purposes of this invention.
[0036] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) shown
in FIG. 1 (SEQ ID NO:1); DNA molecules comprising the coding
sequence for the complete (full-length) and/or mature TR21 protein
shown in FIG. 1 (SEQ ID NO:2); and DNA molecules which comprise a
sequence substantially different from those described above, but
which, due to the degeneracy of the genetic code, still encode the
TR21 protein. Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate
such degenerate variants.
[0037] The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in FIG. 1 (SEQ ID
NO:1), or a nucleic acid molecule having a sequence complementary
thereto. Such isolated molecules, particularly DNA molecules, are
useful, for example, as probes for gene mapping by in situ
hybridization with chromosomes, and for detecting expression of the
TR21 gene in human tissue, for instance, by Northern blot
analysis.
[0038] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated DNA molecule having the nucleotide sequence of the
nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is intended DNA
fragments at least about 15 nt, and more preferably at least about
20 nt, at least about 24 nt, still more preferably at least about
30 nt, at least about 35 nt, and even more preferably, at least
about 40 nt, at least about 45 nt, at least about 50 nt, at least
about 55 nt, at least about 60 nt, at least about 65 nt, at least
about 70 nt, at least about 75 nt, at least about 100 nt, at least
about 150 nt, at least about 200 nt, at least about 250 nt, at
least about 300 nt in length which are useful, for example, as
diagnostic probes and primers as discussed herein. Of course,
larger fragments 350-899 nt in length are also useful according to
the present invention, as are fragments corresponding to most, if
not all, of the nucleotide sequence as shown in FIG. 1 (SEQ ID
NO:1), or the complementary strand thereto. By a fragment at least
20 nt in length, for example, is intended fragments which include
20 or more contiguous bases from the nucleotide sequence of the
nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). In this
context "about" includes the particularly recited size, and sizes
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both termini. In specific embodiments, the
fragments of the invention comprise, or alternatively consist of,
nucleotides 12-50, 120-140, 156-164, 180-188, 315-341, 348-371,
411-419, 444-464, and/or 537-551 of FIG. 1 (SEQ ID NO:1) or the
complementary strand thereto. Polypeptides encoded by these
polynucleotide are also encompassed.
[0039] Representative examples of TR21 polynucleotide fragments of
the invention include, for example, fragments that comprise, or
alternatively, consist of, a sequence from about nucleotide 1 to
11, 12 to 50, 51 to 59, 60 to 83, 84 to 110, 111 to 119, 120 to
140, 141 to 155, 156 to 164, 165 to 179, 180 to 188, 189 to 215,
216 to 248, 249 to 308, 309 to 314, 315 to 341, 342 to 347, 348 to
371, 372 to 411, 412 to 419, 420 to 443, 444 to 464, 465 to 500,
501 to 536, 537 to 551, 552 to 557, 558 to 590, 591 to 649, 650 to
708, 709 to 767, 768 to 826, 827 to 885, and/or 886 to 899 of FIG.
1 (SEQ ID NO: 1), or the complementary strand thereto. In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini.
[0040] In specific embodiments, the polynucleotide fragments of the
invention comprise, or alternatively, consist of, a sequence from
nucleotide 60 to 110, of FIG. 1 (SEQ ID NO:1), or the complementary
strand thereto.
[0041] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a TR21 functional activity.
By a polypeptide demonstrating a TR21 "functional activity" is
meant, a polypeptide capable of displaying one or more known
functional activities associated with a full-length (complete) TR21
protein. Such functional activities include, but are not limited
to, biological activity, antigenicity (ability to bind (or compete
with a TR21 polypeptide for binding) to an anti-TR21 antibody),
immunogenicity (ability to generate antibody which binds to a TR21
polypeptide), ability to form multimers with TR21 polypeptides of
the invention, and ability to bind to a receptor or ligand for a
TR21 polypeptide (e.g., Neutrokine-alpha (International Publication
Number WO 98/18921)).
[0042] The functional activity of TR21 polypeptides, fragments,
variants, derivatives, and analogs thereof, can be assayed by
various methods.
[0043] For example, in one embodiment where one is assaying for the
ability to bind or compete with full-length TR21 polypeptides for
binding to anti-TR21 antibody, various immunoassays known in the
art can be used, including but not limited to, competitive and
non-competitive assay systems using techniques such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ
immunoassays (using colloidal gold, enzyme or radioisotope labels,
for example), western blots, precipitation reactions, agglutination
assays (e.g., gel agglutination assays, hemagglutination assays),
complement fixation assays, immunofluorescence assays, protein A
assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary
antibody. In another embodiment, the primary antibody is detected
by detecting binding of a secondary antibody or reagent to the
primary antibody. In a further embodiment, the secondary antibody
is labeled. Many means are known in the art for detecting binding
in an immunoassay and are within the scope of the present
invention.
[0044] In another embodiment, where a TR21 ligand is identified
(e.g., Neutrokine-alpha), or the ability of a polypeptide fragment,
variant or derivative of the invention to multimerize is being
evaluated, binding can be assayed by means well-known in the art,
such as, for example, reducing and non-reducing gel chromatography,
protein affinity chromatography, and affinity blotting. See
generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995).
In another embodiment, physiological correlates of TR21 binding to
its substrates (signal transduction) can be assayed.
[0045] In addition, assays described herein (and otherwise known in
the art may routinely be applied to measure the ability of TR21
polypeptides and fragments, variants derivatives and analogs
thereof to elicit TR21 related biological activity. For example,
techniques described herein (see e.g., Examples 16, 17 and 18) and
otherwise known in the art may be applied or routinely modified to
assay for the ability of the compositions of the invention (e.g.,
fusion proteins comprising a portion of the extracellular domain of
TR21 and an immunoglobulin Fc domain) to inhibit or stimulate B
cell proliferation (e.g., Neutrokine-alpha mediated B cell
proliferation).
[0046] Other methods will be known to the skilled artisan and are
within the scope of the invention.
[0047] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding a member selected from the
group: a polypeptide comprising or alternatively, consisting of,
the TR21 receptor extracellular domain (amino acid residues from
about 1 to about 81 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising, or alternatively consisting of, the TR21 cysteine rich
domain (amino acid residues from about 19 to about 35 in FIG. 1
(SEQ ID NO:2); a polypeptide comprising, or alternatively
consisting of the TR21 transmembrane domain (amino acid residues
from about 82 to about 101 in FIG. 1 (SEQ ID NO:2); and/or a
polypeptide comprising, or alternatively consisting of, the TR21
intracellular domain (amino acid residues from about 102 to about
184 in FIG. 1 (SEQ ID NO:2)). Since the locations of these domains
have been predicted by computer analysis, one of ordinary skill
would appreciate that the amino acid residues constituting these
domains may vary slightly (e.g., by about 1 to 15 amino acid
residues) depending on the criteria used to define each domain.
[0048] Preferred nucleic acid fragments of the invention encode a
full-length TR21 polypeptide lacking the nucleotides encoding the
amino terminal methionine in FIG. 1 (SEQ ID NO:1), as it is known
that the methionine is cleaved naturally and such sequences may be
useful in genetically engineering TR21 expression vectors.
Polypeptides encoded by such polynucleotides are also contemplated
by the invention.
[0049] Preferred nucleic acid fragments of the present invention
further include nucleic acid molecules encoding epitope-bearing
portions of the TR21 receptor proteins. In particular, such nucleic
acid fragments of the present invention include nucleic acid
molecules encoding: a polypeptide comprising amino acid residues
from about 3 to about 15 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 39 to about 45 in FIG. 1
(SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 51 to about 53 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 59 to about 61 in FIG. 1
(SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 104 to about 112 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 115 to about 122 in FIG.
1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 136 to about 138 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising amino acid residues from about 147 to about 153 in FIG.
1 (SEQ ID NO:2); and a polypeptide comprising amino acid residues
from about 178 to about 182 in FIG. 1 (SEQ ID NO:2). In this
context "about" includes the particularly recited ranges, and
ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,
at either terminus or at both termini. The inventors have
determined that the above polypeptide fragments are antigenic
regions of TR21. Methods for determining other such epitope-bearing
portions of the TR21 proteins are described in detail below.
[0050] It is believed that the extracellular cysteine rich motif of
TR21 disclosed in FIG. 1 are important for interactions between
TR21 and its ligands (e.g., Neutrokine alpha). Accordingly,
specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, the amino acid sequence of amino acid
residues 19 to 35 of FIG. 1 (SEQ ID NO:2). Polypeptides encoded by
these polynucleotides are also encompassed by the invention.
[0051] In additional embodiments, the polynucleotides of the
invention encode functional attributes of TR21. Preferred
embodiments of the invention in this regard include fragments that
comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions and
antigenic regions of TR21.
[0052] The data representing the structural or functional
attributes of TR21 set forth in FIG. 2 and/or Table I, as described
above, was generated using the various modules and algorithms of
the DNA*STAR set on default parameters. In a preferred embodiment,
the data presented in columns VIII, XI, XIII and XIV of Table I can
be used to determine regions of TR21 which exhibit a high degree of
potential for antigenicity. Regions of high antigenicity are
determined from the data presented in columns VIII, XI, XIII and/or
XIV by choosing values which represent regions of the polypeptide
which are likely to be exposed on the surface of the polypeptide in
an environment in which antigen recognition may occur in the
process of initiation of an immune response.
[0053] Certain preferred regions in these regards are set out in
FIG. 2, but may, as shown in Table I, be represented or identified
by using tabular representations of the data presented in FIG. 2.
The DNA*STAR computer algorithm used to generate FIG. 2 (set on the
original default parameters) was used to present the data in FIG. 2
in a tabular format (See Table I). The tabular format of the data
in FIG. 2 may be used to easily determine specific boundaries of a
preferred region.
[0054] The above-mentioned preferred regions set out in FIG. 2 and
in Table I, include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequences set out in FIG. 1. As set out in FIG. 2 and in Table I,
such preferred regions include Garnier-Robson alpha-regions,
beta-regions, turn-regions, and coil-regions, Chou-Fasman
alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle
hydrophilic regions, Hopp-Woods hydrophobic regions, Eisenberg
alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions, Jameson-Wolf regions of high antigenic index and Emini
surface-forming regions.
TABLE-US-00001 TABLE I Res Pos I II III IV V VI VII VIII IX X XI
XII XIII XIV Met 1 . . B . . . . 1.01 -0.67 * * . 0.95 1.41 Arg 2 .
. B . . . . 1.51 -0.67 * * . 1.29 1.70 Arg 3 . . B . . . . 1.60
-1.10 * . . 1.63 2.61 Gly 4 . . . . . T C 1.18 -1.14 * * . 2.37
3.53 Pro 5 . . . . . T C 1.68 -1.07 * * F 2.86 1.49 Arg 6 . . . . T
T . 1.93 -1.07 * * F 3.40 1.49 Ser 7 . . B . . T . 1.93 -0.64 * * F
2.66 1.49 Leu 8 . . B . . . . 1.82 -1.07 * * F 2.46 1.88 Arg 9 . .
B . . . . 1.58 -1.50 . * F 2.46 1.61 Gly 10 . . . . T T . 1.58
-1.00 . * F 3.06 1.21 Arg 11 . . . . T T . 0.88 -0.96 . * F 3.06
2.27 Asp 12 . . . . T T . 0.97 -1.14 . * F 3.40 1.17 Ala 13 . . . .
. T C 1.47 -0.71 . * F 2.86 1.83 Pro 14 . . . . . . C 1.14 -0.66 .
* F 2.32 1.35 Ala 15 . . . . . T C 0.82 -0.23 * . F 1.88 1.25 Pro
16 . . . . . T C -0.14 0.34 * . F 0.79 0.66 Thr 17 . . B . . T .
-0.36 0.49 . . F -0.05 0.32 Pro 18 . . . . T T . -0.36 0.49 . * F
0.35 0.49 Cys 19 . . B . . . . -0.14 0.49 . * . -0.40 0.32 Val 20 .
A B . . . . -0.22 0.06 . . . -0.30 0.38 Pro 21 . A B . . . . -0.71
0.14 . . . -0.30 0.13 Ala 22 . A B . . . . -0.40 0.50 . . . -0.60
0.21 Glu 23 A A . . . . . -1.00 -0.07 * . . 0.30 0.48 Cys 24 A A .
. . . . -1.14 -0.03 * . . 0.30 0.26 Phe 25 A A . . . . . -1.14 0.23
* * . -0.30 0.21 Asp 26 A A . . . . . -0.82 0.37 * * . -0.30 0.09
Leu 27 A A . . . . . -0.27 0.37 * * . -0.30 0.33 Leu 28 A A . . . .
. -0.93 0.30 * * . -0.30 0.52 Val 29 A A . . . . . -1.12 0.09 * * .
-0.30 0.17 Arg 30 A A . . . . . -1.01 0.73 * * . -0.60 0.15 His 31
A A . . . . . -1.68 0.54 * * . -0.60 0.18 Cys 32 . A B . . . .
-1.21 0.43 . * . -0.60 0.13 Val 33 . A B . . . . -1.21 0.21 . * .
-0.30 0.07 Ala 34 . A B . . . . -1.17 0.90 . * . -0.60 0.04 Cys 35
. A B . . . . -1.17 1.09 . * . -0.60 0.06 Gly 36 . A B . . . .
-1.44 0.51 * * . -0.60 0.16 Leu 37 . A B . . . . -0.99 0.36 * . .
0.00 0.23 Leu 38 . A B . . . . -0.02 0.29 * . . 0.30 0.67 Arg 39 .
A B . . . . 0.36 -0.29 * * F 1.50 1.33 Thr 40 . . B . . T . 1.07
-0.29 * * F 2.20 2.50 Pro 41 . . . . . T C 1.20 -0.97 * . F 3.00
6.07 Arg 42 . . . . . T C 1.42 -1.23 * * F 2.70 4.79 Pro 43 . . B .
. T . 1.89 -0.73 * . F 2.20 3.35 Lys 44 . . . . . . C 1.19 -0.79 *
. F 1.90 2.15 Pro 45 . . . . . . C 1.20 -0.71 . * F 1.60 1.11 Ala
46 . . . . T . . 1.11 -0.33 . * F 1.05 0.96 Gly 47 . . B . . T .
0.79 -0.37 . * F 0.85 0.64 Ala 48 . . B . . T . 0.41 0.06 . . F
0.49 0.64 Ser 49 . . . . . T C 0.16 0.13 * . F 0.93 0.64 Ser 50 . .
. . . T C 0.48 0.06 * . F 1.32 1.00 Pro 51 . . . . . . C 0.76 -0.37
* . F 1.96 1.95 Ala 52 . . . . . T C 0.51 -0.39 * * F 2.40 2.10 Pro
53 . . . . . T C 0.29 -0.27 * * F 2.16 1.58 Arg 54 . . B . . T .
0.59 0.03 * * F 0.97 0.84 Thr 55 . . B . . T . 0.68 0.00 * * F 1.48
1.45 Ala 56 . . B . . . . 0.89 -0.07 * * F 1.24 1.45 Leu 57 . . B .
. . . 1.48 -0.10 * * F 1.20 1.28 Gln 58 . . B . . . . 1.39 -0.10 *
* F 1.40 1.53 Pro 59 . . B . . T . 0.42 -0.20 * * F 1.80 2.03 Gln
60 . . B . . T . 0.39 -0.06 . * F 2.00 1.83 Glu 61 . . B . . T .
0.39 -0.31 . * F 1.80 1.05 Ser 62 . . B . . T . 0.86 -0.21 . * F
1.45 0.68 Val 63 . . B . . . . 0.27 -0.21 . . F 1.05 0.39 Gly 64 .
. . . . . C 0.13 -0.11 . . . 0.90 0.23 Ala 65 . . . . . . C 0.13
0.31 . . . 0.10 0.17 Gly 66 . . . . . T C -0.46 -0.07 * . . 0.90
0.39 Ala 67 A . . . . T . -0.74 -0.21 . . F 0.85 0.40 Gly 68 A . .
. . T . -0.70 -0.14 . . F 0.85 0.40 Glu 69 A . . . . T . -0.57 0.04
. * . 0.10 0.33 Ala 70 . A B . . . . -0.79 0.04 . * . -0.30 0.51
Ala 71 . A B . . . . -0.66 0.23 . * . -0.30 0.43 Leu 72 . A B . . .
. -0.41 0.23 . * . -0.30 0.38 Pro 73 . A B . . . . -0.88 0.66 . * .
-0.60 0.37 Leu 74 . . B . . T . -1.69 0.84 . * . -0.20 0.30 Pro 75
. . B . . T . -1.80 1.03 . . F -0.05 0.30 Gly 76 . . B . . T .
-1.56 1.13 . . . -0.20 0.17 Leu 77 . . B . . T . -1.33 1.13 . . .
-0.20 0.20 Leu 78 . . B B . . . -1.33 0.94 . . . -0.60 0.13 Phe 79
. . B B . . . -1.11 0.94 . . . -0.60 0.21 Gly 80 . . B B . . .
-1.71 1.01 . . . -0.60 0.26 Ala 81 . A B . . . . -2.18 1.01 . . .
-0.60 0.26 Pro 82 A A . . . . . -1.71 1.01 . . . -0.60 0.24 Ala 83
A A . . . . . -1.71 0.66 . . . -0.60 0.24 Leu 84 A A . . . . .
-1.60 0.91 . * . -0.60 0.20 Leu 85 A A . . . . . -2.07 0.91 . . .
-0.60 0.13 Gly 86 A A . . . . . -2.33 1.17 . . . -0.60 0.11 Leu 87
A A . . . . . -2.93 1.31 . . . -0.60 0.10 Ala 88 A A . . . . .
-2.93 1.31 . . . -0.60 0.10 Leu 89 A A . . . . . -2.93 1.13 . . .
-0.60 0.10 Val 90 A A . . . . . -2.98 1.39 . . . -0.60 0.10 Leu 91
A A . . . . . -3.44 1.34 . . . -0.60 0.07 Ala 92 A A . . . . .
-3.49 1.53 . . . -0.60 0.07 Leu 93 . A B . . . . -3.24 1.49 . . .
-0.60 0.07 Val 94 . A B . . . . -3.24 1.27 . . . -0.60 0.09 Leu 95
. A B . . . . -3.24 1.27 . . . -0.60 0.07 Val 96 . A B . . . .
-2.73 1.41 . . . -0.60 0.06 Gly 97 . A B . . . . -2.43 1.11 . * .
-0.60 0.11 Leu 98 . . B B . . . -1.51 1.39 * . . -0.60 0.15 Val 99
. . B B . . . -0.54 0.70 * * . -0.60 0.38 Ser 100 . . B B . . .
0.38 0.06 . * . -0.30 0.76 Trp 101 . . B B . . . 1.23 -0.37 * * .
0.45 1.80 Arg 102 . . B B . . . 1.69 -0.66 * * F 0.90 4.20 Arg 103
. . B B . . . 2.61 -1.30 * * F 0.90 6.14 Arg 104 . . . B T . . 2.66
-1.69 * * F 1.60 11.44 Gln 105 . . B B . . . 3.07 -1.91 * * F 1.50
4.82 Arg 106 . . B . . . . 3.01 -1.91 * * F 2.00 4.82 Arg 107 . . B
. . . . 2.31 -1.49 * * F 2.30 2.43 Leu 108 . . . . T . . 1.90 -0.99
* * F 3.00 1.42 Arg 109 . . . . T . . 1.49 -1.00 . * F 2.55 0.97
Gly 110 . . . . . T C 0.90 -0.61 * * F 2.25 0.66 Ala 111 . . . . .
T C 0.79 -0.11 * * F 1.65 0.81 Ser 112 . . . . . T C 0.09 -0.80 . *
F 1.65 0.72 Ser 113 . . . . . T C 0.69 -0.30 . * F 1.05 0.74 Ala
114 . . B . . . . 0.58 -0.30 . * F 1.14 1.13 Glu 115 . . B . . . .
0.58 -0.80 . . F 1.78 1.40 Ala 116 A . . . . T . 1.17 -0.76 . . F
2.32 1.04 Pro 117 A . . . . T . 1.51 -1.14 . . F 2.66 1.71 Asp 118
. . . . T T . 1.81 -1.64 * . F 3.40 1.98 Gly 119 . . . . T T . 1.81
-1.64 * . F 3.06 3.27 Asp 120 . . . . T . . 1.60 -1.64 * . F 2.52
2.13 Lys 121 . . . . . . C 2.19 -1.64 * . F 1.98 1.98 Asp 122 . . .
. . . C 2.19 -1.64 * . F 1.64 3.46 Ala 123 A . . . . T . 1.38 -1.64
* * F 1.30 3.20 Pro 124 A . . . . T . 1.72 -0.96 * . F 1.30 1.32
Glu 125 A . . . . T . 1.77 -0.96 * . F 1.30 1.32 Pro 126 A . . . .
T . 0.87 -0.96 * . F 1.30 2.61 Leu 127 A . . B . . . -0.02 -0.81 *
. F 0.90 1.25 Asp 128 A . . B . . . -0.32 -0.56 * . F 0.75 0.51 Lys
129 A . . B . . . -0.92 0.13 * . F -0.15 0.23 Val 130 . . B B . . .
-1.22 0.39 * . . -0.30 0.23 Ile 131 . . B B . . . -1.22 0.09 . . .
-0.30 0.18 Ile 132 . . B B . . . -0.76 0.51 * . . -0.60 0.14 Leu
133 . . B B . . . -1.64 0.94 . . . -0.39 0.19 Ser 134 . . B . . T .
-1.99 0.99 . . . 0.22 0.19 Pro 135 . . . . . T C -1.13 0.69 * . F
0.78 0.36 Gly 136 . . . . T T . -0.83 0.00 . . F 2.09 0.74 Ile 137
. . . . . T C -0.26 -0.19 * . F 2.10 0.56 Ser 138 . . . . . . C
-0.03 -0.09 * . F 1.69 0.52 Asp 139 . A B . . . . 0.06 -0.01 . . F
1.08 0.53 Ala 140 . A B . . . . -0.32 -0.01 . . F 1.02 1.17 Thr 141
. A B . . . . -0.27 -0.20 . . F 0.66 0.88 Ala 142 . A B . . . .
0.41 0.33 * . . -0.30 0.55 Pro 143 . . . . . . C 0.50 0.76 . . .
-0.20 0.85 Ala 144 . . . . T . . 0.29 0.69 . . . 0.00 0.91 Trp 145
. . . . . . C 0.53 0.63 . . . 0.29 1.39 Pro 146 . . . . . . C 0.84
0.56 . . F 0.63 0.89 Pro 147 . . . . . T C 1.43 0.13 . . F 1.62
1.53 Pro 148 . . . . T T . 1.43 -0.37 . . F 2.76 2.43 Gly 149 . . .
. T T . 1.68 -0.86 . . F 3.40 2.43 Glu 150 . . . . . T C 1.66 -0.86
* . F 2.86 1.55 Asp 151 . . . . . T C 1.56 -0.80 . . F 2.52 1.45
Pro 152 . . . . T T . 1.56 -0.74 . . F 2.38 2.11 Gly 153 . . . . T
T . 1.56 -0.74 . . F 2.20 1.89 Thr 154 . . B . . T . 1.56 -0.31 . .
F 1.32 1.75 Thr 155 . . . . . . C 1.52 0.11 . . F 0.88 1.12 Pro 156
. . . . . T C 1.22 0.19 . . F 1.24 1.54 Pro 157 . . . . T T . 0.58
0.14 . . F 1.60 1.43 Gly 158 . . . . T T . 0.71 0.30 . . F 1.29
0.73 His 159 . . B . . T . 0.17 0.24 . . . 0.58 0.73 Ser 160 . . B
B . . . 0.27 0.46 . . . -0.28 0.35 Val 161 . . B B . . . -0.11 0.46
. * . -0.44 0.55 Pro 162 . . B B . . . -0.21 0.53 . . . -0.60 0.41
Val 163 . . B B . . . 0.13 0.51 * . . -0.60 0.44 Pro 164 . . B . .
. . -0.64 0.13 * . . 0.05 1.03 Ala 165 . . B . . . . -0.69 0.17 . .
F 0.05 0.55 Thr 166 A . . . . . . -0.13 0.17 . . F 0.05 0.73 Glu
167 A . . . . . . -0.23 -0.09 . . F 0.65 0.63 Leu 168 A . . . . . .
0.62 -0.03 . . F 0.65 0.90 Gly 169 A . . . . . . 0.02 -0.53 . . F
1.10 1.09 Ser 170 A . . . . . . -0.24 -0.33 . . F 0.65 0.52 Thr 171
A . . B . . . -0.24 0.31 . . F -0.15 0.47 Glu 172 A . . B . . .
-0.56 0.11 . . F -0.15 0.68 Leu 173 A . . B . . . 0.30 0.17 . . F
-0.15 0.73 Val 174 . . B B . . . 0.33 -0.21 . . F 0.60 1.01 Thr 175
. . B B . . . 0.04 -0.21 . . F 0.45 0.84 Thr 176 . . B B . . . 0.01
0.29 . . F 0.30 1.03 Lys 177 . . . B . . C -0.20 0.03 . . F 0.80
1.38 Thr 178 . . . . . . C 0.61 -0.19 . . F 1.90 1.48 Ala 179 . . .
. . . C 1.47 -0.67 . * F 2.50 1.77 Gly 180 . . . . . T C 1.78 -0.76
. . F 3.00 1.53 Pro 181 . . . . . T C 1.70 -0.36 . . F 2.40 1.84
Glu 182 A . . . . T . 1.27 -0.41 . . F 1.90 2.33 Gln 183 A . . . .
T . 1.19 -0.49 . . . 1.45 3.01 Gln 184 . . B . . . . 1.39 -0.49 . .
. 0.95 2.49
[0055] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the complementary strand of
nucleotides 60 to 110, 249 to 308, 12 to 50, 120 to 140, 156 to
164, 180 to 188, 315 to 341, 348 to 371, 412 to 419, 444 to 464
and/or 537 to 551 of SEQ ID NO:1. By "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20
micrograms/ml denatured, sheared salmon sperm DNA, followed by
washing the filters in 0.1.times.SSC at about 65.degree. C.
Polypeptides encoded by these nucleic acids are also encompassed by
the invention.
[0056] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful, for example, as
diagnostic probes and primers as discussed above and in more detail
below. By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1). In this
context "about" includes the particularly recited size, and sizes
larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at
either terminus or at both termini.
[0057] In specific embodiments, the polynucleotides of the
invention are less than 110000 kb, 50000 kb, 10000 kb, 1000 kb, 500
kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 175 kb, 150 kb, 125 kb,
100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, 7.5
kb, or 5 kb in length.
[0058] In further embodiments, polynucleotides of the invention
comprise at least 15, at least 30, at least 50, at least 100, or at
least 250, at least 500, or at least 800 contiguous nucleotides of
TR21 coding sequence, but consist of less than or equal to 100 kb,
75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic
DNA that flanks the 5' or 3' coding nucleotide set forth in FIG. 1
(SEQ ID NO:1). In further embodiments, polynucleotides of the
invention comprise at least 15, at least 30, at least 50, at least
100, or at least 250, at least 500, or at least 800 contiguous
nucleotides of TR21 and/or coding sequence, but do not comprise all
or a portion of any TR21 intron. In another embodiment, the nucleic
acid comprising TR21 coding sequence does not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the TR21
gene in the genome). In other embodiments, the polynucleotides of
the invention do not contain the coding sequence of more than 1000,
500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic
flanking gene(s).
[0059] As indicated, nucleic acid molecules of the present
invention which encode a TR21 polypeptide may include, but are not
limited to, the coding sequence for the mature polypeptide, by
itself; the coding sequence for the mature polypeptide and
additional sequences, such as those encoding a leader or secretory
sequence, such as a pre-, or pro- or prepro-protein sequence; the
coding sequence of the mature polypeptide, with or without the
aforementioned additional coding sequences, together with
additional, non-coding sequences, including for example, but not
limited to introns and non-coding 5' and 3' sequences, such as the
transcribed, non-translated sequences that play a role in
transcription, mRNA processing--including splicing and
polyadenylation signals, for example--ribosome binding and
stability of mRNA; additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities. Thus, for instance, the polypeptide may be fused
to a marker sequence, such as a peptide, which facilitates
purification of the fused polypeptide. In certain preferred
embodiments of this aspect of the invention, the marker sequence is
a hexa-histidine peptide, such as the tag provided in a pQE vector
(Qiagen, Inc.), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86: 821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. The "HA" tag is
another peptide useful for purification which corresponds to an
epitope derived from the influenza hemagglutinin protein, which has
been described by Wilson et al., Cell 37:767-778 (1984). As
discussed below, other such fusion proteins include the TR21
receptor fused to Fc at the N- or C-terminus.
[0060] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs, or derivatives of the TR21 receptor. Variants
may occur naturally, such as a natural allelic variant. By an
"allelic variant" is intended one of several alternate forms of a
gene occupying a given locus on a chromosome of an organism. Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0061] Such variants include those produced by nucleotide
substitutions, deletions or additions which may involve one or more
nucleotides. The variants may be altered in coding or non-coding
regions or both. Alterations in the coding regions may produce
conservative or non-conservative amino acid substitutions,
deletions, or additions. Especially preferred among these are
silent substitutions, additions, and deletions, which do not alter
the properties and activities of the TR21 receptor or portions
thereof. Also especially preferred in this regard are conservative
substitutions.
[0062] Further embodiments of the invention include isolated
nucleic acid molecules comprising, or alternatively consisting of,
a polynucleotide having a nucleotide sequence at least 80%, 85%, or
90% identical, and more preferably at least 95%, 96%, 97%, 98%, or
99% identical to: (a) a nucleotide sequence encoding the
polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID
NO:2); (b) a nucleotide sequence encoding the polypeptide having
the amino acid sequence in FIG. 1 (SEQ ID NO: 2), but lacking the
amino terminal methionine; (c) a nucleotide sequence encoding the
polypeptide having the amino acid sequence at positions 1 to 184 in
FIG. 1 (SEQ ID NO:2); (d) a nucleotide sequence encoding the TR21
extracellular domain; (e) a nucleotide sequence encoding the TR21
cysteine-rich motif (i.e., amino acid residues about 19 to about 35
in FIG. 1 (SEQ ID NO:2)); (f) a nucleotide sequence encoding the
TR21 transmembrane domain; (g) a nucleotide sequence encoding the
TR21 receptor intracellular domain; (h) a nucleotide sequence
encoding the TR21 receptor extracellular and intracellular domains
with all or part of the transmembrane domain deleted; and (i) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.
Polypeptides encoded by these polynucleotides are also encompassed
by the invention.
[0063] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a TR21 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five mismatches per each 100 nucleotides of the reference
nucleotide sequence encoding the TR21 polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mismatches of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence. The reference (query) sequence may be the entire TR21
encoding nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), or any
TR21 polynucleotide fragment (e.g., a polynucleotide encoding the
amino acid sequence of any of the TR21 N- and/or C-terminal
deletions described herein), variant, derivative or analog, as
described herein.
[0064] As a practical matter, whether any particular nucleic acid
molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to, for instance, the nucleotide sequence shown in FIG. 1
(SEQ ID NO:1) can be determined conventionally using known computer
programs such as the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit
uses the local homology algorithm of Smith and Waterman, Advances
in Applied Mathematics 2: 482-489 (1981), to find the best segment
of homology between two sequences. When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed.
[0065] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch
Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff
Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or
the length of the subject nucleotide sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence because of 5' or 3' deletions, not
because of internal deletions, a manual correction is made to the
results to take into consideration the fact that the FASTDB program
does not account for 5' and 3' truncations of the subject sequence
when calculating percent identity. For subject sequences truncated
at the 5' or 3' ends, relative to the query sequence, the percent
identity is corrected by calculating the number of bases of the
query sequence that are 5' and 3' of the subject sequence, which
are not matched/aligned, as a percent of the total bases of the
query sequence. A determination of whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of this
embodiment. Only bases outside the 5' and 3' bases of the subject
sequence, as displayed by the FASTDB alignment, which are not
matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score. For
example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the
5' end of the subject sequence and therefore, the FASTDB alignment
does not show a matched/alignment of the first 10 bases at 5' end.
The 10 unpaired bases represent 10% of the sequence (number of
bases at the 5' and 3' ends not matched/total number of bases in
the query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90 bases
were perfectly matched the final percent identity would be 90%. In
another example, a 90 base subject sequence is compared with a 100
base query sequence. This time the deletions are internal deletions
so that there are no bases on the 5' or 3' of the subject sequence
which are not matched/aligned with the query. In this case the
percent identity calculated by FASTDB is not manually corrected.
Once again, only bases 5' and 3' of the subject sequence which are
not matched/aligned with the query sequence are manually corrected
for. No other manual corrections are made for the purposes of this
embodiment.
[0066] The present application is directed to nucleic acid
molecules comprising, or alternatively consisting of a nucleotide
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identical to the nucleic acid sequence for example, shown in FIG. 1
(SEQ ID NO:1), irrespective of whether they encode a polypeptide
having TR21 receptor activity. This is because even where a
particular nucleic acid molecule does not encode a polypeptide
having TR21 functional activity, one of skill in the art would
still know how to use the nucleic acid molecule, for instance, as a
hybridization probe or a polymerase chain reaction (PCR) primer.
Uses of the nucleic acid molecules of the present invention that do
not encode a polypeptide having TR21 receptor activity include,
inter alia: (1) isolating the TR21 gene or allelic variants thereof
in a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase chromosomal spreads to provide precise chromosomal
location of the TR21 receptor gene, as described in Verma et al.,
Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,
New York (1988); and (3) Northern Blot analysis for detecting TR21
receptor mRNA expression in specific tissues.
[0067] Preferred, however, are nucleic acid molecules comprising,
or alternatively consisting of, a nucleotide sequence at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to for example,
the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1), which do,
in fact, encode a polypeptide having TR21 functional activity. By
"a polypeptide having TR21 functional activity" is intended
polypeptides exhibiting activity similar, but not necessarily
identical, to an activity of the TR21 receptor of the invention
(either the full-length protein or, preferably, the mature
protein), as measured in a particular biological assay.
[0068] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to, for
example, the nucleic acid shown in FIG. 1 (SEQ ID NO:1), will
encode a polypeptide "having TR21-short functional activity."
Similarly, a large number of the nucleic acid molecules having a
sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%
identical to, for example, a nucleic acid sequence shown in FIG. 1,
will encode a polypeptide "having TR21 functional activity." In
fact, since degenerate variants of these nucleotide sequences all
encode the same polypeptide, this will be clear to the skilled
artisan even without performing a biological assay. It will be
further recognized in the art that, for such nucleic acid molecules
that are not degenerate variants, a reasonable number will also
encode a polypeptide having TR21 functional activity. This is
because the skilled artisan is fully aware of amino acid
substitutions that are either less likely or not likely to
significantly effect protein function (e.g., replacing one
aliphatic amino acid with a second aliphatic amino acid).
[0069] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in J. U. Bowie et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
TR21 Polynucleotide Assays
[0070] This invention is also related to the use of TR21
polynucleotides to detect complementary polynucleotides such as,
for example, as a diagnostic reagent. Detection of a normal and
mutated form of TR21 associated with a dysfunction will provide a
diagnostic tool that can add or define a diagnosis of a disease or
susceptibility to a disease which results from under-expression
over-expression or altered expression of TR21 (or a soluble form
thereof), such as, for example, tumors or autoimmune disease.
[0071] Individuals carrying mutations in the TR21 gene may be
detected at the DNA level by a variety of techniques. Nucleic acids
for diagnosis may be obtained from a biological sample from a
patient (e.g., a patient's cells, such as from blood, urine,
saliva, tissue biopsy and autopsy material). The genomic DNA may be
used directly for detection or may be amplified enzymatically by
using PCR prior to analysis. (Saiki et al., Nature 324:163-166
(1986)). RNA or cDNA may also be used in the same ways. As an
example, PCR primers complementary to the nucleic acid encoding
TR21 can be used to identify and analyze TR21 expression and
mutations. For example, deletions and insertions can be detected by
a change in size of the amplified product in comparison to the
normal genotype. Point mutations can be identified by hybridizing
amplified DNA to radiolabeled TR21 RNA or alternatively,
radiolabeled TR21 antisense DNA sequences. Perfectly matched
sequences can routinely be distinguished from mismatched duplexes
by techniques known in the art, such as, for example, RNase A
digestion or by differences in melting temperatures.
[0072] Sequence differences between a reference gene and genes
having mutations also may be revealed by direct DNA sequencing. In
addition, cloned DNA segments may be employed as probes to detect
specific DNA segments. The sensitivity of such methods can be
greatly enhanced by appropriate use of PCR or another amplification
method. For example, a sequencing primer is used with
double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is
performed by conventional procedures with radiolabeled nucleotide
or by automatic sequencing procedures with fluorescent-tags.
[0073] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents. Small
sequence deletions and insertions can be visualized by
high-resolution gel electrophoresis using techniques known in the
art. DNA fragments of different sequences may be distinguished on
denaturing formamide gradient gels in which the mobilities of
different DNA fragments are retarded in the gel at different
positions according to their specific melting or partial melting
temperatures (see, e.g., Myers et al., Science 230:1242
(1985)).
[0074] Sequence changes at specific locations also may be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., Proc. Natl.
Acad. Sci. USA 85: 4397-4401 (1985)).
[0075] Thus, the detection of a specific DNA sequence may be
achieved by methods which include, but are not limited to,
hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or the use of restriction enzymes, (e.g., restriction
fragment length polymorphisms ("RFLP") and Southern blotting of
genomic DNA.
[0076] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
[0077] The invention also encompasses isolated nucleic acids
encoding the above-described TR21 polypeptides and proteins. Such
polynucleotide sequences can routinely be determined using
techniques known in the art. For example, the amino acid sequence
of the TR21 polypeptides of the invention can be routinely
determined using techniques known in the art, such as via the Edman
degradation technique. (See, e.g., Creighton, 1983, "Proteins:
Structures and Molecular Principles", W.H. Freeman & Co., N.Y.,
pp. 34-49). The amino acid sequence obtained may be used as a guide
for the generation of oligonucleotide mixtures that can be used to
screen for polynucleotide sequences encoding TR21 polypeptides.
Screening may be accomplished, for example, by standard
hybridization or PCR techniques. For example, polynucleotides
encoding TR21 polypeptides of the invention may be isolated by
techniques known in the art, such as, for example, by performing
PCR using two degenerate oligonucleotide primer pools designed on
the basis of amino acid sequence of the TR21 polypeptide of
interest. Techniques for the generation of oligonucleotide mixtures
and the screening are well known. (See, e.g., Ausubel, supra., and
PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M.
et al., eds. Academic Press, Inc., New York). The template for the
reaction may be cDNA obtained by reverse transcription of mRNA
prepared from, for example, human or non-human cell lines or
tissue, such as B cells, known or suspected to express a TR21
polypeptide.
[0078] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences encode a TR21 polypeptide. The PCR
fragment may then be used to isolate a full-length cDNA clone by a
variety of methods. For example, the amplified fragment may be
labeled and used to screen a cDNA library, such as a bacteriophage
cDNA library. Alternatively, the labeled fragment may be used to
isolate genomic clones via the screening of a genomic library.
[0079] PCR technology may also be utilized to isolate full-length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source
(i.e., one known, or suspected, to express the TR21 gene, such as,
for example, B cells). A reverse transcription reaction may be
performed on the RNA using an oligonucleotide primer specific for
the most 5' end of the amplified fragment for the priming of first
strand synthesis. The resulting RNA/DNA hybrid may then be "tailed"
with guanines using a standard terminal transferase reaction, the
hybrid may be digested with RNAase H, and second strand synthesis
may then be primed with a poly-C primer. Thus, cDNA sequences
upstream of the amplified fragment may easily be isolated. For a
review of cloning strategies which may be used, see e.g., Sambrook
et al., 1989, infra.
[0080] Additionally, an expression library can be constructed
utilizing cDNA synthesized from, for example, RNA isolated from a
tissue known, or suspected, to express a TR21 polypeptide.
According to this strategy, polypeptides expressed by the cloned
cDNA are screened using standard antibody screening techniques in
conjunction with antibodies raised against the TR21 polypeptides of
the invention. (For screening techniques, see, for example, Harlow,
E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold
Spring Harbor Press, Cold Spring Harbor.) Additionally, screening
can be accomplished by screening with labeled Neutrokine-alpha
proteins or fusion proteins, such as, for example, those described
herein. Library clones detected via their reaction with such
labeled compounds can be purified and subjected to sequence
analysis according to methods well known to those of skill in the
art.
TR21 Vectors and Host Cells
[0081] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors
and/or nucleic acids of the invention and the production of TR21
polypeptides or fragments thereof by recombinant techniques.
[0082] Host cells can be genetically engineered to incorporate
nucleic acid molecules and express polypeptides of the present
invention. The polynucleotides may be introduced alone or with
other polynucleotides. Such other polynucleotides may be introduced
independently, co-introduced or introduced joined to the
polynucleotides of the invention.
[0083] In accordance with the present invention the vector may be,
for example, a plasmid vector, a single or double-stranded phage
vector, a single or double-stranded RNA or DNA viral vector. Such
vectors may be introduced into cells as polynucleotides, preferably
DNA, by well-known techniques for introducing DNA and RNA into
cells. Viral vectors may be replication competent or replication
defective. In the latter case viral propagation generally will
occur only in complementing host cells.
[0084] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate trans-acting
factors either are supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0085] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0086] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0087] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells, such as Saccharomyces or Pichia; insect cells
such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such
as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate
culture mediums and conditions for the above-described host cells
are known in the art.
[0088] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. The
availability of drugs which inhibit the function of the enzymes
encoded by these selectable markers allows for selection of cell
lines in which the vector sequences have been amplified after
integration into the host cell's DNA. An advantage of glutamine
synthase based vectors are the availability of cell lines (e.g.,
the murine myeloma cell line, NS0) which are glutamine synthase
negative. Glutamine synthase expression systems can also function
in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary
(CHO) cells) by providing additional inhibitor to prevent the
functioning of the endogenous gene. A glutamine synthase expression
system and components thereof are detailed in PCT publications:
WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657
which are hereby incorporated in their entireties by reference
herein. Additionally, glutamine synthase expression vectors that
may be used according to the present invention are commercially
available from suppliers including, for example, Lonza Biologics,
Inc. (Portsmouth, N.H.). Expression and production of monoclonal
antibodies using a GS expression system in murine myeloma cells is
described in Bebbington et al., Bio/technology 10:169(1992) and in
Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein
incorporated by reference.
[0089] Among vectors preferred for use in bacteria include pHE4-5
(ATCC.TM. Accession No. 209311; and variations thereof), pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, PBLUESCRIPT.TM. vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from STRATAGENE.TM.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from PHARMACIA.TM.. Preferred expression
vectors for use in yeast systems include, but are not limited to,
pYES2, pY01, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,
pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and pA0815 (all
available from Invitrogen, Carlsbad, Calif.). Among preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG
available from STRATAGENE.TM.; and pSVK3, pBPV, pMSG and pSVL
available from PHARMACIA.TM.. Other suitable vectors will be
readily apparent to the skilled artisan.
[0090] In one embodiment, the yeast Pichia pastoris is used to
express TR21 protein in a eukaryotic system. Pichia pastoris is a
methylotrophic yeast which can metabolize methanol as its sole
carbon source. A main step in the metabolism of methanol is the
oxidation of methanol to formaldehyde using O.sub.2. This reaction
is catalyzed by the enzyme alcohol oxidase. In order to metabolize
methanol as its sole carbon source, Pichia pastoris must generate
high levels of alcohol oxidase due, in part, to the relatively low
affinity of alcohol oxidase for O.sub.2. Consequently, in a growth
medium depending on methanol as a main carbon source, the promoter
region of one of the two alcohol oxidase genes (AOX1) is highly
active. In the presence of methanol, alcohol oxidase produced from
the AOX1 gene comprises up to approximately 30% of the total
soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol.
Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77
(1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987).
Thus, a heterologous coding sequence, such as, for example, a TR21
polynucleotide of the present invention, under the transcriptional
regulation of all or part of the AOX1 regulatory sequence is
expressed at exceptionally high levels in Pichia yeast grown in the
presence of methanol.
[0091] In one example, the plasmid vector pPIC9K is used to express
DNA encoding a TR21 polypeptide of the invention, as set forth
herein, in a Pichia yeast system essentially as described in
"Pichia Protocols: Methods in Molecular Biology," D. R. Higgins and
J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This
expression vector allows expression and secretion of a TR21 protein
of the invention by virtue of the strong AOX1 promoter linked to
the Pichia pastoris alkaline phosphatase (PHO) secretory signal
peptide (i.e., leader) located upstream of a multiple cloning
site.
[0092] Many other yeast vectors could be used in place of pPIC9K,
such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,
pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PA0815,
as one skilled in the art would readily appreciate, as long as the
proposed expression construct provides appropriately located
signals for transcription, translation, secretion (if desired), and
the like, including an in-frame AUG as required.
[0093] In one embodiment, high-level expression of a heterologous
coding sequence, such as, for example, a TR21 polynucleotide of the
present invention, may be achieved by cloning the heterologous
polynucleotide of the invention into an expression vector such as,
for example, pGAPZ or pGAPZalpha, and growing the yeast culture in
the absence of methanol.
[0094] The present invention also relates to host cells containing
the above-described vector constructs described herein, and
additionally encompasses host cells containing nucleotide sequences
of the invention that are operably associated with one or more
heterologous control regions (e.g., promoter and/or enhancer) using
techniques known of in the art. The host cell can be a higher
eukaryotic cell, such as a mammalian cell (e.g., a human derived
cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a prokaryotic cell, such as a bacterial cell. The
host strain may modulate the expression of the inserted gene
sequences, or modify and process the gene product in the specific
fashion desired. Expression from certain promoters can be elevated
in the presence of certain inducers; thus expression of the
genetically engineered polypeptide may be controlled. Furthermore,
different host cells have characteristics and specific mechanisms
for the translational and post-translational processing and
modification (e.g., phosphorylation, cleavage) of proteins.
Appropriate cell lines can be chosen to ensure the desired
modifications and processing of the foreign protein expressed.
[0095] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0096] In addition to encompassing host cells containing the vector
constructs discussed herein, the invention also encompasses
primary, secondary, and immortalized host cells of vertebrate
origin, particularly mammalian origin, that have been engineered to
delete or replace endogenous genetic material (e.g., TR21 coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide sequences) that is operably associated with TR21
polynucleotides of the invention, and which activates, alters,
and/or amplifies endogenous TR21 polynucleotides. For example,
techniques known in the art may be used to operably associate
heterologous control regions (e.g., promoter and/or enhancer) and
endogenous TR21 polynucleotide sequences via homologous
recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24,
1997; International Publication Number WO 96/29411; International
Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), the disclosures of each of which are
incorporated by reference in their entireties).
[0097] The TR21 polypeptide may be expressed in a modified form,
such as a fusion protein (comprising the polypeptide joined via a
peptide bond to a heterologous protein sequence (of a different
protein)), and may include not only secretion signals but also
additional heterologous functional regions. Alternatively, such a
fusion protein can be made by protein synthetic techniques, e.g.,
by use of a peptide synthesizer. Thus, a region of additional amino
acids, particularly charged amino acids, may be added to the
N-terminus of the polypeptide to improve stability and persistence
in the host cell, during purification or during subsequent handling
and storage. Also, peptide moieties may be added to the polypeptide
to facilitate purification. Such regions may be removed prior to
final preparation of the polypeptide. The addition of peptide
moieties to polypeptides to engender secretion or excretion, to
improve stability and to facilitate purification, among others, are
familiar and routine techniques in the art.
[0098] In one embodiment, polynucleotides encoding TR21
polypeptides of the invention may be fused to signal sequences
which will direct the localization of a protein of the invention to
particular compartments of a prokaryotic or eukaryotic cell and/or
direct the secretion of a protein of the invention from a
prokaryotic or eukaryotic cell. For example, in E. coli, one may
wish to direct the expression of the protein to the periplasmic
space. Examples of signal sequences or proteins (or fragments
thereof) to which the polypeptides of the invention may be fused in
order to direct the expression of the polypeptide to the
periplasmic space of bacteria include, but are not limited to, the
pelB signal sequence, the maltose binding protein (MBP) signal
sequence, MBP, the ompA signal sequence, the signal sequence of the
periplasmic E. coli heat-labile enterotoxin B-subunit, and the
signal sequence of alkaline phosphatase. Several vectors are
commercially available for the construction of fusion proteins
which will direct the localization of a protein, such as the pMAL
series of vectors (particularly the pMAL-p series) available from
New England Biolabs. In a specific embodiment, polynucleotides
encoding TR21 polypeptides of the invention may be fused to the
pelB pectate lyase signal sequence to increase the efficiency of
expression and purification of such polypeptides in Gram-negative
bacteria. See, U.S. Pat. Nos. 5,576,195 and 5,846,818, the contents
of which are herein incorporated by reference in their
entireties.
[0099] Examples of signal peptides that may be fused to a
polypeptide of the invention in order to direct its secretion in
mammalian cells include, but are not limited to, the MPIF-1 signal
sequence (amino acids 1-21 of GenBank Accession number AAB51134),
the stanniocalcin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:4),
and a consensus signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID
NO:5). A suitable signal sequence that may be used in conjunction
with baculoviral expression systems is the gp67 signal sequence,
(amino acids 1-19 of GenBank Accession Number AAA72759).
[0100] A preferred fusion protein comprises a heterologous region
from immunoglobulin that is useful to solubilize proteins. For
example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses
fusion proteins comprising various portions of constant region of
immunoglobulin molecules together with another human protein or
part thereof. In many cases, the Fc part in a fusion protein is
thoroughly advantageous for use in therapy and diagnosis and thus
results, for example, in improved pharmacokinetic properties (EP-A
0232 262). On the other hand, for some uses, it would be desirable
to be able to delete the Fc part after the fusion protein has been
expressed, detected and purified in the advantageous manner
described. This is the case when the Fc portion proves to be a
hindrance to use in therapy and diagnosis, for example, when the
fusion protein is to be used as an antigen for immunizations. In
drug discovery, for example, human proteins, such as the
hIL5-receptor, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., Journal of Molecular Recognition 8:52-58
(1995) and K. Johanson et al., The Journal of Biological Chemistry
270:16:9459-9471 (1995).
[0101] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or non-glycosylated. In
addition, polypeptides of the invention may also include an initial
modified methionine residue, in some cases as a result of
host-mediated processes.
[0102] In addition, proteins of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
Proteins: Structures and Molecular Principles, W.H. Freeman &
Co., N.Y. (1983), and Hunkapiller, et al., Nature 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
the TR21 polypeptides of the invention can be synthesized by use of
a peptide synthesizer. Furthermore, if desired, nonclassical amino
acids or chemical amino acid analogs can be introduced as a
substitution or addition into the TR21 polypeptide sequence.
Non-classical amino acids include, but are not limited to, to the
D-isomers of the common amino acids, 2,4-diaminobutyric acid,
a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric
acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid, 3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-butylglycine, t-butylalanine, phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino
acids such as b-methyl amino acids, Ca-methyl amino acids,
Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0103] The invention additionally, encompasses TR21 polypeptides
which are differentially modified during or after translation,
e.g., by glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited to, specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4, acetylation, formylation, oxidation,
reduction, metabolic synthesis in the presence of tunicamycin;
etc.
[0104] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0105] In specific embodiments, TR21 polypeptides of the invention
are attached to macrocyclic chelators useful for conjugating
radiometal ions, including but not limited to, .sup.111In,
.sup.177Lu, .sup.90Y, .sup.166Ho, and .sup.153Sm, to polypeptides.
In a preferred embodiment, the radiometal ion associated with the
macrocyclic chelators attached to TR21 polypeptides of the
invention is .sup.111In. In another preferred embodiment, the
radiometal ion associated with the macrocyclic chelator attached to
TR21 polypeptides of the invention is .sup.90Y. In specific
embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to TR21
polypeptide of the invention via a linker molecule. Examples of
linker molecules useful for conjugating DOTA to a polypeptide are
commonly known in the art--see, for example, DeNardo et al., Clin
Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem.
10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol.
26(8):943-50, 1999 which are hereby incorporated by reference in
their entirety. In addition, U.S. Pat. Nos. 5,652,361 and
5,756,065, which disclose chelating agents that may be conjugated
to antibodies, and methods for making and using them, are hereby
incorporated by reference in their entireties. Though U.S. Pat.
Nos. 5,652,361 and 5,756,065 focus on conjugating chelating agents
to antibodies, one skilled in the art could readily adapt the
method disclosed therein in order to conjugate chelating agents to
other polypeptides.
[0106] Also provided by the invention are chemically modified
derivatives of TR21 which may provide additional advantages such as
increased solubility, stability and circulating time of the
polypeptide, or decreased immunogenicity (see U.S. Pat. No.
4,179,337). The chemical moieties for derivatization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0107] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog). For example, the
polyethylene glycol may have an average molecular weight of about
200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,
5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000,
18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,
50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,
90,000, 95,000, or 100,000 kDa.
[0108] As noted above, the polyethylene glycol may have a branched
structure. Branched polyethylene glycols are described, for
example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl.
Biochem. Biotechnol 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are
incorporated herein by reference.
[0109] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0110] As suggested above, polyethylene glycol may be attached to
proteins via linkage to any of a number of amino acid residues. For
example, polyethylene glycol can be linked to a protein via
covalent bonds to lysine, histidine, aspartic acid, glutamic acid,
or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid
residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or
cysteine) of the protein or to more than one type of amino acid
residue (e.g., lysine, histidine, aspartic acid, glutamic acid,
cysteine and combinations thereof) of the protein.
[0111] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective proteins chemically modified at the
N-terminus modification may be accomplished by reductive alkylation
which exploits differential reactivity of different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0112] As indicated above, pegylation of the proteins of the
invention may be accomplished by any number of means. For example,
polyethylene glycol may be attached to the protein either directly
or by an intervening linker. Linkerless systems for attaching
polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et
al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No.
4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,
the disclosures of each of which are incorporated herein by
reference.
[0113] One system for attaching polyethylene glycol directly to
amino acid residues of proteins without an intervening linker
employs tresylated MPEG, which is produced by the modification of
monomethoxy polyethylene glycol (MPEG) using tresylchloride
(ClSO.sub.2CH.sub.2CF.sub.3). Upon reaction of protein with
tresylated MPEG, polyethylene glycol is directly attached to amine
groups of the protein. Thus, the invention includes
protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule
having a 2,2,2-trifluoreothane sulphonyl group.
[0114] Polyethylene glycol can also be attached to proteins using a
number of different intervening linkers. For example, U.S. Pat. No.
5,612,460, the entire disclosure of which is incorporated herein by
reference, discloses urethane linkers for connecting polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein
the polyethylene glycol is attached to the protein by a linker can
also be produced by reaction of proteins with compounds such as
MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,
MPEG-p-nitrophenolcarbonate, and various MPEG-succinate
derivatives. A number additional polyethylene glycol derivatives
and reaction chemistries for attaching polyethylene glycol to
proteins are described in WO 98/32466, the entire disclosure of
which is incorporated herein by reference. Pegylated protein
products produced using the reaction chemistries set out herein are
included within the scope of the invention.
[0115] The number of polyethylene glycol moieties attached to each
protein of the invention (i.e., the degree of substitution) may
also vary. For example, the pegylated proteins of the invention may
be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20, or more polyethylene glycol molecules. Similarly, the
average degree of substitution within ranges such as 1-3, 2-4, 3-5,
4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule. Methods for determining the degree of
substitution are discussed, for example, in Delgado et al., Crit.
Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[0116] As mentioned, the TR21 proteins of the invention may be
modified by either natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. It will be appreciated that the same type of
modification may be present in the same or varying degrees at
several sites in a given TR21 polypeptide. TR21 polypeptides may be
branched, for example, as a result of ubiquitination, and they may
be cyclic, with or without branching. Cyclic, branched, and
branched cyclic TR21 polypeptides may result from natural
posttranslational processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation,
amidation, covalent attachment of flavin, covalent attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphatidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to
proteins such as arginylation, and ubiquitination. (See, for
instance, PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T.
E. Creighton, W.H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al.,
Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci
663:48-62 (1992)).
[0117] The TR21 polypeptides of the invention can be recovered and
purified from chemical synthesis and recombinant cell cultures by
standard methods which include, but are not limited to, ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxyapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Well-known techniques for refolding
protein may be employed to regenerate active conformation when the
polypeptide is denatured during isolation and/or purification.
[0118] TR21 receptor polynucleotides and polypeptides may be used
in accordance with the present invention for a variety of
applications, particularly those that make use of the chemical and
biological properties of TR21. Among these are applications in
treatment of autoimmune diseases. Also contemplated are
applications in the treatment of tumors, resistance to parasites,
bacteria and viruses, to inhibit proliferation of B cells, to
induce proliferation of T-cells, endothelial cells and certain
hematopoietic cells, to treat restenosis, graft vs. host disease,
to regulate anti-viral responses and to prevent certain autoimmune
diseases after stimulation of TR21 by an agonist. Additional
applications relate to diagnosis and to treatment of disorders of
cells, tissues and organisms. These aspects of the invention are
discussed further below.
TR21 Transgenics and "Knock-Outs"
[0119] The TR21 proteins of the invention can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[0120] Any technique known in the art may be used to introduce the
transgene (i.e., nucleic acids of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol Biotechnol 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, Mol Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pluripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety. Further,
the contents of each of the documents recited in this paragraph is
herein incorporated by reference in its entirety. Gordon,
"Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989), which
is incorporated by reference herein in its entirety. See also, U.S.
Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative Selection
Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi, et al.,
Cells and Non-Human Organisms Containing Predetermined Genomic
Modifications and Positive-Negative Selection Methods and Vectors
for Making Same); U.S. Pat. No. 4,736,866 (Leder, et al.,
Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner,
et al., Genetic Transformation of Zygotes); each of which is hereby
incorporated by reference in its entirety.
[0121] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campbell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)), each of which is herein incorporated by
reference in its entirety).
[0122] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric animals. The transgene may be integrated as a
single transgene or as multiple copies such as in concatamers,
e.g., head-to-head tandems or head-to-tail tandems. The transgene
may also be selectively introduced into and activated in a
particular cell type by following, for example, the teaching of
Lasko et al. (Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred. Briefly, when such a technique is to be utilized,
vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via
homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleotide sequence of the
endogenous gene. The transgene may also be selectively introduced
into a particular cell type, thus inactivating the endogenous gene
in only that cell type, by following, for example, the teaching of
Gu et al. (Science 265:103-106 (1994)). The regulatory sequences
required for such a cell-type specific inactivation will depend
upon the particular cell type of interest, and will be apparent to
those of skill in the art. The contents of each of the documents
recited in this paragraph is herein incorporated by reference in
its entirety.
[0123] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[0124] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[0125] Transgenic and "knock-out" animals of the invention have
uses which include, but are not limited to, animal model systems
useful in elaborating the biological function of TR21 polypeptides,
studying conditions and/or disorders associated with aberrant TR21
expression, and in screening for compounds effective in
ameliorating such conditions and/or disorders.
[0126] In further embodiments of the invention, cells that are
genetically engineered to express the proteins of the invention, or
alternatively, that are genetically engineered not to express the
proteins of the invention (e.g., knockouts) are administered to a
patient in vivo. Such cells may be obtained from the patient (i.e.,
animal, including human) or an MHC compatible donor and can
include, but are not limited to fibroblasts, bone marrow cells,
blood cells (e.g., lymphocytes), adipocytes, muscle cells,
endothelial cells, etc. The cells are genetically engineered in
vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and
implanted in the body, e.g., genetically engineered fibroblasts can
be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each
of which is incorporated by reference herein in its entirety).
[0127] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well-known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
TR21 Receptor Polypeptides and Fragments
[0128] The TR21 proteins (polypeptides) of the invention may be in
monomers or multimers (i.e., dimers, trimers, tetramers, and higher
multimers). Accordingly, the present invention relates to monomers
and multimers of the TR21 proteins (polypeptides) of the invention,
their preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the
polypeptides of the invention are monomers, dimers, trimers or
tetramers. In additional embodiments, the multimers of the
invention are at least dimers, at least trimers, or at least
tetramers.
[0129] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only TR21 proteins of the invention (including TR21
fragments, variants, and fusion proteins, as described herein).
These homomers may contain TR21 proteins having identical or
different polypeptide sequences. In a specific embodiment, a
homomer of the invention is a multimer containing only TR21
proteins having an identical polypeptide sequence. In another
specific embodiment, a homomer of the invention is a multimer
containing TR21 proteins having different polypeptide sequences
(e.g., TR21 mutations containing proteins have polypetide
sequences. In specific embodiments, the multimer of the invention
is a homodimer (e.g., containing TR21 proteins having identical or
different polypeptide sequences) or a homotrimer (e.g., containing
TR21 proteins having identical or different polypeptide sequences).
In additional embodiments, the homomeric multimer of the invention
is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
[0130] As used herein, the term heteromer refers to a multimer
containing heterologous proteins (i.e., proteins containing only
polypeptide sequences that do not correspond to polypeptide
sequences encoded by the TR21 gene) in addition to the TR21
proteins of the invention. In a specific embodiment, the multimer
of the invention is a heterodimer, a heterotrimer, or a
heterotetramer. In additional embodiments, the heteromeric multimer
of the invention is at least a heterodimer, at least a
heterotrimer, or at least a heterotetramer.
[0131] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked, by for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when proteins of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when proteins of the
invention contact antibodies to the polypeptides of the invention
(including antibodies to the heterologous polypeptide sequence in a
fusion protein of the invention) in solution. In other embodiments,
multimers of the invention are formed by covalent associations with
and/or between the TR21 proteins of the invention. Such covalent
associations may involve one or more amino acid residues contained
in the polypeptide sequence of the protein (e.g., the polypeptide
sequence shown in FIG. 1 (SEQ ID NO:2)). In one instance, the
covalent associations are cross-linking between cysteine residues
located within the polypeptide sequences of the proteins which
interact in the native (i.e., naturally occurring) polypeptide. In
another instance, the covalent associations are the consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may involve one or more amino acid residues contained
in the heterologous polypeptide sequence in a TR21 fusion protein.
In one example, covalent associations are mediated by the
heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are mediated by the heterologous
sequence contained in a TR21-Fc fusion protein of the invention (as
described herein). In another specific example, covalent
associations of fusion proteins of the invention are mediated by
heterologous polypeptide sequences from other TNF family
ligand/receptor members that are capable of forming covalently
associated multimers, such as for example, osteoprotegerin (see,
e.g., International Publication No. WO 98/49305, the contents of
which are herein incorporated by reference in its entirety). In
another embodiment, two or more TR21 polypeptides of the invention
are joined through synthetic linkers (e.g., peptide, carbohydrate
or soluble polymer linkers). Examples include those peptide linkers
described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference). Proteins comprising multiple TR21 polypeptides
separated by peptide linkers may be produced using conventional
recombinant DNA technology.
[0132] Another method for preparing multimeric TR21 polypeptides of
the invention involves use of TR21 polypeptides fused to a leucine
zipper or isoleucine zipper polypeptide sequence. Leucine zipper
domains and isoleucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been
found in a variety of different proteins. Among the known leucine
zippers are naturally occurring peptides and derivatives thereof
that dimerize or trimerize. Examples of leucine zipper domains
suitable for producing soluble multimeric TR21 proteins are those
described in PCT application WO 94/10308, hereby incorporated by
reference. Recombinant fusion proteins comprising a soluble TR21
polypeptide fused to a peptide that dimerizes or trimerizes in
solution are expressed in suitable host cells, and the resulting
soluble multimeric TR21 is recovered from the culture supernatant
using techniques known in the art.
[0133] Certain members of the TNF family of proteins are believed
to exist in trimeric form (Beutler and Huffel, Science 264:667,
1994; Banner et al., Cell 73:431, 1993). Thus, trimeric TR21 may
offer the advantage of enhanced biological activity. Preferred
leucine zipper moieties are those that preferentially form trimers.
One example is a leucine zipper derived from lung surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters
344:191, (1994)) and in U.S. patent application Ser. No.
08/446,922, hereby incorporated by reference. Other peptides
derived from naturally occurring trimeric proteins may be employed
in preparing trimeric TR21.
[0134] In another example, proteins of the invention are associated
by interactions between Flag.RTM. polypeptide sequences contained
in Flag.RTM.-TR21 fusion proteins of the invention. In a further
embodiment, associated proteins of the invention are associated by
interactions between heterologous polypeptide sequence contained in
Flag.RTM.-TR21 fusion proteins of the invention and anti-Flag.RTM.
antibody.
[0135] The multimers of the invention may be generated using
chemical techniques known in the art. For example, proteins desired
to be contained in the multimers of the invention may be chemically
cross-linked using linker molecules and linker molecule length
optimization techniques known in the art (see, e.g., U.S. Pat. No.
5,478,925, which is herein incorporated by reference in its
entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the polypeptide sequence of the proteins desired to be
contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
Further, proteins of the invention may be routinely modified by the
addition of cysteine or biotin to the C terminus or N-terminus of
the polypeptide sequence of the protein and techniques known in the
art may be applied to generate multimers containing one or more of
these modified proteins (see, e.g., U.S. Pat. No. 5,478,925, which
is herein incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the protein components desired to be contained in the
multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0136] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, proteins contained in multimers of the invention are
produced recombinantly using fusion protein technology described
herein or otherwise known in the art (see, e.g., U.S. Pat. No.
5,478,925, which is herein incorporated by reference in its
entirety). In a specific embodiment, polynucleotides coding for a
homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain and which can be incorporated by membrane
reconstitution techniques into liposomes (see, e.g., U.S. Pat. No.
5,478,925, which is herein incorporated by reference in its
entirety).
[0137] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell. For example, a recombinantly produced version of the
TR21 polypeptide can be substantially purified by the one-step
method described in Smith and Johnson, Gene 67:31-40 (1988).
[0138] Accordingly, in one embodiment, the invention provides an
isolated TR21 polypeptide having the amino acid sequence encoded by
the amino acid sequence in FIG. 1 (SEQ ID NO:2), or a polypeptide
comprising, or alternatively consisting of, a portion of the above
polypeptides, such as for example, a mature TR21, the TR21
extracellular domain (amino acids 1 to 81 of FIG. 1 (SEQ ID NO:2)),
the TR21 cysteine rich motif (amino acids 19 to 35 of FIG. 1 (SEQ
ID NO:2)), and/or the TR21 intracellular domain (amino acids 102 to
184 of FIG. 1 (SEQ ID NO:2)).
[0139] Polypeptide fragments of the present invention include
polypeptides comprising or alternatively, consisting of: an amino
acid sequence contained in FIG. 1 (SEQ ID NO:2); and encoded by a
nucleic acid which hybridizes (e.g., under stringent hybridization
conditions) to the complementary strand of the nucleotide sequence
shown in FIG. 1 (SEQ ID NO: 1), or a fragment thereof.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0140] Protein fragments may be "free-standing," or comprised
within a larger polypeptide of which the fragment forms a part or
region, most preferably as a single continuous region.
Representative examples of polypeptide fragments of the invention,
include, for example, fragments that comprise or alternatively,
consist of about amino acid residues: 1 to 2, 3 to 15, 16 to 18, 19
to 26, 27 to 35, 36 to 38, 39 to 45, 46 to 50, 51 to 53, 54 to 58,
59 to 61, 62 to 70, 71 to 81, 82 to 101, 102 to 103, 104 to 112,
113 to 114, 115 to 122, 123 to 135, 136 to 138, 139 to 146, 147 to
153, 154 to 177, 178 to 182 and/or 183 to 184, of SEQ ID NO:2 or
FIG. 1. In this context "about" includes the particularly recited
ranges, ranges larger or smaller by several (5, 4, 3, 2, or 1)
amino acids, at either extreme or at both extremes. Moreover,
polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, 140, 150 or amino acids in length.
Polynucleotides encoding these polypeptides are also encompassed by
the invention. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0141] In additional embodiments, the polypeptide fragments of the
invention comprise, or alternatively consist of, one or more TR21
domains. Preferred polypeptide fragments of the present invention
include one, two, three or more members selected from the group:
(a) a polypeptide comprising or alternatively, consisting of, the
TR21 extracellular domain (predicted to constitute amino acid
residues 1 to 81 FIG. 1 (SEQ ID NO:2)); (b) a polypeptide
comprising or alternatively, consisting of, the TR21 cysteine rich
domain (predicted to constitute amino acid residues 19 to 35 FIG. 1
(SEQ ID NO:2)); (c) a polypeptide comprising or alternatively,
consisting of, the TR21 transmembrane domain (predicted to
constitute amino acid residues 82 to 101 FIG. 1 (SEQ ID NO:2)); (d)
a polypeptide comprising or alternatively, consisting of, the TR21
intracellular domain (predicted to constitute amino acid residues
102 to 184 FIG. 1 (SEQ ID NO:2)); (e) a polypeptide comprising, or
alternatively, consisting of, one, two, three, four or more,
epitope bearing portions of the TR21 protein; or (f) any
combination of polypeptides (a)-(e). Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0142] As discussed above, it is believed that the extracellular
cysteine rich motif of TR21 is important for interactions between
TR21 and its ligands (e.g., Neutrokine-alpha). Accordingly, in
preferred embodiments, polypeptides of the invention comprise, or
alternatively consist of amino acid residues 19 to 35 of FIG. 1
(SEQ ID NO:2). Proteins comprising or alternatively consisting of a
polypeptide sequence which is at least 80%, 85%, 90%, 92%, 95%,
96%, 97%, 98% or 99% identical to the polypeptide sequences of the
cysteine rich motif are also encompassed by the invention.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0143] Among the especially preferred fragments of the invention
are fragments characterized by structural or functional attributes
of TR21. Such fragments include amino acid residues that comprise
alpha-helix and alpha-helix forming regions ("alpha-regions"),
beta-sheet and beta-sheet-forming regions ("beta-regions"), turn
and turn-forming regions ("turn-regions"), coil and coil-forming
regions ("coil-regions"), hydrophilic regions, hydrophobic regions,
alpha amphipathic regions, beta amphipathic regions, surface
forming regions, and high antigenic index regions (i.e., containing
three or more contiguous amino acids having an antigenic index of
greater than or equal to 1.5, as identified using the default
parameters of the Jameson-Wolf program) of complete (i.e.,
full-length) TR21 (FIG. 1 (SEQ ID NO:2)). Certain preferred regions
are those set out in FIG. 2 and Table 1 and include, but are not
limited to, regions of the aforementioned types identified by
analysis of the amino acid sequence depicted in FIG. 1 (SEQ ID
NO:2), such preferred regions include; Garnier-Robson predicted
alpha-regions, beta-regions, turn-regions, and coil-regions;
Chou-Fasman predicted alpha-regions, beta-regions, and
turn-regions; Kyte-Doolittle predicted hydrophilic; Hopp-Woods
predicted hydrophobic regions; Eisenberg alpha and beta amphipathic
regions; Emini surface-forming regions; and Jameson-Wolf high
antigenic index regions, as predicted using the default parameters
of these computer programs. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0144] As mentioned above, even if deletion of one or more amino
acids from the N-terminus of a protein results in modification of
loss of one or more biological functions of the protein, other
functional activities (e.g., biological activities, ability to
multimerize, ability to bind TR21 ligand (e.g., Neutrokine-alpha))
may still be retained. For instance, Ron et al., J. Biol. Chem.,
268:2984-2988 (1993) reported modified KGF proteins that had
heparin binding activity even if 3, 8, or 27 amino-terminal amino
acid residues were missing. The ability of shortened TR21 "muteins"
to induce and/or bind to antibodies which recognize the complete or
mature forms of the polypeptides generally will be retained when
less than the majority of the residues of the complete or mature
polypeptide are removed from the N-terminus. As used herein, a
"mutein" is a mutant protein including single or multiple amino
acid substitutions, deletions, or additions (including fusion
proteins). Whether a particular polypeptide lacking N-terminal
residues of a complete full-length polypeptide retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art. It is not unlikely
that a TR21 mutein with a large number of deleted N-terminal amino
acid residues may retain some biological or immunogenic activities.
In fact, peptides composed of as few as six TR21 amino acid
residues may often evoke an immune response.
[0145] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the TR21 amino acid sequence shown in FIG. 1, up to the
alanine residue at position number 179 and polynucleotides encoding
such polypeptides. In particular, the present invention provides
polypeptides comprising, or alternatively consisting of, the amino
acid sequence of residues n.sup.1-184 of FIG. 1, where n.sup.1 is
an integer from 2 to 179 corresponding to the position of the amino
acid residue in FIG. 1 (SEQ ID NO:2).
[0146] More in particular, N-terminal deletions of the TR21
polypeptide of the invention shown as SEQ ID NO:2 include
polypeptides comprising the amino acid sequence of residues: R-2 to
Q-184; R-3 to Q-184; G-4 to Q-184; P-5 to Q-184; R-6 to Q-184; S-7
to Q-184; L-8 to Q-184; R-9 to Q-184; G-10 to Q-184; R-11 to Q-184;
D-12 to Q-184; A-13 to Q-184; P-14 to Q-184; A-15 to Q-184; P-16 to
Q-184; T-17 to Q-184; P-18 to Q-184; C-19 to Q-184; V-20 to Q-184;
P-21 to Q-184; A-22 to Q-184; E-23 to Q-184; C-24 to Q-184; F-25 to
Q-184; D-26 to Q-184; L-27 to Q-184; L-28 to Q-184; V-29 to Q-184;
R-30 to Q-184; H-31 to Q-184; C-32 to Q-184; V-33 to Q-184; A-34 to
Q-184; C-35 to Q-184; G-36 to Q-184; L-37 to Q-184; L-38 to Q-184;
R-39 to Q-184; T-40 to Q-184; P-41 to Q-184; R-42 to Q-184; P-43 to
Q-184; K-44 to Q-184; P-45 to Q-184; A-46 to Q-184; G-47 to Q-184;
A-48 to Q-184; S-49 to Q-184; S-50 to Q-184; P-51 to Q-184; A-52 to
Q-184; P-53 to Q-184; R-54 to Q-184; T-55 to Q-184; A-56 to Q-184;
L-57 to Q-184; Q-58 to Q-184; P-59 to Q-184; Q-60 to Q-184; E-61 to
Q-184; S-62 to Q-184; V-63 to Q-184; G-64 to Q-184; A-65 to Q-184;
G-66 to Q-184; A-67 to Q-184; G-68 to Q-184; E-69 to Q-184; A-70 to
Q-184; A-71 to Q-184; L-72 to Q-184; P-73 to Q-184; L-74 to Q-184;
P-75 to Q-184; G-76 to Q-184; L-77 to Q-184; L-78 to Q-184; F-79 to
Q-184; G-80 to Q-184; A-81 to Q-184; P-82 to Q-184; A-83 to Q-184;
L-84 to Q-184; L-85 to Q-184; G-86 to Q-184; L-87 to Q-184; A-88 to
Q-184; L-89 to Q-184; V-90 to Q-184; L-91 to Q-184; A-92 to Q-184;
L-93 to Q-184; V-94 to Q-184; L-95 to Q-184; V-96 to Q-184; G-97 to
Q-184; L-98 to Q-184; V-99 to Q-184; S-100 to Q-184; W-101 to
Q-184; R-102 to Q-184; R-103 to Q-184; R-104 to Q-184; Q-105 to
Q-184; R-106 to Q-184; R-107 to Q-184; L-108 to Q-184; R-109 to
Q-184; G-110 to Q-184; A-111 to Q-184; S-112 to Q-184; S-113 to
Q-184; A-114 to Q-184; E-115 to Q-184; A-116 to Q-184; P-117 to
Q-184; D-118 to Q-184; G-119 to Q-184; D-120 to Q-184; K-121 to
Q-184; D-122 to Q-184; A-123 to Q-184; P-124 to Q-184; E-125 to
Q-184; P-126 to Q-184; L-127 to Q-184; D-128 to Q-184; K-129 to
Q-184; V-130 to Q-184; I-131 to Q-184; I-132 to Q-184; L-133 to
Q-184; S-134 to Q-184; P-135 to Q-184; G-136 to Q-184; I-137 to
Q-184; S-138 to Q-184; D-139 to Q-184; A-140 to Q-184; T-141 to
Q-184; A-142 to Q-184; P-143 to Q-184; A-144 to Q-184; W-145 to
Q-184; P-146 to Q-184; P-147 to Q-184; P-148 to Q-184; G-149 to
Q-184; E-150 to Q-184; D-151 to Q-184; P-152 to Q-184; G-153 to
Q-184; T-154 to Q-184; T-155 to Q-184; P-156 to Q-184; P-157 to
Q-184; G-158 to Q-184; H-159 to Q-184; S-160 to Q-184; V-161 to
Q-184; P-162 to Q-184; V-163 to Q-184; P-164 to Q-184; A-165 to
Q-184; T-166 to Q-184; E-167 to Q-184; L-168 to Q-184; G-169 to
Q-184; S-170 to Q-184; T-171 to Q-184; E-172 to Q-184; L-173 to
Q-184; V-174 to Q-184; T-175 to Q-184; T-176 to Q-184; K-177 to
Q-184; T-178 to Q-184; and/or A-179 to Q-184 of the TR21 sequence
disclosed as SEQ ID NO:2 and shown in FIG. 1. Polypeptides encoded
by these polynucleotides are also encompassed by the invention.
[0147] In another embodiment, N-terminal deletions of the TR21
polypeptide can be described by the general formula n.sup.2-81,
where n.sup.2 is a number from 2 to 76, corresponding to the
position of amino acid identified in FIG. 1 (SEQ ID NO:2).
Preferably, N-terminal deletions of the TR21 polypeptide of the
invention shown as FIG. 1 (SEQ ID NO:2) include polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues R-2 to A-81; R-3 to A-81; G-4
to A-81; P-5 to A-81; R-6 to A-81; S-7 to A-81; L-8 to A-81; R-9 to
A-81; G-10 to A-81; R-11 to A-81; D-12 to A-81; A-13 to A-81; P-14
to A-81; A-15 to A-81; P-16 to A-81; T-17 to A-81; P-18 to A-81;
C-19 to A-81; V-20 to A-81; P-21 to A-81; A-22 to A-81; E-23 to
A-81; C-24 to A-81; F-25 to A-81; D-26 to A-81; L-27 to A-81; L-28
to A-81; V-29 to A-81; R-30 to A-81; H-31 to A-81; C-32 to A-81;
V-33 to A-81; A-34 to A-81; C-35 to A-81; G-36 to A-81; L-37 to
A-81; L-38 to A-81; R-39 to A-81; T-40 to A-81; P-41 to A-81; R-42
to A-81; P-43 to A-81; K-44 to A-81; P-45 to A-81; A-46 ti A-81;
G-47 to A-81; A-48 to A-81; S-49 to A-81; S-50 to A-81; P-51 to
A-81; A-52 to A-81; P-53 to A-81; R-54 to A-81; T-55 to A-81; A-56
to A-81; L-57 to A-81; Q-58 to A-81; P-59 to A-81; Q-60 to A-81;
E-61 to A-81; S-62 to A-81; V-63 to A-81; G-64 to A-81; A-65 to
A-81; G-66 to A-81; A-67 to A-81; G-68 to A-81; E-69 to A-81; A-70
to A-81; A-71 to A-81; L-72 to A-81; P-73 to A-81; L-74 to A-81;
P-75 to A-81; and/or G-76 to A-81 of the TR21 extracellular domain
sequence shown in FIG. 1 (SEQ ID NO:2). Polypeptides encoded by
these polynucleotides are also encompassed by the invention.
[0148] In a most preferred embodiment, the polypeptides of the
invention comprise, or alternatively consist of amino acids P-16 to
L-38 as shown in FIG. 1 (SEQ ID NO:2). Polypeptides at least 90%,
at least 95%, at least 96%, at least 97%, and/or at least 99%
identical to amino acids P-16 to L-38 as shown as FIG. 1 (SEQ ID
NO:2) are also encompassed by the invention. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0149] In another most preferred embodiment, the polypeptides of
the invention comprise, or alternatively consist of amino acids
P-16 to A-81 as shown in FIG. 1 (SEQ ID NO:2). Polypeptides at
least 90%, at least 95%, at least 96%, at least 97%, and/or at
least 99% identical to amino acids P-16 to A-81 as shown as FIG. 1
(SEQ ID NO:2) are also encompassed by the invention.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0150] In another most preferred embodiment, the polypeptides of
the invention comprise, or alternatively consist of amino acids
P-16 to A-83 as shown in FIG. 1 (SEQ ID NO:2). Polypeptides at
least 90%, at least 95%, at least 96%, at least 97%, and/or at
least 99% identical to amino acids P-16 to A-83 as shown as FIG. 1
(SEQ ID NO:2) are also encompassed by the invention.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0151] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other functional activities (e.g., biological activities,
ability to multimerize, ability to bind TR21 ligands (e.g.,
Neutrokine alpha) may still be retained). For example the ability
of the shortened TR21 mutein to induce and/or bind to antibodies
which recognize the complete or mature forms of the polypeptide
generally will be retained when less than the majority of the
residues of the complete or mature polypeptide are removed from the
C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete polypeptide retains such immunologic
activities can readily be determined by routine methods described
herein and otherwise known in the art. It is not unlikely that a
TR21 mutein with a large number of deleted C-terminal amino acid
residues may retain some biological or immunogenic activities. In
fact, peptides composed of as few as six TR21 amino acid residues
may often evoke an immune response.
[0152] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the TR21 polypeptide shown
in FIG. 1, up to the arginine residue at position number 6, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues 1-m.sup.1 of FIG. 1, where m.sup.1 is an
integer from 6 to 183 corresponding to the position of the amino
acid residue in FIG. 1 (SEQ ID NO:2).
[0153] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues: M-1 to Q-183; M-1 to E-182;
M-1 to P-181; M-1 to G-180; M-1 to A-179; M-1 to T-178; M-1 to
K-177; M-1 to T-176; M-1 to T-175; M-1 to V-174; M-1 to L-173; M-1
to E-172; M-1 to T-171; M-1 to S-170; M-1 to G-169; M-1 to L-168;
M-1 to E-167; M-1 to T-166; M-1 to A-165; M-1 to P-164; M-1 to
V-163; M-1 to P-162; M-1 to V-161; M-1 to S-160; M-1 to H-159; M-1
to G-158; M-1 to P-157; M-1 to P-156; M-1 to T-155; M-1 to T-154;
M-1 to G-153; M-1 to P-152; M-1 to D-151; M-1 to E-150; M-1 to
G-149; M-1 to P-148; M-1 to P-147; M-1 to P-146; M-1 to W-145; M-1
to A-144; M-1 to P-143; M-1 to A-142; M-1 to T-141; M-1 to A-140;
M-1 to D-139; M-1 to S-138; M-1 to I-137; M-1 to G-136; M-1 P-135;
M-1 to S-134; M-1 to L-133; M-1 to I-132; M-1 to I-131; M-1 to
V-130; M-1 to K-129; M-1 to D-128; M-1 to L-127; M-1 to P-126; M-1
to E-125; M-1 to P-124; M-1 to A-123; M-1 to D-122; M-1 to K-121;
M-1 to D-120; M-1 to G-119; M-1 to D-118; M-1 to P-117; M-1 to
A-116; M-1 to E-115; M-1 to A-114; M-1 to S-113; M-1 to S-112; M-1
to A-111; M-1 to G-110; M-1 to R-109; M-1 to L-108; M-1 to R-107;
M-1 to R-106; M-1 to Q-105; M-1 to R-104; M-1 to R-103; M-1 to
R-102; M-1 to W-101; M-1 to S-100; M-1 to V-99; M-1 to L-98; M-1 to
G-97; M-1 to V-96; M-1 to L-95; M-1 to V-94; M-1 to L-93; M-1 to
A-92; M-1 to L-91; M-1 to V-90; M-1 to L-89; M-1 to A-88; M-1 to
L-87; M-1 to G-86; M-1 to L-85; M-1 to L-84; M-1 to A-83; M-1 to
P-82; M-1 to A-81; M-1 to G-80; M-1 to F-79; M-1 to L-78; M-1 to
L-77; M-1 to G-76; M-1 to P-75; M-1 to L-74; M-1 to P-73; M-1 to
L-72; M-1 to A-71; M-1 to A-70; M-1 to E-69; M-1 to G-68; M-1 to
A-67; M-1 to G-66; M-1 to A-65; M-1 to G-64; M-1 to V-63; M-1 to
S-62; M-1 to E-61; M-1 to Q-60; M-1 to P-59; M-1 to Q-58; M-1 to
L-57; M-1 to A-56; M-1 to T-55; M-1 to R-54; M-1 to P-53; M-1 to
A-52; M-1 to P-51; M-1 to S-50; M-1 to S-49; M-1 to A-48; M-1 to
G-47; M-1 to A-46; M-1 P-45; M-1 to K-44; M-1 to P-43; M-1 to R-42;
M-1 to P-41; M-1 to T-40; M-1 to R-39; M-1 to L-38; M-1 to L-37;
M-1 to G-36; M-1 to C-35; M-1 to A-34; M-1 to V-33; M-1 to C-32;
M-1 to H-31; M-1 to R-30; M-1 to V-29; M-1 to L-28; M-1 to L-27;
M-1 to D-26; M-1 to F-25; M-1 to C-24; M-1 to E-23; M-1 to A-22;
M-1 to P-21; M-1 to V-20; M-1 to C-19; M-1 to P-18; M-1 to T-17;
M-1 to P-16; M-1 to A-15; M-1 to P14; M-1 to A-13; M-1 to D-12; M-1
to R-11; M-1 to G-10; M-1 to R-9; M-1 to L-8; M-1 to S-7; and/or
M-1 to R-6 of the TR21 sequence disclosed as SEQ ID NO:2 and shown
in FIG. 1. Polypeptides encoded by these polynucleotides are also
encompassed by the invention.
[0154] The invention also provides polynucleotides encoding
polypeptides having one or more amino acids deleted from both the
amino and the carboxyl termini, which may be described generally as
having residues n.sup.1-m.sup.1 and/or n.sup.2-m.sup.1 of FIG. 1
(i.e., SEQ ID NO:2), where n.sup.1, n.sup.2, and m.sup.1 are
integers as described above. Thus, any of the above listed N- or
C-terminal deletions can be combined to produce a polynucleotide
encoding an N- and C-terminal deleted TR21 polypeptide.
[0155] In a most preferred embodiment, the polypeptides of the
invention comprise, or alternatively consist of amino acids P-16 to
L-38, or P-16 to A-81, P-16 to A-84, as shown in FIG. 1 (SEQ ID
NO:2). Polypeptides at least 90%, at least 95%, at least 96%, at
least 97%, and/or at least 99% identical to amino acids P-16 to
L-38, or P-16 to A-81, P-16 to A-84, as shown in FIG. 1 (SEQ ID
NO:2) are also encompassed by the invention. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0156] The present invention encompasses TR21 polypeptides
comprising, or alternatively consisting of, an epitope of the
polypeptide having an amino acid sequence of FIG. 1 (SEQ ID NO:2),
or an epitope of a polypeptide sequence encoded by a polynucleotide
that hybridizes to the complement of the sequence of SEQ ID NO:1
(e.g., under stringent hybridization conditions or lower stringency
hybridization conditions as defined herein). The present invention
further encompasses polynucleotide sequences encoding an epitope of
a TR21 polypeptide sequence of the invention (such as, for example,
the sequence disclosed in SEQ ID NO:2), polynucleotide sequences of
the complementary strand of a polynucleotide sequence encoding an
epitope of the invention, and polynucleotide sequences which
hybridize to this complementary strand (e.g., under stringent
hybridization conditions or lower stringency hybridization
conditions defined herein).
[0157] The term "epitopes," as used herein, refers to portions of a
polypeptide having antigenic or immunogenic activity in an animal,
preferably a mammal, and most preferably in a human. In a preferred
embodiment, the present invention encompasses a polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An "immunogenic epitope," as used herein, is defined
as a portion of a protein that elicits an antibody response in an
animal, as determined by any method known in the art, for example,
by the methods for generating antibodies described herein. (See,
e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). Further still, U.S. Pat. No. 5,194,392 to Geysen (1990)
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092 to
Geysen (1989) describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand-binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide
Mixtures discloses linear C1-C7-alkyl peralkylated oligopeptides
and sets and libraries of such peptides, as well as methods for
using such oligopeptide sets and libraries for determining the
sequence of a peralkylated oligopeptide that preferentially binds
to an acceptor molecule of interest. Thus, non-peptide analogs of
the epitope-bearing peptides of the invention also can be made
routinely by these methods. Antibodies that specifically bind TR21
are also encompassed by the invention.
[0158] The term "antigenic epitope," as used herein, is defined as
a portion of a protein to which an antibody can immunospecifically
bind its antigen as determined by any method well known in the art,
for example, by the immunoassays described herein. Immunospecific
binding excludes non-specific binding but does not necessarily
exclude cross-reactivity with other antigens. Antigenic epitopes
need not necessarily be immunogenic.
[0159] Fragments which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci.
USA 82:5131-5135 (1985), further described in U.S. Pat. No.
4,631,211).
[0160] In the present invention, antigenic epitopes preferably
contain a sequence of at least 3, at least 4, at least 5, at least
6, at least 7, more preferably at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at
least 20, at least 25, at least 30, at least 40, at least 50, and,
most preferably, between about 15 to about 30 amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 amino acid residues in length.
[0161] Non-limiting examples of antigenic polypeptides of the
invention include one, two, three, four, five, or more members
selected from the group: a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Arg-3 to about Ala-15
in FIG. 1 (SEQ ID NO:2); a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Arg-39 to about
Pro-45 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising, or
alternatively consisting of, amino acid residues from about Pro-51
to about Pro-53 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising,
or alternatively consisting of, amino acid residues from about
Pro-59 to Glu-61 in FIG. 1 (SEQ ID NO:2); a polypeptide comprising,
or alternatively consisting of, amino acid residues from about
Arg-104 to about Ser-112 in FIG. 1 (SEQ ID NO:2); a polypeptide
comprising, or alternatively consisting of, amino acid residues
from about Glu-115 to about Asp-122 in FIG. 1 (SEQ ID NO:2); a
polypeptide comprising, or alternatively consisting of, amino acid
residues from about Gly-136 to about Ser-138 in FIG. 1 (SEQ ID
NO:2); a polypeptide comprising, or alternatively consisting of,
amino acid residues from about Pro-147 to about Gly-153 in FIG. 1
(SEQ ID NO:2); and a polypeptide comprising, or alternatively
consisting of, amino acid residues from about Thr-178 to Glu-182 in
FIG. 1 (SEQ ID NO:2). In this context, "about" means the
particularly recited ranges and ranges that are larger or smaller
by several, a few, 5, 4, 3, 2 or 1 amino acid residues at either or
both the amino- and carboxy-termini. These polypeptide fragments
have been determined to bear antigenic epitopes of the TR21
polypeptide by the analysis of the Jameson-Wolf antigenic index, as
shown in FIG. 1 and Table I, above. Additional non-exclusive
preferred antigenic epitopes include the antigenic epitopes
disclosed herein, as well as portions thereof. Antigenic epitopes
are useful, for example, to raise antibodies, including monoclonal
antibodies that specifically bind the epitope. Preferred antigenic
epitopes include the antigenic epitopes disclosed herein, as well
as any combination of two, three, four, five or more of these
antigenic epitopes. Antigenic epitopes can be used as the target
molecules in immunoassays. (See, e.g., Wilson et al., Cell
37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Polynucleotides encoding these polypeptides are encompassed by the
invention. Additionally, antibodies that bind to one or more of
these polypeptides are also encompassed by the invention.
[0162] Additional polypeptides of the invention include one, two,
three, four, five, six, seven or more members selected from the
group consisting of, amino acids 3 to 15 in SEQ ID NO:2; amino
acids 39 to 45 in SEQ ID NO:2; amino acids 51 to 53 in SEQ ID NO:2;
amino acids 59 to 61 in SEQ ID NO:2; amino acids 104 to 112 in SEQ
ID NO:2; amino acids 136 to 138 in SEQ ID NO:2; amino acids 147 to
153 in SEQ ID NO:2; and amino acids 178 to 182 in SEQ ID NO:2.
Polynucleotides encoding these polypeptides are encompassed by the
invention. Additionally, antibodies that bind to one or more of
these polypeptides are also encompassed by the invention.
[0163] Similarly, immunogenic epitopes can be used, for example, to
induce antibodies according to methods well known in the art. (See,
for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow
et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al.,
J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes
include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five, six, seven or more of these
immunogenic epitopes. The polypeptides comprising one or more
immunogenic epitopes may be presented for eliciting an antibody
response together with a carrier protein, such as an albumin, to an
animal system (such as rabbit or mouse), or, if the polypeptide is
of sufficient length (at least about 25 amino acids), the
polypeptide may be presented without a carrier. However,
immunogenic epitopes comprising as few as 8 to 10 amino acids have
been shown to be sufficient to raise antibodies capable of binding
to, at the very least, linear epitopes in a denatured polypeptide
(e.g., in Western blotting).
[0164] Epitope-bearing polypeptides of the present invention may be
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe et
al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-2354 (1985). If in vivo immunization is used,
animals may be immunized with free peptide; however, anti-peptide
antibody titer may be boosted by coupling the peptide to a
macromolecular carrier, such as keyhole limpet Hemocyanin (KLH) or
tetanus toxoid. For instance, peptides containing cysteine residues
may be coupled to a carrier using a linker such as
maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g of peptide or carrier protein
and Freund's adjuvant or any other adjuvant known for stimulating
an immune response. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0165] As one of skill in the art will appreciate, and as discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to other polypeptide
sequences. For example, the polypeptides of the present invention
may be fused with the constant domain of immunoglobulins (IgA, IgE,
IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination
thereof and portions thereof), or albumin (including, but not
limited to, recombinant human albumin and fragments or variants
thereof (see, e.g., U.S. Pat. No. 5,876,969, EP Patent 0413622, and
U.S. Pat. No. 5,766,883, herein incorporated by reference in their
entirety). Such fusion proteins may facilitate purification and may
increase half-life in vivo. This has been shown for chimeric
proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. See, e.g., EP
394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced
delivery of an antigen across the epithelial barrier to the immune
system has been demonstrated for antigens (e.g., insulin)
conjugated to an FcRn binding partner such as IgG or Fc fragments
(see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG
Fusion proteins that have a disulfide-linked dimeric structure due
to the IgG portion disulfide bonds have also been found to be more
efficient in binding and neutralizing other molecules than
monomeric polypeptides or fragments thereof alone. See, e.g.,
Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic
acids encoding the above epitopes can also be recombined with a
gene of interest as an epitope tag (e.g., the hemagglutinin ("HA")
tag or flag tag) to aid in detection and purification of the
expressed polypeptide. For example, a system described by Janknecht
et al. allows for the ready purification of non-denatured fusion
proteins expressed in human cell lines (Janknecht et al., Proc.
Natl. Acad. Sci. USA 88:8972-897(1991)). In this system, the gene
of interest is subcloned into a vaccinia recombination plasmid such
that the open reading frame of the gene is translationally fused to
an amino-terminal tag consisting of six histidine residues. The tag
serves as a matrix-binding domain for the fusion protein. Extracts
from cells infected with the recombinant vaccinia virus are loaded
onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged
proteins can be selectively eluted with imidazole-containing
buffers.
[0166] Additional fusion proteins of the invention may be generated
through the techniques of gene shuffling, motif shuffling, exon
shuffling and/or codon-shuffling (collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to modulate the
activities of polypeptides of the invention, such methods can be
used to generate polypeptides with altered activity, as well as
agonists and antagonists of the polypeptides. See, generally, U.S.
Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and
5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson,
et al., J. Mol. Biol. J. Mol. Biol. 287:265-76 (1999); and Lorenzo
and Blasco, Biotechniques 24(2):308-13 (1998) (each of these
patents and publications are hereby incorporated by reference in
its entirety). In one embodiment, alteration of TR21
polynucleotides corresponding to FIG. 1 (SEQ ID NO:1) and the
polypeptides encoded by these polynucleotides may be achieved by
DNA shuffling. DNA shuffling involves the assembly of two or more
DNA segments by homologous or site-specific recombination to
generate variation in the polynucleotide sequence. In another
embodiment polynucleotides of the invention, or polypeptides
encoded thereby, may be altered by random mutagenesis by
error-prone PCR, random nucleotide insertion or other methods prior
to recombination. In another embodiment, one or more components,
motifs, sections, parts, domains, fragments, etc., of a
polynucleotide encoding a polypeptide of the invention may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0167] It will be recognized in the art that some amino acid
sequences of TR21 can be varied without significant effect on the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity. Thus,
the invention further includes variations of the TR21 receptor,
which show substantial TR21 receptor activity or which include
regions of TR21 proteins, such as the protein portions discussed
herein. Such mutants include deletions, insertions, inversions,
repeats, and type substitutions. As indicated above, guidance
concerning which amino acid changes are likely to be phenotypically
silent can be found in J. U. Bowie et al., Science 247:1306-1310
(1990).
[0168] Thus, the fragment, derivative, or analog of the polypeptide
of FIG. 1 (SEQ ID NO:2), may be (i) one in which at least one or
more of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue(s), and more preferably at least one but less than ten
conserved amino acid residues) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the
mature polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0169] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the TR21
receptor protein. The prevention of aggregation is highly
desirable. Aggregation of proteins not only results in a loss of
activity but can also be problematic when preparing pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al.,
Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes
36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug
Carrier Systems 10:307-377 (1993)).
[0170] The replacement of amino acids can also change the
selectivity of binding to cell surface receptors. Ostade et al.,
Nature 361:266-268 (1993), describes certain mutations resulting in
selective binding of TNF-.alpha. to only one of the two known types
of TNF receptors. Thus, the TR21 polypeptide receptors of the
present invention may include one or more amino acid substitutions,
deletions, or additions, either from natural mutations or human
manipulation.
[0171] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table II).
TABLE-US-00002 TABLE II Conservative Amino Acid Substitutions
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0172] In specific embodiments, the number of substitutions,
additions or deletions in the amino acid sequence of FIG. 1 and/or
any of the polypeptide fragments described herein (e.g., the
cysteine rich motif, the extracellular domain and/or intracellular
domain) is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6,
5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3
or 1-2.
[0173] In another embodiment, site directed changes at the amino
acid level of TR21 can be made by replacing a particular amino acid
with a conservative substitution. Preferred conservative
substitution mutations of the TR21 amino acid sequence provided in
SEQ ID NO:2 include: M1 replaced with A, G, I, L, S, T, or V; R2
replaced with H, or K; R3 replaced with H, or K; G4 replaced with
A, I, L, S, T, M, or V; R6 replaced with H, or K; S7 replaced with
A, G, I, L, T, M, or V; L8 replaced with A, G, I, S, T, M, or V; R9
replaced with H, or K; G10 replaced with A, I, L, S, T, M, or V;
R11 replaced with H, or K; D12 replaced with E; A13 replaced with
G, I, L, S, T, M, or V; A15 replaced with G, I, L, S, T, M, or V;
T17 replaced with A, G, I, L, S, M, or V; V20 replaced with A, G,
I, L, S, T, or M; A22 replaced with G, I, L, S, T, M, or V; E23
replaced with D; F25 replaced with W, or Y; D26 replaced with E;
L27 replaced with A, G, I, S, T, M, or V; L28 replaced with A, G,
I, S, T, M, or V; V29 replaced with A, G, I, L, S, T, or M; R30
replaced with H, or K; H31 replaced with K, or R; V33 replaced with
A, G, I, L, S, T, or M; A34 replaced with G, I, L, S, T, M, or V;
G36 replaced with A, I, L, S, T, M, or V; L37 replaced with A, G,
I, S, T, M, or V; L38 replaced with A, G, I, S, T, M, or V; R39
replaced with H, or K; T40 replaced with A, G, I, L, S, M, or V;
R42 replaced with H, or K; K44 replaced with H, or R; A46 replaced
with G, I, L, S, T, M, or V; G47 replaced with A, I, L, S, T, M, or
V; A48 replaced with G, I, L, S, T, M, or V; S49 replaced with A,
G, I, L, T, M, or V; S50 replaced with A, G, I, L, T, M, or V; A52
replaced with G, I, L, S, T, M, or V; R54 replaced with H, or K;
T55 replaced with A, G, I, L, S, M, or V; A56 replaced with G, I,
L, S, T, M, or V; L57 replaced with A, G, I, S, T, M, or V; Q58
replaced with N; Q60 replaced with N; E61 replaced with D; S62
replaced with A, G, I, L, T, M, or V; V63 replaced with A, G, I, L,
S, T, or M; G64 replaced with A, I, L, S, T, M, or V; A65 replaced
with G, I, L, S, T, M, or V; G66 replaced with A, I, L, S, T, M, or
V; A67 replaced with G, I, L, S, T, M, or V; G68 replaced with A,
I, L, S, T, M, or V; E69 replaced with D; A70 replaced with G, I,
L, S, T, M, or V; A71 replaced with G, I, L, S, T, M, or V; L72
replaced with A, G, I, S, T, M, or V; L74 replaced with A, G, I, S,
T, M, or V; G76 replaced with A, I, L, S, T, M, or V; L77 replaced
with A, G, I, S, T, M, or V; L78 replaced with A, G, I, S, T, M, or
V; F79 replaced with W, or Y; G80 replaced with A, I, L, S, T, M,
or V; A81 replaced with G, I, L, S, T, M, or V; A83 replaced with
G, I, L, S, T, M, or V; L84 replaced with A, G, I, S, T, M, or V;
L85 replaced with A, G, I, S, T, M, or V; G86 replaced with A, I,
L, S, T, M, or V; L87 replaced with A, G, I, S, T, M, or V; A88
replaced with G, I, L, S, T, M, or V; L89 replaced with A, G, I, S,
T, M, or V; V90 replaced with A, G, I, L, S, T, or M; L91 replaced
with A, G, I, S, T, M, or V; A92 replaced with G, I, L, S, T, M, or
V; L93 replaced with A, G, I, S, T, M, or V; V94 replaced with A,
G, I, L, S, T, or M; L95 replaced with A, G, I, S, T, M, or V; V96
replaced with A, G, I, L, S, T, or M; G97 replaced with A, I, L, S,
T, M, or V; L98 replaced with A, G, I, S, T, M, or V; V99 replaced
with A, G, I, L, S, T, or M; S100 replaced with A, G, I, L, T, M,
or V; W101 replaced with F, or Y; R102 replaced with H, or K; R103
replaced with H, or K; R104 replaced with H, or K; Q105 replaced
with N; R106 replaced with H, or K; R107 replaced with H, or K;
L108 replaced with A, G, I, S, T, M, or V; R109 replaced with H, or
K; G110 replaced with A, I, L, S, T, M, or V; A111 replaced with G,
I, L, S, T, M, or V; S112 replaced with A, G, I, L, T, M, or V;
S113 replaced with A, G, I, L, T, M, or V; A114 replaced with G, I,
L, S, T, M, or V; E115 replaced with D; A116 replaced with G, I, L,
S, T, M, or V; D118 replaced with E; G119 replaced with A, I, L, S,
T, M, or V; D120 replaced with E; K121 replaced with H, or R; D122
replaced with E; A123 replaced with G, I, L, S, T, M, or V; E125
replaced with D; L127 replaced with A, G, I, S, T, M, or V; D128
replaced with E; K129 replaced with H, or R; V130 replaced with A,
G, I, L, S, T, or M; I131 replaced with A, G, L, S, T, M, or V;
I132 replaced with A, G, L, S, T, M, or V; L133 replaced with A, G,
I, S, T, M, or V; S134 replaced with A, G, I, L, T, M, or V; G136
replaced with A, I, L, S, T, M, or V; I137 replaced with A, G, L,
S, T, M, or V; S138 replaced with A, G, I, L, T, M, or V; D139
replaced with E; A140 replaced with G, I, L, S, T, M, or V; T141
replaced with A, G, I, L, S, M, or V; A142 replaced with G, I, L,
S, T, M, or V; A144 replaced with G, I, L, S, T, M, or V; W145
replaced with F, or Y; G149 replaced with A, I, L, S, T, M, or V;
E150 replaced with D; D151 replaced with E; G153 replaced with A,
I, L, S, T, M, or V; T154 replaced with A, G, I, L, S, M, or V;
T155 replaced with A, G, I, L, S, M, or V; G158 replaced with A, I,
L, S, T, M, or V; H159 replaced with K, or R; S160 replaced with A,
G, I, L, T, M, or V; V161 replaced with A, G, I, L, S, T, or M;
V163 replaced with A, G, I, L, S, T, or M; A165 replaced with G, I,
L, S, T, M, or V; T166 replaced with A, G, I, L, S, M, or V; E167
replaced with D; L168 replaced with A, G, I, S, T, M, or V; G169
replaced with A, I, L, S, T, M, or V; S170 replaced with A, G, I,
L, T, M, or V; T171 replaced with A, G, I, L, S, M, or V; E172
replaced with D; L173 replaced with A, G, I, S, T, M, or V; V174
replaced with A, G, I, L, S, T, or M; T175 replaced with A, G, I,
L, S, M, or V; T176 replaced with A, G, I, L, S, M, or V; K177
replaced with H, or R; T178 replaced with A, G, I, L, S, M, or V;
A179 replaced with G, I, L, S, T, M, or V; G180 replaced with A, I,
L, S, T, M, or V; E182 replaced with D; Q183 replaced with N;
and/or Q184 replaced with N. Polynucleotides encoding these
polypeptides are also encompassed by the invention. The resulting
TR21 of the invention may be routinely screened for TR21 functional
activity and/or physical properties (such as, for example, enhanced
or reduced stability and/or solubility). Preferably, the resulting
proteins of the invention have an increased and/or a decreased TR21
functional activity. More preferably, the resulting TR21 proteins
of the invention have more than one increased and/or decreased TR21
functional activity and/or physical property.
[0174] Amino acids in the TR21 proteins of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in vitro
proliferative activity. Sites that are critical for ligand-receptor
binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos
et al. Science 255:306-312 (1992)).
[0175] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids that may produce proteins
with highly desirable improved characteristics, such as less
aggregation. Aggregation may not only reduce activity but also be
problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993).
[0176] In another embodiment, the invention provides for
polypeptides having amino acid sequences containing
non-conservative substitutions of the amino acid sequence provided
in SEQ ID NO:2. For example, non-conservative substitutions of the
TR21 protein sequence provided in SEQ ID NO:2 include: M1 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R2 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R3 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G4 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P5 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R6 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S7 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; L8 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R9 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; G10 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; R11 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; D12 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; A13 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P14 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; A15 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; P16 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T17 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P18 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C19 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V20
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P21 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
A22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E23
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; C24 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or P; F25 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; D26 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; L27 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; L28 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; V29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; R30 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; H31 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; C32 replaced with D, E, H, K, R, A, G, , L, S, T, M,
V, N, Q, F, W, Y, or P; V33 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A34 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; C35 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; G36 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L37 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R39
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
T40 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P41
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R42 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; P43 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; K44 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; P45 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A46 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G47 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A48 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; S49 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; S50 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P51
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; A52 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P53
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R54 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; T55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L57
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q58 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P59
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; Q60 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; E61 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; S62 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; V63 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G64 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A65
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G66 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A67 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G68 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; E69 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A71 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L72 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P73 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; L74 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P75 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; G76 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F79
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
G80 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A81
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P82 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
A83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L84
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L85 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G86 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L87 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A88 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L89 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V90 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L91
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A92 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L93 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; V94 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L95 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; V96 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L98
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V99 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S100 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; W101 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; R102 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R103 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R104 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q105
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; R106 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; R107 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; L108 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; R109 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; G110 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A111 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S112
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S113 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A114 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E115 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A116 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P117 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; D118 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G119
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D120 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K121
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
D122 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; A123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P124 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; E125 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; P126 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; L127 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; D128 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K129 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; V130 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; I131 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; I132 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L133 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P135
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S138
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D139 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A140
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T141 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A142 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P143 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A144 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; W145 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P146 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P147
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; P148 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; G149 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; E150 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; D151 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; P152 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; G153 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T154 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; T155 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; P157 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; G158 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; H159 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; S160 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; V161 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; P162 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; V163 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P164 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; A165 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; T166 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; E167 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; L168 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G169 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T171
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E172 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L173
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V174 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T175 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T176 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K177 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; T178 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A179 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; G180 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P181 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; E182 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; Q183 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; and/or Q184 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C. Polynucleotides
encoding these polypeptides are also encompassed by the invention.
The resulting TR21 proteins of the invention may be routinely
screened for TR21 functional activities and/or physical properties
(such as, for example, enhanced or reduced stability and/or
solubility) described throughout the specification and known in the
art. Preferably, the resulting proteins of the invention have an
increased and/or a decreased TR21 functional activity. More
preferably, the resulting TR21 proteins of the invention have more
than one increased and/or decreased TR21 functional activity and/or
physical property.
[0177] To improve or alter the characteristics of TR21
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or "muteins including single or multiple
amino acid substitutions, deletions, additions or fusion proteins.
Such modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
[0178] Non-naturally occurring variants may be produced using
art-known mutagenesis techniques, which include, but are not
limited to oligonucleotide mediated mutagenesis, alanine scanning,
PCR mutagenesis, site directed mutagenesis (see e.g., Carter et
al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl.
Acids Res. 10:6487 (1982)), cassette mutagenesis (see e.g., Wells
et al., Gene 34:315 (1985)), restriction selection mutagenesis (see
e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415
(1986)).
[0179] Thus, the invention also encompasses TR21 derivatives and
analogs that have one or more amino acid residues deleted, added,
or substituted to generate TR21 polypeptides that are better suited
for expression, scale up, etc., in the host cells chosen. For
example, cysteine residues can be deleted or substituted with
another amino acid residue in order to eliminate disulfide bridges;
N-linked glycosylation sites can be altered or eliminated to
achieve, for example, expression of a homogeneous product that is
more easily recovered and purified from yeast hosts which are known
to hyperglycosylate N-linked sites. To this end, a variety of amino
acid substitutions at one or both of the first or third amino acid
positions on any one or more of the glycosylation recognitions
sequences in the TR21 polypeptides of the invention, and/or an
amino acid deletion at the second position of any one or more such
recognition sequences will prevent glycosylation of the TR21 at the
modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J
5(6): 1193-1197). Additionally, one or more of the amino acid
residues of the polypeptides of the invention (e.g., arginine and
lysine residues) may be deleted or substituted with another residue
to eliminate undesired processing by proteases such as, for
example, furins or kexins.
[0180] The polypeptides of the present invention include a
polypeptide comprising, or alternatively, consisting of: amino
acids 1 to 184 in FIG. 1 (SEQ ID NO:2); amino acids 2 to 184 in
FIG. 1 (SEQ ID NO:2); the TR21 extracellular domain; the TR21
cysteine rich motif; the TR21 transmembrane domain; the
intracellular domain of TR21; and the TR21 extracellular domain and
the TR21 intracellular domain with all or part of the transmembrane
domain deleted; as well as polypeptides which are at least 80%
identical, more preferably at least 90% or 95% identical, still
more preferably at least 96%, 97%, 98%, 99% or 100% identical to
the polypeptides described above (e.g., the polypeptide of FIG. 1
(SEQ ID NO:2)), and also include portions of such polypeptides with
at least 30 amino acids and more preferably at least 50 or at least
100 amino acids. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0181] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
TR21 polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the TR21
receptor. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a reference amino acid
sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0182] As a practical matter, whether any particular polypeptide is
at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to,
for instance, the amino acid sequence shown in FIG. 1 (SEQ ID
NO:2), can be determined conventionally using known computer
programs such the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711). When using
Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a
reference sequence according to the present invention, the
parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference amino acid
sequence and that gaps in homology of up to 5% of the total number
of amino acid residues in the reference sequence are allowed.
[0183] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0184] In additional embodiments, polynucleotides of the invention
comprise, or alternatively consist of, a polynucleotide sequence at
least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical
to the polynucleotide sequence encoding the extracellular cysteine
rich motif of TR21 disclosed in FIG. 1 (amino acid residues from 19
to 35). In another embodiment, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide that hybridizes
under stringent hybridization conditions to DNA complementary to
the polynucleotide sequence encoding the TR21 extracellular
cysteine rich motif. The present invention also encompasses the
above polynucleotide/nucleic acid sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded by these nucleic
acids and/or polynucleotide sequences are also encompassed by the
invention.
[0185] The present application is also directed to proteins
containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
98% or 99% identical to the TR21 polypeptide sequence set forth as
n.sup.1-m.sup.1, and/or n.sup.2-m.sup.1 as described herein. In
preferred embodiments, the application is directed to proteins
comprising or alternatively consisting of, polypeptides at least
80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to
polypeptides having the amino acid sequence of the specific TR21 N-
and C-terminal deletions recited herein. Polynucleotides encoding
these polypeptides are also encompassed by the invention.
[0186] In certain preferred embodiments, TR21 proteins of the
invention comprise fusion proteins as described above wherein the
TR21 polypeptides are those described as n.sup.1-m.sup.1, and/or
n.sup.2-m.sup.1 as described herein. In preferred embodiments, the
application is directed to nucleic acid molecules at least 80%,
85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequences encoding polypeptides having the amino acid sequence
of the specific N- and C-terminal deletions recited herein.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0187] In preferred embodiments, the TR21 polypeptide or fragment
thereof binds a Neutrokine-alpha polypeptide or splice variants
thereof. The ability of a TR21 polypeptide (e.g., fragment) of the
invention to bind a Neutrokine-alpha polypeptide, or a splice
variant thereof, can routinely be determined using techniques
described herein or otherwise known in the art. In another
non-exclusive preferred embodiment, a TR21 polypeptide, or fragment
thereof, of the invention antagonizes B cell proliferation and/or
differentiation merdiated by Neutokine-alpha or a splice variant
thereof. The ability of a TR21 polypeptide to antagonize B cell
proliferation and/or differentiation mediated by Neutrokine-alpha
or a splice variant thereof can routinely be determined using
techniques described herein or otherwise known in the art.
[0188] In preferred embodiments, the TR21 polypeptide or fragment
thereof binds a heteromultimeric polypeptide complex comprising one
or more Neutrokine-alpha polypeptides or splice avariants thereof.
The ability of a TR21 polypeptide (e.g., fragment) of the invention
to bind a heteromultimeric polypeptide complex comprising one or
more Neutrokine alpha polypeptides or splice variants thereof can
routinely be determined using techniques described herein or
otherwise known in the art. In another non-exclusive preferred
embodiment, a TR21 polypeptide or fragment thereof of the invention
antagonizes B cell proliferation and/or differentiation mediated by
a heteromultimeric polypeptide complex comprising one or more
Neutrokine-alpha polypeptides or splice variants thereof. The
ability of a TR21 polypeptide to antagonize B cell proliferation
and/or differentiation mediated by a heteromultimeric polypeptide
complex comprising one or more Neutrokine-alpha polypeptides or
splice variants thereof can routinely be determined using
techniques described herein or otherwise known in the art.
[0189] In one embodiment, one or more of the TR21 polypeptides of
the invention are expressed at relatively high levels in mature B
cells. In a specific embodiment, expression of one or more of the
TR21 polypeptides of the invention is restricted to mature B
cells.
[0190] In one embodiment, the B cell proliferation assay described
herein may be modified for use in screening for TR21 related
proteins or agonists or antagonists thereof. In this instance, a
baseline level of B cell proliferation and/or differentiation
mediated by Neutrokine-alpha or Neutrokine-alpha SV is determined.
Potential TR21 related proteins or polypeptide(s) are added to an
experimental well and the resultant level of B cell proliferation
and/or differentiation mediated by Neutrokine-alpha or
Neutrokine-alpha SV is assessed and compared to the baseline level.
An increase in B cell proliferation and/or differentiation in the
experimental well will indicate that the potential TR21 related
proteins(s) or polypeptide(s) is either (or both) a TR21 related
protein or an agonist. Decreased B cell proliferation and/or
differentiation will indicate that the potential TR21 protein(s) or
polypeptide(s) is an antagonist.
[0191] In another embodiment, the B cell proliferation assay may be
modified for use in screening for TR21 related proteins or agonists
or antagonists thereof using heteromultimeric polypeptide complexes
comprising one or more Neutrokine-alpha or Neutrokine-alpha SV
proteins. The baseline level of B cell proliferation and/or
differentiation mediated by a heteromultimeric polypeptide complex
comprising one or more Neutrokine-alpha or Neutrokine-alpha SV
proteins may be determined by routinely modifying the techniques
described herein. Potential TR21 related proteins or polypeptide(s)
are added to an experimental well and the resultant level of B cell
proliferation and/or differentiation is assessed and compared to
the baseline level. An increase in B cell proliferation and/or
differentiation in the experimental well will indicate that the
potential TR21 related proteins(s) or polypeptide(s) is either (or
both) a TR21 related protein or an agonist. Decreased B cell
proliferation and/or differentiation will indicate that the
potential TR21 protein(s) or polypeptide(s) is an antagonist
[0192] In another embodiment, TR21 polypeptides (including TR21
soluble fragments) may be identified by means of a functional
screen using the modified B cell proliferation assay as described
above.
[0193] Moreover, the TR21 polypeptides described herein may be used
as a means of detecting and/or quantifying levels of
Neutrokine-alpha or Neutrokine-alpha SV or heteromultimeric
polypeptide complexes comprising Neutrokine-alpha or
Neutrokine-alpha SV in a sample (e.g., a biological sample) by for
example using, or routinely modifying immunoassays known in the
art, and/or using or routinely modifying, the B cell proliferation
assay described herein.
TR21 Antibodies
[0194] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
TR21 polypeptide, polypeptide fragment, or variant of SEQ ID NO:2,
and/or a TR21 epitope (as determined by immunoassays well known in
the art for assaying specific antibody-antigen binding). Antibodies
of the invention include, but are not limited to, polyclonal,
monoclonal, multispecific, human, humanized or chimeric antibodies,
single chain antibodies, Fab fragments, F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), and epitope-binding fragments of any of the above.
The term "antibody," as used herein, refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen-binding site
that immunospecifically binds an antigen. The immunoglobulin
molecules of the invention can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass of immunoglobulin molecule. In specific
embodiments, the immunoglobulin molecules of the invention are
IgG1. In other specific embodiments, the immunoglobulin molecules
of the invention are IgG4.
[0195] Most preferably the antibodies are human antigen-binding
antibody fragments of the present invention and include, but are
not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdfv) and fragments
comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the
variable region(s) alone or in combination with the entirety or a
portion of the following: hinge region, CH1, CH2, and CH3 domains.
Also included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine (e.g., mouse and rat),
donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used herein, "human" antibodies include antibodies having the amino
acid sequence of a human immunoglobulin and include antibodies
isolated from human immunoglobulin libraries or from animals
transgenic for one or more human immunoglobulin and that do not
express endogenous immunoglobulins, as described infra and, for
example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.
[0196] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., PCT publications WO 93/17715; WO
92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553
(1992).
[0197] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which they recognize or specifically bind.
The epitope(s) or polypeptide portion(s) may be specified as
described herein, e.g., by N-terminal and C-terminal positions, by
size in contiguous amino acid residues, or listed in the Tables and
Figures. Antibodies which specifically bind any epitope or
polypeptide of the present invention may also be excluded.
Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0198] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of a polypeptide of
the present invention are included. Antibodies that bind
polypeptides with at least 95%, at least 90%, at least 85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%,
at least 55%, and at least 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
In specific embodiments, antibodies of the present invention
cross-react with murine, rat and/or rabbit homologs of human
proteins and the corresponding epitopes thereof. Antibodies that do
not bind polypeptides with less than 95%, less than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%,
less than 60%, less than 55%, and less than 50% identity (as
calculated using methods known in the art and described herein) to
a polypeptide of the present invention are also included in the
present invention. In a specific embodiment, the above-described
cross-reactivity is with respect to any single specific antigenic
or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or
more of the specific antigenic and/or immunogenic polypeptides
disclosed herein. Further included in the present invention are
antibodies which bind polypeptides encoded by polynucleotides which
hybridize to a polynucleotide of the present invention under
stringent hybridization conditions (as described herein).
Antibodies of the present invention may also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2 M,
10.sup.-2 M, 5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.7 M, 5.times.10.sup.-8 M
or 10.sup.-8 M. Even more preferred binding affinities include
those with a dissociation constant or Kd less than
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11M, 5.times.10.sup.-12 M,
.sup.10-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-5 M.
[0199] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of the invention as
determined by any method known in the art for determining
competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively
inhibits binding to the epitope by at least 95%, at least 90%, at
least 85 %, at least 80%, at least 75%, at least 70%, at least 60%,
or at least 50%.
[0200] Antibodies of the present invention may act as agonists or
antagonists of the polypeptides of the present invention. For
example, the present invention includes antibodies which disrupt
the receptor/ligand interactions with the polypeptides of the
invention either partially or fully. Preferably, antibodies of the
present invention bind an antigenic epitope disclosed herein (e.g.,
amino acid residues 3 to 15, 39 to 45, 51 to 53, 59 to 61, 104 to
112, 115 to 122, 136 to 138, 147 to 153 and 178 to 182 of SEQ ID
NO:2), or a portion thereof. The invention features both
receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor-specific antibodies that do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. For example,
receptor activation can be determined by detecting activation of
the transcription factors NF-AT, AP-1, and/or NF-KAPPAB using
techniques known in the art, and/or the phosphorylation (e.g.,
tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example,
as described supra). In specific embodiments, antibodies are
provided that inhibit ligand activity or receptor activity by at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody. In other specific embodiments, antibodies
are provided that promote ligand activity or receptor activity by
at least 95%, at least 90%, at least 85%, at least 80%, at least
75%, at least 70%, at least 60%, or at least 50% of the activity in
absence of the antibody.
[0201] The invention also features receptor-specific antibodies
which both prevent ligand binding and receptor activation as well
as antibodies that recognize the receptor-ligand complex, and,
preferably, do not specifically recognize the unbound receptor or
the unbound ligand. Likewise, included in the invention are
neutralizing antibodies which bind the ligand and prevent binding
of the ligand to the receptor, as well as antibodies which bind the
ligand, thereby preventing receptor activation, but do not prevent
the ligand from binding the receptor. Further included in the
invention are antibodies which activate the receptor. These
antibodies may act as receptor agonists, i.e., potentiate or
activate either all or a subset of the biological activities of the
ligand-mediated receptor activation, for example, by inducing
multimerization and/or aggregation (i.e., via antibody
cross-linking) of the receptor (i.e., TR21). The antibodies may be
specified as agonists, antagonists or inverse agonists for
biological activities comprising the specific biological activities
of the peptides of the invention disclosed herein. The above
antibody agonists can be made using methods known in the art. See,
e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et
al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214
(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et
al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine
9(4):233-241 (1997); Carlson et al., J. Biol. Chem.
272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762
(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et
al., Cytokine 8(1):14-20 (1996) (which are all incorporated by
reference herein in their entireties).
[0202] Antibodies of the present invention may be used, for
example, but not limited to, to purify, detect, and target the
polypeptides of the present invention, including both in vitro and
in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and
quantitatively measuring levels of the polypeptides of the present
invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its
entirety).
[0203] By way of another non-limiting example, antibodies of the
invention may be administered to individuals as a form of passive
immunization. Alternatively, antibodies of the present invention
may be used for epitope mapping to identify the epitope(s) bound by
the antibody. Epitopes identified in this way may, in turn, for
example, be used as vaccine candidates, i.e., to immunize an
individual to elicit antibodies against the naturally occurring
forms of TR21.
[0204] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- or C-terminus or
chemically conjugated (including covalently and non-covalently
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays and
effector molecules such as heterologous polypeptides, drugs,
radionuclides, or toxins. See, e.g., PCT publications WO 92/08495;
WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP
396,387.
[0205] The antibodies of the invention include derivatives that are
modified, i.e., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from generating an anti-idiotypic response. For example,
but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other protein, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical
amino acids.
[0206] The antibodies of the present invention may be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, etc. to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
Such adjuvants are also well known in the art.
[0207] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N. Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0208] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art
and are discussed in detail in the Examples (e.g., Example 3). In a
non-limiting example, mice can be immunized with a polypeptide of
the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well-known
techniques to any suitable myeloma cells, for example cells from
cell line SP20 available from the ATCC.TM.. Hybridomas are selected
and cloned by limited dilution. The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide of the invention.
Ascites fluid, which generally contains high levels of antibodies
can be generated by immunizing mice with positive hybridoma
clones.
[0209] Accordingly, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention.
[0210] Protocols for generating EBV-transformed B cell lines are
commonly known in the art, such as, for example, the protocol
outlined in Chapter 7.22 of Current Protocols in Immunology,
Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is
hereby incorporated in its entirety by reference herein. The source
of B cells for transformation is commonly human peripheral blood,
but B cells for transformation may also be derived from other
sources including, but not limited to, lymph nodes, tonsil, spleen,
tumor tissue, and infected tissues. Tissues are generally made into
single cell suspensions prior to EBV transformation. Investigators
may also choose to perform selection procedures that will enrich
the sample for B cells that are antigen-reactive. For example, one
method of enriching for antigen-reactive B cells is panning on a
plastic dish that has been coated with antigen. Antigen reactive B
cells may then be eluted from the plastic dish and used for
transformation. Alternatively, it is possible to enrich for
antigen-reactive B cells using fluorescence activated cells sorting
(FACS). In this method, one might use fluorescently labeled antigen
to sort out a population of antigen reactive B-cells from
non-antigen reactive B cells an other cells types. Both FACS
analysis and panning, may also be performed in a manner so as to
enrich for B cells as opposed to antigen-reactive B cells. The
advantage of selecting for total B cells populations is that one is
more likely to include plasma cells, or B cells actively secreting
immunoglobulin, that might be missed in procedures that require the
presence of cell-surface immunoglobulin for detection. Growth of
EBV-infected cells is promoted by monocytes, so investigators may
wish to take care not to exclude these form culture, or to resupply
monocytes after selection procedures. Additionally, steps may be
taken to either physically remove or inactivate T cells (e.g., by
treatment with cyclosporin A) in B cell-containing samples, because
T cells from individuals seropositive for anti-EBV antibodies can
suppress B cell immortalization by EBV.
[0211] In general, the sample containing human B cells is
innoculated with EBV, and cultured for 3-4 weeks. A typical source
of EBV is the culture supernatant of the B95-8 cell line (ATCC.TM.
#VR-1492). Physical signs of EBV transformation can generally be
seen towards the end of the 3-4 week culture period. By
phase-contrast microscopy, transformed cells may appear large,
clear, hairy and tend to aggregate in tight clusters of cells.
Initially, EBV lines are generally polyclonal. However, over
prolonged periods of cell cultures, EBV lines may become monoclonal
or polyclonal as a result of the selective outgrowth of particular
B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned (e.g., by limiting dilution culture) or fused with a
suitable fusion partner and plated at limiting dilution to obtain
monoclonal B cell lines. Suitable fusion partners for EBV
transformed cell lines include mouse myeloma cell lines (e.g.,
SP2/0, X63-Ag8.653), heteromyeloma cell lines (human.times.mouse;
e.g., SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM
1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention
also provides a method of generating polyclonal or monoclonal human
antibodies against polypeptides of the invention or fragments
thereof, comprising EBV-transformation of human B cells.
[0212] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
[0213] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles that carry the
polynucleotide sequences encoding them. In a particular embodiment,
such phage can be utilized to display antigen-binding domains
expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labeled antigen or antigen bound or
captured to a solid surface or bead. Phages used in these methods
are typically filamentous phage including fd and M13 binding
domains expressed from phage with Fab, Fv or disulfide stabilized
Fv antibody domains recombinantly fused to either the phage gene
III or gene VIII protein. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50
(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology
57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT
publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO
93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108; each of which is incorporated herein by reference in
its entirety.
[0214] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988) (said
references incorporated by reference in their entireties).
[0215] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entirety. Humanized antibodies are antibody molecules from
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332).
[0216] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0217] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring which express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar,
Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO
96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;
5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which
are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0218] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/technology 12:899-903 (1988)).
[0219] Further, antibodies to the polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan & Bona,
FASEB J. 7(5): 437-444; (1989) and Nissinoff, J. Immunol. 147(8):
2429-2438 (1991)). For example, antibodies which bind to and
competitively inhibit polypeptide multimerization and/or binding of
a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes that "mimic" the polypeptide multimerization and/or
binding domain and, as a consequence, bind to and neutralize
polypeptide and/or its ligand (e.g., Neutrokine-alpha). Such
neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes
can be used in therapeutic regimens to neutralize polypeptide
ligand. For example, such anti-idiotypic antibodies can be used to
bind a polypeptide of the invention and/or to bind its
ligands/receptors, and thereby block its biological activity.
[0220] Intrabodies of the invention can be produced using methods
known in the art, such as those disclosed and reviewed in Chen et
al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther.
4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol.
51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998);
Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J.
Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol.
291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250
(1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and
references cited therein. In particular, a CCR5 intrabody has been
produced by Steinberger et al, Proc. Natl. Acad. Sci. USA
97:805-810 (2000).
Polynucleotides Encoding TR21 Antibodies
[0221] The invention further provides polynucleotides comprising a
nucleotide sequence encoding an antibody of the invention and
fragments thereof. The invention also encompasses polynucleotides
that hybridize under stringent or lower stringency hybridization
conditions, e.g., as defined supra, to polynucleotides that encode
an antibody, preferably, that specifically binds to a TR21
polypeptide of the invention, preferably, an antibody that binds to
a polypeptide having the amino acid sequence of SEQ ID NO:2.
[0222] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. For example, if the nucleotide sequence of the antibody is
known, a polynucleotide encoding the antibody may be assembled from
chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding the antibody, annealing and
ligating of those oligonucleotides, and then amplification of the
ligated oligonucleotides by PCR.
[0223] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0224] Once the nucleotide sequence and corresponding amino acid
sequence of the antibody is determined, the nucleotide sequence of
the antibody may be manipulated using methods well known in the art
for the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., 1990, Molecular
Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley &
Sons, NY, which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0225] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody, as
described supra. The framework regions may be naturally occurring
or consensus framework regions, and preferably human framework
regions (See, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing of human framework regions). Preferably, the
polynucleotide generated by the combination of the framework
regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one
or more amino acid substitutions may be made within the framework
regions, and, preferably, the amino acid substitutions improve
binding of the antibody to its antigen. Additionally, such methods
may be used to make amino acid substitutions or deletions of one or
more variable region cysteine residues participating in an
intrachain disulfide bond to generate antibody molecules lacking
one or more intrachain disulfide bonds. Other alterations to the
polynucleotide are encompassed by the present invention and within
the skill of the art.
[0226] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As described supra, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine mAb and a human immunoglobulin constant region, e.g.,
humanized antibodies.
[0227] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423- 42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038- 1041 (1988)).
Methods of Producing TR21 Antibodies
[0228] The antibodies of the invention can be produced by any
method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques. Methods of producing antibodies include, but
are not limited to, hybridoma technology, EBV transformation, and
other methods discussed herein as well as through the use
recombinant DNA technology, as discussed below.
[0229] Recombinant expression of an antibody of the invention, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), requires construction of an expression vector
containing a polynucleotide that encodes the antibody. Once a
polynucleotide encoding an antibody molecule or a heavy or light
chain of an antibody, or portion thereof (preferably containing the
heavy or light chain variable domain), of the invention has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT Publication WO
86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain.
[0230] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention, or a heavy or light chain
thereof, or a single chain antibody of the invention, operably
linked to a heterologous promoter. In preferred embodiments for the
expression of double-chained antibodies, vectors encoding both the
heavy and light chains may be co-expressed in the host cell for
expression of the entire immunoglobulin molecule, as detailed
below.
[0231] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be produced and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express an
antibody molecule of the invention in situ. These include but are
not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with recombinant bacteriophage DNA, plasmid
DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with
recombinant yeast expression vectors containing antibody coding
sequences; insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus) containing antibody coding
sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid
expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells, especially for the expression of
whole recombinant antibody molecule, are used for the expression of
a recombinant antibody molecule. For example, mammalian cells such
as Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2 (1990)).
[0232] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione-agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0233] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0234] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts. (See e.g., Logan & Shenk,
Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544 (1987)).
[0235] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and
normal mammary gland cell line such as, for example, CRL7030 and
Hs578Bst.
[0236] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compounds that interact directly or indirectly
with the antibody molecule.
[0237] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
1993, TIB TECH 11(5):155-215); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981), which are incorporated by reference herein in their
entireties.
[0238] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0239] Vectors which use glutamine synthase (GS) or DHFR as the
selectable markers can be amplified in the presence of the drugs
methionine sulphoximine or methotrexate, respectively. An advantage
of glutamine synthase based vectors are the availability of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine
synthase negative. Glutamine synthase expression systems can also
function in glutamine synthase expressing cells (e.g. Chinese
Hamster Ovary (CHO) cells) by providing additional inhibitor to
prevent the functioning of the endogenous gene. A glutamine
synthase expression system and components thereof are detailed in
PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404;
and WO91/06657 which are incorporated in their entireties by
reference herein. Additionally, glutamine synthase expression
vectors that may be used according to the present invention are
commercially available from suppliers, including, for example Lonza
Biologics, Inc. (Portsmouth, N.H.). Expression and production of
monoclonal antibodies using a GS expression system in murine
myeloma cells is described in Bebbington et al., Bio/technology
10:169(1992) and in Biblia and Robinson Biotechnol. Prog. 11:1
(1995) which are incorporated in their entireties by reference
herein.
[0240] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy
and light chains may comprise cDNA or genomic DNA.
[0241] Once an antibody molecule of the invention has been produced
by an animal, chemically synthesized, or recombinantly expressed,
it may be purified by any method known in the art for purification
of an immunoglobulin molecule, for example, by chromatography
(e.g., ion exchange, affinity, particularly by affinity for the
specific antigen after Protein A, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins. In
addition, the antibodies of the present invention or fragments
thereof can be fused to heterologous polypeptide sequences
described herein or otherwise known in the art, to facilitate
purification.
[0242] The present invention encompasses antibodies recombinantly
fused or chemically conjugated (including both covalently and
non-covalently conjugations) to a polypeptide (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention to generate
fusion proteins. The fusion does not necessarily need to be direct,
but may occur through linker sequences. The antibodies may be
specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino
acids of the polypeptide) of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095;
Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No.
5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al.,
J. Immunol. 146:2446-2452(1991), which are incorporated by
reference in their entireties.
[0243] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region or a portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the constant region, hinge region, CH1 domain, CH2
domain, and CH3 domain or any combination of whole domains or
portions thereof. The polypeptides may also be fused or conjugated
to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can
form dimers through disulfide bonding between the Fc portions.
Higher multimeric forms may be made by fusing polypeptides to
portions of IgA and IgM. Methods for fusing or conjugating the
polypeptides of the present invention to antibody portions are
known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;
5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166;
PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc.
Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad.
Sci. USA 89:11337- 11341(1992) (said references incorporated by
reference in their entireties).
[0244] As discussed, supra, the polypeptides corresponding to a
TR21 polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2
may be fused or conjugated to the above antibody portions to
increase the in vivo half life of the polypeptides or for use in
immunoassays using methods known in the art. Further, the
polypeptides corresponding to SEQ ID NO:2 may be fused or
conjugated to the above antibody portions to facilitate
purification. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. (EP 394,827; Traunecker et
al., Nature 331:84-86 (1988). The polypeptides of the present
invention fused or conjugated to an antibody having
disulfide-linked dimeric structures (due to the IgG) may also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many
cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and thus can result in, for example, improved
pharmacokinetic properties. (EP A 232,262). Alternatively, deleting
the Fc part after the fusion protein has been expressed, detected,
and purified, would be desired. For example, the Fc portion may
hinder therapy and diagnosis if the fusion protein is used as an
antigen for immunizations. In drug discovery, for example, human
proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[0245] Moreover, the antibodies or fragments thereof of the present
invention can be fused to marker sequences, such as a peptide to
facilitate purification. In preferred embodiments, the marker amino
acid sequence is a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86:821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Other peptide tags
useful for purification include, but are not limited to, the "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the
"Flag.TM." tag.
[0246] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically to, for example, monitor
the development or progression of a tumor as part of a clinical
testing procedure to, e.g., determine the efficacy of a given
treatment regimen. Detection can be facilitated by coupling the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials,
radioactive materials, positron emitting metals using various
positron emission tomographies, and nonradioactive paramagnetic
metal ions. The detectable substance may be coupled or conjugated
either directly to the antibody (or fragment thereof) or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include iodine (.sup.121I, .sup.123I, .sup.125I,
.sup.131I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.111In, .sup.112In, .sup.113mIn,
.sup.115mIn), technetium (.sup.99Tc,.sup.99mTc), thallium
(.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133 Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, and .sup.97Ru.
[0247] Further, an antibody or fragment thereof may be conjugated
to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.211At, .sup.103Pd,
.sup.133Xe, .sup.131I, .sup.125I, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd,
.sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se, .sup.113Sn, .sup.90Y,
.sup.117Tin, .sup.186Re, .sup.188Re and .sup.166Ho. In specific
embodiments, an antibody or fragment thereof is attached to
macrocyclic chelators useful for conjugating radiometal ions,
including but not limited to, .sup.177Lu, .sup.90Y, .sup.166Ho,
.sup.111In, and .sup.153Sm, to polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic
chelators attached to TR21 polypeptides of the invention is
.sup.111In. In another preferred embodiment, the radiometal ion
associated with the macrocyclic chelator attached to TR21
polypeptides of the invention is .sup.90Y. In specific embodiments,
the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA). In other specific embodiments, the DOTA is attached to an
antibody of the invention or fragment thereof via a linker
molecule. Examples of linker molecules useful for conjugating DOTA
to a polypeptide are commonly known in the art--see, for example,
DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et
al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al,
Nucl. Med. Biol. 26(8):943-50 (1999) which are hereby incorporated
by reference in their entirety. In addition, U.S. Pat. Nos.
5,652,361 and 5,756,065, which disclose chelating agents that may
be conjugated to antibodies, and methods for making and using them,
are hereby incorporated by reference in their entireties.
[0248] A cytotoxin or cytotoxic agent includes any agent that is
detrimental to cells and includes such molecules as small molecule
toxins and enzymatically active toxins of bacterial, fungal, plant,
or animal origin, or fragments thereof. Examples include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide,
emetine, mitomycin, etoposide (VP-16), tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum(II) (DDP) (cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), anti-mitotic agents (e.g.,
vincristine and vinblastine), improsulfan, piposulfan, benzodopa,
carboquone, meturedopa, uredopa, altretamine, triethylenemelamine,
trietylenephosphoramide, triethylenethiophosphaoramide
trimethylolomelamine, chlomaphazine, cholophosphamide,
estramustine, ifosfamide, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard, chlorozotocin, fotemustine,
nimustine, ranimustine, aclacinomysins, azaserine, cactinomycin,
calicheamicin, carabicin, carminomycin, carzinophilin,
chromomycins, detorubicin, 6-diazo-5-oxo-L-norleucine, epirubicin,
esorubicin, idarubicin, marcellomycin, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, quelamycin,
rodorubicin, streptonigrin, tubercidin, ubenimex, zinostatin,
zorubicin, denopterin, pteropterin, trimetrexate, fludarabine,
thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur,
dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU,
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone, aminoglutethimide, mitotane, trilostane, frolinic
acid, aceglatone, aldophosphamide glycoside, aminolevulinic acid,
amsacrine, bestrabucil, bisantrene, edatraxate, defofamine,
demecolcine, diaziquone, elfomithine, elliptinium acetate,
etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine,
mitoguazone, mopidamol, nitracrine, pentostatin, phenamet,
pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine,
PSKO, razoxane, sizofiran, spirogermanium, tenuazonic acid,
triaziquone, 2,2',2''-trichlorotriethylamine, urethan, vindesine,
dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,
gacytosine, arabinoside ("Ara-C"), taxoids, e.g. paclitaxel
(TAXOL'', Bristol-Myers Squibb Oncology, Princeton, N.J.) doxetaxel
(TAXOTERE'', Rh6ne-Poulenc Rorer, Antony, France), gemcitabine,
ifosfamide, vinorelbine, navelbine, novantrone, teniposide,
aminopterin, XELODA.TM., ibandronate, CPT-I 1, topoisomerase
inhibitor RFS 2000, difluoromethylomithine (DMFO), retinoic acid,
esperamicins, capecitabine, and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Also included in this
definition are anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens including for
example tamoxifen, raloxifene, aromatase inhibiting
4(5)-imidazoles, 4 hydroxytamoxifen, trioxifene, keoxifene, LY
117018, onapristone, toremifene (Fareston), and anti-androgens such
as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin,
and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
[0249] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, a-interferon, .beta.-interferon, nerve growth
factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I
(See, International Publication No. WO 97/33899), AIM II (See,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See,
International Publication No. WO 99/23105), CD40-ligand, a
thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or
endostatin; or, biological response modifiers such as, for example,
lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),
interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"),
or other growth factors.
[0250] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the target
antigen. Such solid supports include, but are not limited to,
glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or polypropylene.
[0251] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0252] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980, which is incorporated herein by
reference in its entirety.
[0253] An antibody, with or without a therapeutic moiety conjugated
to it, administered alone or in combination with cytotoxic
factor(s) and/or cytokine(s) can be used as a therapeutic.
Immunophenotyping Using TR21 Antibodies
[0254] The antibodies of the invention may be utilized for
immunophenotyping of cell lines and biological samples. The
translation product of the gene of the present invention may be
useful as a cell specific marker, or more specifically as a
cellular marker that is differentially expressed at various stages
of differentiation and/or maturation of particular cell types.
Monoclonal antibodies directed against a specific epitope, or
combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be
utilized using monoclonal antibodies to screen for cellular
populations expressing the marker(s), and include magnetic
separation using antibody-coated magnetic beads, "panning" with
antibody attached to a solid matrix (i.e., plate), and flow
cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al.,
Cell, 96:737-49 (1999)).
[0255] These techniques allow for the screening of particular
populations of cells, such as might be found with hematological
malignancies (i.e. minimal residual disease (MRD) in acute leukemic
patients) and "non-self" cells in transplantations to prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for the screening of hematopoietic stem and progenitor cells
capable of undergoing proliferation and/or differentiation, as
might be found in human umbilical cord blood.
Assays For TR21 Antibody Binding
[0256] The antibodies of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al, eds, 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety). Exemplary immunoassays are described briefly below (but
are not intended by way of limitation).
[0257] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trayslol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.16.1.
[0258] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%- 20% SDS-PAGE depending on the molecular weight of
the antigen), transferring the protein sample from the
polyacrylamide gel to a membrane such as nitrocellulose, PVDF or
nylon, blocking the membrane in blocking solution (e.g., PBS with
3% BSA or non-fat milk), washing the membrane in washing buffer
(e.g., PBS-Tween 20), blocking the membrane with primary antibody
(the antibody of interest) diluted in blocking buffer, washing the
membrane in washing buffer, blocking the membrane with a secondary
antibody (which recognizes the primary antibody, e.g., an
anti-human antibody) conjugated to an enzyme (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e g.,
.sup.32P or .sup.125I) diluted in blocking buffer, washing the
membrane in wash buffer, and detecting the presence of the antigen.
One of skill in the art would be knowledgeable as to the parameters
that can be modified to increase the signal detected and to reduce
the background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
[0259] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzyme
(e.g., horseradish peroxidase or alkaline phosphatase) to the well
and incubating for a period of time, and detecting the presence of
the antigen. In ELISAs the antibody of interest does not have to be
conjugated to a detectable compound; instead, a second antibody
(which recognizes the antibody of interest) conjugated to a
detectable compound may be added to the well. Further, instead of
coating the well with the antigen, the antibody may be coated to
the well. In this case, a second antibody conjugated to a
detectable compound may be added following the addition of the
antigen of interest to the coated well. One of skill in the art
would be knowledgeable as to the parameters that can be modified to
increase the signal detected as well as other variations of ELISAs
known in the art. For further discussion regarding ELISAs see,
e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York at
11.2.1.
[0260] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the antibody of interest
in the presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest conjugated to a labeled
compound (e.g., .sup.3H or .sup.125I) in the presence of increasing
amounts of an unlabeled second antibody.
Therapeutic Uses of TR21 Antibodies
[0261] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein).
[0262] The present invention is further directed to antibody-based
therapies which involve administering antibodies of the invention
to an animal, preferably a mammal, and most preferably a human,
patient for treating one or more of the disclosed diseases,
disorders, or conditions. Therapeutic compounds of the invention
include, but are not limited to, antibodies of the invention
(including fragments, analogs and derivatives thereof as described
herein) and nucleic acids encoding antibodies of the invention
(including fragments, analogs and derivatives thereof and
anti-idiotypic antibodies as described herein). The antibodies of
the invention can be used to treat, inhibit or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions described herein.
[0263] For example, antibody antagonists of the invention may be
used to treat, inhibit or prevent autoimmune diseases, disorders,
or conditions associated with such diseases or disorders,
including, but not limited to, autoimmune hemolytic anemia
(including, but not limited to cryoglobinemia or Coombs positive
anemia), autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, autoimmune neutropenia,
autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,
dermatitis (e.g. atopic dermatitis), gluten-sensitive enteropathy,
allergic encephalomyelitis, myocarditis, relapsing polychondritis,
rheumatic heart disease, Multiple Sclerosis, Neuritis, Uveitis
Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein
purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune
Pulmonary Inflammation, IgA glomerulonephritis, dense deposit
disease, Guillain-Barre Syndrome, diabetes mellitus (e.g. Type I
diabetes mellitus or insulin dependent diabetes mellitis), juvenile
onset diabetes, and autoimmune inflammatory eye, autoimmune
thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,
systemic lupus erhythematosus, discoid lupus, Goodpasture's
syndrome, Pemphigus, receptor autoimmunities such as, for example,
(a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin
resistance, autoimmune thrombocytopenic purpura, rheumatoid
arthritis, scleroderma with anti-collagen antibodies, mixed
connective tissue disease, polymyositis/dermatomyositis, pernicious
anemia (Addison's disease), idiopathic Addison's disease,
infertility, glomerulonephritis such as primary glomerulonephritis
and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, and
adrenergic drug resistance (including adrenergic drug resistance
with asthma or cystic fibrosis), chronic active hepatitis, primary
biliary cirrhosis, other endocrine gland failure, vitiligo,
vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic
dermatitis, asthma, inflammatory myopathies, and other
inflammatory, granulamatous, degenerative, atrophic disorders, and
other disorders such as inflammatory skin diseases including
psoriasis and sclerosis, responses associated with inflammatory
bowel disease (such as Crohn's disease and ulcerative colitis),
respiratory distress syndrome (including adult respiratory distress
syndrome, ARDS), meningitis, encephalitis, colitis, allergic
conditions such as eczema and other conditions involving
infiltration of T cells and chronic inflammatory responses,
atherosclerosis, leukocyte adhesion deficiency, Reynaud's syndrome,
and immune responses associated with acute and delayed
hypersensitivity mediated by cytokines and T-lymphocytes typically
found in tuberculosis, sarcoidosis, granulomatosis and diseases
involving leukocyte diapedesis, central nervous system (CNS)
inflammatory disorder, multiple organ injury syndrome,
antigen-antibody complex mediated diseases, anti-glomerular
basement membrane disease, Lambert-Eaton myasthenic syndrome,
Beheet disease, giant cell arteritis, immune complex nephritis, IgA
nephropathy, IgM polyneuropathies or autoimmune thrombocytopenia
etc.
[0264] In a specific embodiment, antibodies of the invention are
used to treat, inhibit, prognose, diagnose or prevent rheumatoid
arthritis. In a specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent advanced
rheumatoid arthritis. In another specific embodiment, antibodies of
the invention are used to treat, inhibit, prognose, diagnose or
prevent systemic lupus erythematosis.
[0265] For example, an antibody, or antibodies, of the present
invention are used to treat patients with clinical diagnosis of
rheumatoid arthritis (RA). The patient treated will not have a B
cell malignancy. Moreover, the patient is optionally further
treated with any one or more agents employed for treating RA such
as salicylate; nonsteroidal anti-inflammatory drugs such as
indomethacin, phenylbutazone, phenylacetic acid derivatives (e.g.
ibuprofen and fenoprofen), naphthalene acetic acids (naproxen),
pyrrolealkanoic acid (tometin), indoleacetic acids (sulindac),
halogenated anthranilic acid (meclofenamate sodium), piroxicam,
zomepirac and diflunisal; antimalarials such as chloroquine; gold
salts; penicillamine; or immunosuppressive agents such as
methotrexate or corticosteroids in dosages known for such drugs or
reduced dosages. Preferably however, the patient is only treated
with an antibody, or antibodies, of the present invention.
Antibodies of the present invention are administered to the RA
patient according to a dosing schedule as described infra, which
may be readily determined by one of ordinary skill in the art. The
primary response is determined by the Paulus index (Paulus et al.
Athritis Rheum. 33:477-484 (1990)), i.e. improvement in morning
stiffness, number of painful and inflamed joints, erythrocyte
sedimentation (ESR), and at least a 2-point improvement on a
5-point scale of disease severity assessed by patient and by
physician. Administration of an antibody, or antibodies, of the
present invention will alleviate one or more of the symptoms of RA
in the patient treated as described above.
[0266] In a further specific embodiment, antibodies of the
invention are used to treat, inhibit, prognose, diagnose or prevent
hemolytic anemia. For example, patients diagnosed with autoimmune
hemolytic anemia (AIHA), e.g., cryoglobinemia or Coombs positive
anemia, are treated with an antibody, or antibodies, of the present
invention. AIHA is an acquired hemolytic anemia due to
auto-antibodies that react with the patient's red blood cells. The
patient treated will not have a B cell malignancy. Further adjunct
therapies (such as glucocorticoids, prednisone, azathioprine,
cyclophosphamide, vinca-laden platelets or Danazol) may be combined
with the antibody therapy, but preferably the patient is treated
with an antibody, or antibodies, of the present invention as a
single-agent throughout the course of therapy. Antibodies of the
present invention are administered to the hemolytic anemia patient
according to a dosing schedule as described infra, which may be
readily determined by one of ordinary skill in the art. Overall
response rate is determined based upon an improvement in blood
counts, decreased requirement for transfusions, improved hemoglobin
levels and/or a decrease in the evidence of hemolysis as determined
by standard chemical parameters. Administration of an antibody, or
antibodies of the present invention will improve any one or more of
the symptoms of hemolytic anemia in the patient treated as
described above. For example, the patient treated as described
above will show an increase in hemoglobin and an improvement in
chemical parameters of hemolysis or return to normal as measured by
serum lactic dehydrogenase and/or bilirubin.
[0267] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent adult
immune thrombocytopenic purpura. Adult immune thrombocytopenic
purpura (ITP) is a relatively rare hematologic disorder that
constitutes the most common of the immune-mediated cytopenias. The
disease typically presents with severe thrombocytopenia that may be
associated with acute hemorrhage in the presence of normal to
increased megakaryocytes in the bone marrow. Most patients with ITP
have an IgG antibody directed against target antigens on the outer
surface of the platelet membrane, resulting in platelet
sequestration in the spleen and accelerated reticuloendothelial
destruction of platelets (Bussell, J. B. Hematol. Oncol. Clin.
North Am. (4):179 (1990)). A number of therapeutic interventions
have been shown to be effective in the treatment of ITP. Steroids
are generally considered first-line therapy, after which most
patients are candidates for intravenous immunoglobulin (IVIG),
splenectomy, or other medical therapies including vincristine or
immunosuppressive/cytotoxic agents. Up to 80% of patients with ITP
initially respond to a course of steroids, but far fewer have
complete and lasting remissions. Splenectomy has been recommended
as standard second-line therapy for steroid failures, and leads to
prolonged remission in nearly 60% of cases yet may result in
reduced immunity to infection. Splenectomy is a major surgical
procedure that may be associated with substantial morbidity (15%)
and mortality (2%). IVIG has also been used as second line medical
therapy, although only a small proportion of adult patients with
ITP achieve remission. Therapeutic options that would interfere
with the production of autoantibodies by activated B cells without
the associated morbidities that occur with corticosteroids and/or
splenectomy would provide an important treatment approach for a
proportion of patients with ITP. Patients with clinical diagnosis
of ITP are treated with an antibody, or antibodies of the present
invention, optionally in combination with steroid therapy. The
patient treated will not have a B cell malignancy. Antibodies of
the present invention are administered to the RA patient according
to a dosing schedule as described infra, which may be readily
determined by one of ordinary skill in the art. Overall patient
response rate is determined based upon a platelet count determined
on two consecutive occasions two weeks apart following treatments
as described above. See, George et al. "Idiopathic Thrombocytopenic
Purpura: A Practice Guideline Developed by Explicit Methods for The
American Society of Hematology", Blood 88:3-40 (1996), expressly
incorporated herein by reference.
[0268] In other embodiments, antibody agonists of the invention are
be used to treat, inhibit or prevent immunodeficiencies, and/or
disorders, or conditions associated with immunodeficiencies. Such
immunodeficiencies include, but are not limited to, severe combined
immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine
deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia
(XLA), Bruton's disease, congenital agammaglobulinemia, X-linked
infantile agammaglobulinemia, acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, transient
hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0269] In another specific embodiment, antibodies of the invention
are used to treat, inhibit, prognose, diagnose or prevent CVID, or
a subgroup of individuals having CVID.
[0270] In another specific embodiment, antibody agonists of the
invention are used as an adjuvant to stimulate B cell
proliferation, immunoglobulin production, and/or to enhance B cell
survival.
[0271] The treatment and/or prevention of diseases, disorders, or
conditions associated with aberrant expression and/or activity of a
polypeptide of the invention includes, but is not limited to,
alleviating symptoms associated with those diseases, disorders or
conditions. The antibodies of the invention may also be used to
target and kill cells expressing TR21 on their surface (e.g., B
cells) and/or cells having TR21 bound to their surface. Antibodies
of the invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0272] A summary of the ways in which the antibodies of the present
invention may be used therapeutically includes binding
polynucleotides or polypeptides of the present invention locally or
systemically in the body or by direct cytotoxicity of the antibody,
e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some of these approaches are described in more detail below. Armed
with the teachings provided herein, one of ordinary skill in the
art will know how to use the antibodies of the present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[0273] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors
(such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to
increase the number or activity of effector cells which interact
with the antibodies.
[0274] The antibodies of the invention may be administered alone or
in combination with other types of treatments (e.g., radiation
therapy, chemotherapy, hormonal therapy, immunotherapy and
anti-tumor agents). Generally, administration of products of a
species origin or species reactivity (in the case of antibodies)
that is the same species as that of the patient is preferred. Thus,
in a preferred embodiment, human antibodies, fragments derivatives,
analogs, or nucleic acids, are administered to a human patient for
therapy or prophylaxis.
[0275] It is preferred to use high affinity and/or potent in vivo
inhibiting and/or neutralizing antibodies against polypeptides or
polynucleotides of the present invention, fragments or regions
thereof, for both immunoassays directed to and therapy of disorders
related to polynucleotides or polypeptides, including fragments
thereof, of the present invention. Such antibodies, fragments, or
regions, will preferably have an affinity for polynucleotides or
polypeptides of the invention, including fragments thereof.
Preferred binding affinities include those with a dissociation
constant or Kd less than 5.times.10.sup.-2 M, 10.sup.-2 M,
5.times.10.sup.-3 M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M,
5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M,
5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M, or 10.sup.-8
M. Even more preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M.
Gene Therapy Using TR21 Antibodies
[0276] In a specific embodiment, nucleic acids comprising sequences
encoding antibodies or functional derivatives thereof, are
administered to treat, inhibit or prevent a disease or disorder
associated with aberrant expression and/or activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded protein that
mediates a therapeutic effect.
[0277] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0278] For general reviews of the methods of gene therapy, see
Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May,
TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[0279] In a preferred aspect, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being
part of expression vectors that express the antibody or fragments
or chimeric proteins or heavy or light chains thereof in a suitable
host. In particular, such nucleic acid sequences have promoters
operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In
another particular embodiment, nucleic acid molecules are used in
which the antibody coding sequences and any other desired sequences
are flanked by regions that promote homologous recombination at a
desired site in the genome, thus providing for intrachromosomal
expression of the antibody encoding nucleic acids (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al., Nature 342:435-438 (1989). In specific embodiments, the
expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences
encoding both heavy and light chains or fragments thereof, of the
antibody.
[0280] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0281] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989)).
[0282] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an antibody of the invention are used. For
example, a retroviral vector can be used (see Miller et al., Meth.
Enzymol. 217:581-599 (1993)). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding the antibody to be used in gene therapy are cloned into
one or more vectors, which facilitate delivery of the gene into a
patient. More detail about retroviral vectors can be found in
Boesen et al., Biotherapy 6:291-302 (1994), which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons
and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and
Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
[0283] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). In a preferred embodiment, adenovirus vectors are used.
[0284] Adeno-associated virus (AAV) has also been proposed for use
in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med.
204:289-300 (1993); U.S. Pat. No. 5,436,146).
[0285] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0286] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen
et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther.
29:69-92m (1985) and may be used in accordance with the present
invention, provided that the necessary developmental and
physiological functions of the recipient cells are not disrupted.
The technique should provide for the stable transfer of the nucleic
acid to the cell, so that the nucleic acid is expressible by the
cell and preferably heritable and expressible by its progeny.
[0287] The resulting recombinant cells can be delivered to a
patient by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, patient state, etc., and can be
determined by one skilled in the art.
[0288] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0289] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0290] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding an antibody are introduced
into the cells such that they are expressible by the cells or their
progeny, and the recombinant cells are then administered in vivo
for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which
can be isolated and maintained in vitro can potentially be used in
accordance with this embodiment of the present invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985
(1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0291] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[0292] The compounds or pharmaceutical compositions of the
invention are preferably tested in vitro, and then in vivo for the
desired therapeutic or prophylactic activity, prior to use in
humans. For example, in vitro assays to demonstrate the therapeutic
or prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
Therapeutic/Prophylactic Administration and Composition
[0293] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention, such
as, for example, an antibody of the invention. In a preferred
aspect, the compound is substantially purified (e.g., substantially
free from substances that limit its effect or produce undesired
side-effects). The subject is preferably an animal, including but
not limited to animals such as cows, pigs, horses, chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably
human.
[0294] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid or an
immunoglobulin are described above; additional appropriate
formulations and routes of administration can be selected from
among those described herein below.
[0295] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, and oral routes. The compounds or
compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, e.g., by use of an
inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0296] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, e.g., in conjunction with a wound
dressing after surgery, by injection, by means of a catheter, by
means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
adsorb.
[0297] In another embodiment, the compound or composition can be
delivered in a vesicle, in particular a liposome (see Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353- 365 (1989);
Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
[0298] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. In one embodiment, a
pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed.
Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek
et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); Controlled Drug Bioavailability, Drug Product Design
and Performance, Smolen and Ball (eds.), Wiley, New York (1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61
(1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)). In yet another embodiment, a controlled release
system can be placed in proximity of the therapeutic target, i.e.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)). Other controlled release
systems are discussed in the review by Langer (Science
249:1527-1533 (1990)).
[0299] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0300] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopoeia or other
generally recognized pharmacopoeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0301] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0302] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0303] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0304] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Further, the dosage
and frequency of administration of antibodies of the invention may
be reduced by enhancing uptake and tissue penetration (e.g., into
the brain) of the antibodies by modifications such as, for example,
lipidation.
[0305] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. Diagnosis and Imaging
[0306] Labeled antibodies, and derivatives and analogs thereof,
which specifically bind to a polypeptide of interest can be used
for diagnostic purposes to detect, diagnose, or monitor diseases
and/or disorders associated with the aberrant expression and/or
activity of a polypeptide of the invention. The invention provides
for the detection of aberrant expression of a polypeptide of
interest, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of aberrant expression.
[0307] The invention provides a diagnostic assay for diagnosing a
disorder, comprising (a) assaying the expression of the polypeptide
of interest in cells or body fluid of an individual using one or
more antibodies specific to the polypeptide interest and (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a particular disorder. With
respect to cancer, the presence of a relatively high amount of
transcript in biopsied tissue from an individual may indicate a
predisposition for the development of the disease, or may provide a
means for detecting the disease prior to the appearance of actual
clinical symptoms. A more definitive diagnosis of this type may
allow health professionals to employ preventative measures or
aggressive treatment earlier thereby preventing the development or
further progression of the cancer.
[0308] Antibodies of the invention can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell.
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein gene expression include immunoassays, such as
the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase;
radioisotopes, such as radioisotopes, such as iodine (.sup.131I,
.sup.125I, .sup.123I, .sup.121I), carbon (.sup.14C), sulfur
(.sup.35S), tritium (3 H), indium (.sup.115mIn, .sup.113mIn,
.sup.112In, .sup.111In), and technetium (.sup.99Tc, .sup.99mTc),
thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru;
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0309] One aspect of the invention is the detection and diagnosis
of a disease or disorder associated with aberrant expression of a
polypeptide of interest in an animal, preferably a mammal and most
preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled
molecule which specifically binds to the polypeptide of interest;
b) waiting for a time interval following the administering for
permitting the labeled molecule to preferentially concentrate at
sites in the subject where the polypeptide is expressed (and for
unbound labeled molecule to be cleared to background level); c)
determining background level; and d) detecting the labeled molecule
in the subject, such that detection of labeled molecule above the
background level indicates that the subject has a particular
disease or disorder associated with aberrant expression of the
polypeptide of interest. Background level can be determined by
various methods including, comparing the amount of labeled molecule
detected to a standard value previously determined for a particular
system.
[0310] It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of 99mTc. The labeled antibody or antibody fragment
will then preferentially accumulate at the location of cells which
contain the specific protein. In vivo tumor imaging is described in
S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled
Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes,
eds., Masson Publishing Inc. (1982).
[0311] Depending on several variables, including the type of label
used and the mode of administration, the time interval following
the administration for permitting the labeled molecule to
preferentially concentrate at sites in the subject and for unbound
labeled molecule to be cleared to background level is 6 to 48 hours
or 6 to 24 hours or 6 to 12 hours. In another embodiment the time
interval following administration is 5 to 20 days or 5 to 10
days.
[0312] In an embodiment, monitoring of the disease or disorder is
carried out by repeating the method for diagnosing the disease or
disease, for example, one month after initial diagnosis, six months
after initial diagnosis, one year after initial diagnosis, etc.
[0313] Presence of the labeled molecule can be detected in the
patient using methods known in the art for in vivo scanning. These
methods depend upon the type of label used. Skilled artisans will
be able to determine the appropriate method for detecting a
particular label. Methods and devices that may be used in the
diagnostic methods of the invention include, but are not limited
to, computed tomography (CT), whole body scan such as position
emission tomography (PET), magnetic resonance imaging (MRI), and
sonography.
[0314] In a specific embodiment, the molecule is labeled with a
radioisotope and is detected in the patient using a radiation
responsive surgical instrument (Thurston et al., U.S. Pat. No.
5,441,050). In another embodiment, the molecule is labeled with a
fluorescent compound and is detected in the patient using a
fluorescence responsive scanning instrument. In another embodiment,
the molecule is labeled with a positron emitting metal and is
detected in the patent using positron emission-tomography. In yet
another embodiment, the molecule is labeled with a paramagnetic
label and is detected in a patient using magnetic resonance imaging
(MRI).
Kits
[0315] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises an antibody of
the invention, preferably a purified antibody, in one or more
containers. In a specific embodiment, the kits of the present
invention contain a substantially isolated polypeptide comprising
an epitope which is specifically immunoreactive with an antibody
included in the kit. Preferably, the kits of the present invention
further comprise a control antibody which does not react with the
polypeptide of interest. In another specific embodiment, the kits
of the present invention contain a means for detecting the binding
of an antibody to a polypeptide of interest (e.g., the antibody may
be conjugated to a detectable substrate such as a fluorescent
compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the
first antibody may be conjugated to a detectable substrate).
[0316] In another specific embodiment of the present invention, the
kit is a diagnostic kit for use in screening serum containing
antibodies specific against proliferative and/or cancerous
polynucleotides and polypeptides. Such a kit may include a control
antibody that does not react with the polypeptide of interest. Such
a kit may include a substantially isolated polypeptide antigen
comprising an epitope which is specifically immunoreactive with at
least one anti-polypeptide antigen antibody. Further, such a kit
includes means for detecting the binding of said antibody to the
antigen (e.g., the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In specific embodiments, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0317] In a more specific embodiment the detecting means of the
above-described kit includes a solid support to which said
polypeptide antigen is attached. Such a kit may also include a
non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can
be detected by binding of the said reporter-labeled antibody.
[0318] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum containing antigens of
the polypeptide of the invention. The diagnostic kit includes a
substantially isolated antibody specifically immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the
binding of the polynucleotide or polypeptide antigen to the
antibody. In one embodiment, the antibody is attached to a solid
support. In a specific embodiment, the antibody may be a monoclonal
antibody. The detecting means of the kit may include a second,
labeled monoclonal antibody. Alternatively, or in addition, the
detecting means may include a labeled, competing antigen.
[0319] In one diagnostic configuration, test serum is reacted with
a solid phase reagent having a surface-bound antigen obtained by
the methods of the present invention. After binding with specific
antigen antibody to the reagent and removing unbound serum
components by washing, the reagent is reacted with reporter-labeled
anti-human antibody to bind reporter to the reagent in proportion
to the amount of bound anti-antigen antibody on the solid support.
The reagent is again washed to remove unbound labeled antibody, and
the amount of reporter associated with the reagent is determined.
Typically, the reporter is an enzyme which is detected by
incubating the solid phase in the presence of a suitable
fluorometric, luminescent or calorimetric substrate (SIGMA.TM., St.
Louis, Mo.).
[0320] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material, such as polymeric beads, dip sticks, 96-well plate or
filter material. These attachment methods generally include
non-specific adsorption of the protein to the support or covalent
attachment of the protein, typically through a free amine group, to
a chemically reactive group on the solid support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively,
streptavidin coated plates can be used in conjunction with
biotinylated antigen(s).
[0321] Thus, the invention provides an assay system or kit for
carrying out this diagnostic method. The kit generally includes a
support with surface-bound recombinant antigens, and a
reporter-labeled anti-human antibody for detecting surface-bound
anti-antigen antibody.
Diagnosis of Immune System-Related Disorders
[0322] TR21 is preferentially expressed in mature B lymphocytes.
For a number of immune system-related disorders, substantially
altered (increased or decreased) levels of TR21 gene expression may
be detected in immune system tissue or other cells or bodily fluids
(e.g., sera, plasma, urine, synovial fluid or spinal fluid) taken
from an individual having such a disorder, relative to a "standard"
TR21 gene expression level, that is, the TR21 expression level in
immune system tissues or bodily fluids from an individual not
having the immune system disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of an immune system
disorder, which involves measuring the expression level of the gene
encoding the TR21 polypeptide in immune system tissue or other
cells or body fluid from an individual and comparing the measured
gene expression level with a standard TR21 gene expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of an immune system disorder
or abnormal activation, proliferation, differentiation, and/or
death.
[0323] In particular, it is believed that certain tissues in
mammals with cancer of cells or tissue of the immune system express
significantly enhanced or reduced levels of the TR21 polypeptide
and mRNA encoding the TR21 polypeptide when compared to a
corresponding "standard" level. Further, it is believed that
enhanced or depressed levels of the TR21 polypeptide can be
detected in certain body fluids (e.g., sera, plasma, urine, and
spinal fluid) or cells or tissue from mammals with such a cancer
when compared to sera from mammals of the same species not having
the cancer.
[0324] For example, as disclosed herein, TR21 is highly expressed
primarily in cells of B cell lineage. Accordingly, polynucleotides
of the invention (e.g., polynucleotide sequences complementary to
all or a portion of TR21 mRNA) and antibodies (and antibody
fragments) directed against the polypeptides of the invention may
be used to quantitate or qualitate concentrations of cells of B
cell lineage (e.g., B cell leukemia and lymphoma cells) expressing
TR21 on their cell surfaces. These antibodies additionally have
diagnostic applications in detecting abnormalities in the level of
TR21 gene expression, or abnormalities in the structure and/or
temporal, tissue, cellular, or subcellular location of TR21. These
diagnostic assays may be performed in vivo or in vitro, such as,
for example, on blood samples, biopsy tissue or autopsy tissue.
[0325] Additionally, as disclosed herein, TR21 ligand (e.g.,
Neutrokine-alpha) is expressed primarily on cells of monocytic
lineage. Accordingly, TR21 polypeptides of the invention (including
labeled TR21 polypeptides and TR21 fusion proteins), and anti-TR21
antibodies (including anti-TR21 antibody fragments) against the
polypeptides of the invention may be used to quantitate or
qualitate concentrations of cells of monocytic lineage (e.g.,
monocyte cell lineage related leukemias or lymphomas) expressing
Neutrokine-alpha on their cell surfaces. These TR21 polypeptides
and antibodies additionally have diagnostic applications in
detecting abnormalities in the level of Neutrokine-alpha gene
expression, or abnormalities in the structure and/or temporal,
tissue, cellular, or subcellular location of Neutrokine-alpha,
and/or diagnosing activity/defects in signaling pathways associated
with TR21. These diagnostic assays may be performed in vivo or in
vitro, such as, for example, on blood samples or biopsy tissue
using techniques described herein or otherwise known in the
art.
[0326] In one embodiment, TR21 polynucleotides or polypeptides or
TR21 agonists (e.g., anti-TR21 antibodies) or antagonists (e.g.,
anti-TR21 antibodies) of the invention are used to treat, diagnose,
or prognose an individual having an autoimmune disease or
disorder.
[0327] Autoimmune diseases or disorders that may be treated,
diagnosed, or prognosed using TR21 polynucleotides or polypeptides
(e.g., TR21 extracellular domain-Fc fusion polypeptides) or TR21
agonists (e.g., anti-TR21 antibodies) or antagonists (e.g.,
anti-TR21 antibodies) of the invention include, but are not limited
to, one or more of the following: autoimmune hemolytic anemia,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia
purpura, autoimmune neutropenia, autoimmunocytopenia, hemolytic
anemia, antiphospholipid syndrome, dermatitis, gluten sensitive
enteropathy, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g.,
IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, myocarditis, IgA glomerulonephritis, dense deposit
disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin
dependent diabetes mellitis, and autoimmune inflammatory eye,
autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's
thyroiditis, systemic lupus erhythematosus, discoid lupus,
Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as,
for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c)
insulin resistance, autoimmune hemolytic anemia, autoimmune
thrombocytopenic purpura, rheumatoid arthritis, scleroderma with
anti-collagen antibodies, mixed connective tissue disease,
polymyositis/dermatomyositis, pernicious anemia, idiopathic
Addison's disease, infertility, glomerulonephritis such as primary
glomerulonephritis and IgA nephropathy, bullous pemphigoid,
Sjogren's syndrome, diabetes mellitus, and adrenergic drug
resistance (including adrenergic drug resistance with asthma or
cystic fibrosis), chronic active hepatitis, primary biliary
cirrhosis, other endocrine gland failure, vitiligo, vasculitis,
post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma,
inflammatory myopathies, and other inflammatory, granulamatous,
degenerative, and atrophic disorders.
[0328] According to this embodiment, an individual having an
autoimmune disease or disorder may express aberrantly high levels
of TR21 ligand (e.g., Neutrokine-alpha) and/or TR21 when compared
to an individual not having an autoimmune disease or disorder. Any
means described herein or otherwise known in the art may be applied
to detect TR21 polynucleotides or polypeptides of the invention
(e.g., FACS analysis or ELISA detection of TR21 polypeptides of the
invention and hybridization or PCR detection of TR21
polynucleotides of the invention) and to determine the expression
profile of, for example, TR21, polynucleotides and/or polypeptides
of the invention, in a biological sample.
[0329] A biological sample of persons afflicted with an autoimmune
disease or disorder may be characterized by high levels of
expression of TR21 when compared to that observed in individuals
not having an autoimmune disease or disorder. Thus, TR21
polynucleotides and/or polypeptides (e.g., anti-TR21 antibodies and
TR21-extracellular domain-Fc fusion polypeptides) of the invention
and/or agonists or antagonists thereof, may be used according to
the methods of the invention in the diagnosis and/or prognosis of
an autoimmune disease or disorder. For example, a biological sample
obtained from a person suspected of being afflicted with an
autoimmune disease or disorder ("the subject") may be analyzed for
the relative expression level(s) of TR21 polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of the TR21 molecules of the invention is (are) then compared
to the expression level(s) of the same molecules of the invention
as expressed in a person known not to be afflicted with an
autoimmune disease or disorder. According to this example, a
significant difference in expression level(s) of TR21,
polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, between samples obtained from
the subject and the control suggests that the subject is afflicted
with an autoimmune disease or disorder.
[0330] In another embodiment, TR21 polynucleotides or polypeptides
or TR21 agonists (such as, for example, anti-TR21 antibodies) or
TR21 antagonists (such as, for example, anti-TR21 antibodies) of
the invention are used to treat, diagnose, or prognose an
individual having systemic lupus erythematosus or a subset of this
disease. According to this embodiment, an individual having
systemic lupus erythematosus or a subset of individuals having
systemic lupus erythematosus may express aberrantly high levels of
TR21 when compared to an individual not having systemic lupus
erythematosus or this subset of systemic lupus. TR21
polynucleotides or polypeptides of the invention (e.g., FACS
analysis or ELISA detection of TR21 polypeptides of the invention
and hybridization or PCR detection of TR21 polynucleotides of the
invention) may be used to determine the expression profile of TR21,
polynucleotides and/or polypeptides of the invention in a
biological sample.
[0331] A biological sample of persons afflicted with systemic lupus
erythematosus may be characterized by high levels of expression of
TR21 when compared to that observed in individuals not having
systemic lupus erythematosus. Thus, TR21 polynucleotides and/or
polypeptides of the invention and/or agonists or antagonists
thereof, may be used according to the methods of the invention in
the diagnosis and/or prognosis of systemic lupus erythematosus or a
subset of systemic lupus erythematosus. For example, a biological
sample obtained from a person suspected of being afflicted with
systemic lupus erythematosus ("the subject") may be analyzed for
the relative expression level(s) of TR21 polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of these molecules of the invention is (are) then compared to
the expression level(s) of the same molecules of the invention as
expressed in a person known not to be afflicted with systemic lupus
erythematosus. According to this example, a significant difference
in expression level(s) of TR21, polynucleotides and/or polypeptides
of the invention, and/or agonists (e.g., agonistic antibodies)
and/or antagonists thereof, between samples obtained from the
subject and the control suggests that the subject is afflicted with
systemic lupus erythematosus or a subset thereof.
[0332] Furthermore, there may be a direct correlation between the
severity of systemic lupus erythematosus, or a subset of this
disease, and the concentration of TR21 polynucleotides (RNA) and/or
polypeptides of the invention. Thus, TR21 polynucleotides (RNA)
and/or polypeptides and/or agonists or antagonists of the
invention, may be used according to the methods of the invention in
prognosis of the severity of systemic lupus erythematosus or a
subset of systemic lupus erythematosus. For example, a biological
sample obtained from a person suspected of being afflicted with
systemic lupus erythematosus ("the subject") may be analyzed for
the relative expression level(s) of TR21 polynucleotides and/or
polypeptides of the invention. The expression level(s) of one or
more of these molecules of the invention is (are) then compared to
the expression level(s) of the same molecules of the invention as
expressed in a panel of persons known to represent a range in
severities of this disease. According to this example, the match of
expression level with a characterized member of the panel indicates
the severity of the disease.
[0333] In another embodiment, TR21 polynucleotides or polypeptide
(e.g., anti-TR21 antibodies and TR21 extracellular domain-Fc fusion
polypeptides) or TR21 agonists (such as, for example, anti-TR21
antibodies) or TR21 antagonists (such as, for example, anti-TR21
antibodies) of the invention are used to treat, diagnose, or
prognose an individual having rheumatoid arthritis or a subset of
this disease. According to this embodiment, an individual having
rheumatoid arthritis or a subset of individuals having rheumatoid
arthritis may express aberrantly high levels of TR21 when compared
to an individual not having rheumatoid arthritis or this subset of
rheumatoid arthritis. Any means described herein or otherwise known
in the art may be applied to detect TR21 polynucleotides or
polypeptides of the invention (e.g., FACS analysis or ELISA
detection of TR21 polypeptides of the invention and hybridization
or PCR detection of TR21 polynucleotides of the invention) and to
determine the expression profile of TR21 polynucleotides and/or
polypeptides of the invention) in a biological sample.
[0334] A biological sample of persons afflicted with rheumatoid
arthritis may be characterized by high levels of expression of TR21
when compared to that observed in individuals not having rheumatoid
arthritis. Thus, TR21 polynucleotides and/or polypeptides of the
invention, and/or agonists or antagonists thereof, may be used
according to the methods of the invention in the diagnosis and/or
prognosis of rheumatoid arthritis or a subset of rheumatoid
arthritis. For example, a biological sample obtained from a person
suspected of being afflicted with rheumatoid arthritis ("the
subject") may be analyzed for the relative expression level(s) of
TR21 polynucleotides and/or polypeptides of the invention. The
expression level(s) of one or more of these molecules of the
invention is (are) then compared to the expression level(s) of the
same molecules of the invention as expressed in a person known not
to be afflicted with rheumatoid arthritis. According to this
example, a significant difference in expression level(s) of TR21,
polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, between samples obtained from
the subject and the control suggests that the subject is afflicted
with rheumatoid arthritis or a subset thereof.
[0335] In another embodiment, TR21 polynucleotides or polypeptides
or TR21 agonists (e.g., anti-TR21 antibodies) or antagonists (e.g.,
anti-TR21 antibodies) of the invention are used to treat, prevent,
diagnose, or prognose an individual having an immunodeficiency.
[0336] Immunodeficiencies that may be treated, prevented,
diagnosed, and/or prognosed with the TR21 polynucleotides or
polypeptides or TR21 agonists (e.g., anti-TR21 antibodies) or
antagonists (e.g., anti-TR21 antibodies) of the invention, include,
but are not limited to one or more immunodeficiencies selected
from: severe combined immunodeficiency (SCID)-X linked,
SCID-autosomal, adenosine deaminase deficiency (ADA deficiency),
X-linked agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), chronic granulomatous disease,
Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with
hyper IgM, non X-linked immunodeficiency with hyper IgM, selective
IgA deficiency, IgG subclass deficiency (with or without IgA
deficiency), antibody deficiency with normal or elevated Igs,
immunodeficiency with thymoma, Ig heavy chain deletions, kappa
chain deficiency, B cell lymphoproliferative disorder (BLPD),
selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss
type), reticular dysgenesis, neonatal neutropenia, severe
congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia
with immunodeficiency, ataxia-telangiectasia, short limbed
dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0337] According to this embodiment, an individual having an
immunodeficiency may express aberrantly low levels of TR21 when
compared to an individual not having an immunodeficiency. Any means
described herein or otherwise known in the art may be applied to
detect TR21 polynucleotides or polypeptides of the invention (e.g.,
FACS analysis or ELISA detection of TR21 polypeptides of the
invention and hybridization or PCR detection of TR21
polynucleotides of the invention) and to determine the expression
profile of TR21 polynucleotides and/or polypeptides of the
invention in a biological sample.
[0338] A biological sample of a person afflicted with an
immunodeficiency may be characterized by low levels of expression
of TR21 when compared to that observed in individuals not having an
immunodeficiency. Thus, TR21 polynucleotides and/or polypeptides of
the invention, and/or agonists or antagonists thereof, may be used
according to the methods of the invention in the diagnosis and/or
prognosis of an immunodeficiency. For example, a biological sample
obtained from a person suspected of being afflicted with an
immunodeficiency ("the subject") may be analyzed for the relative
expression level(s) of TR21 polynucleotides and/or polypeptides of
the invention. The expression level(s) of one or more of these
molecules of the invention is (are) then compared to the expression
level(s) of the same molecules of the invention as expressed in a
person known not to be afflicted with an immunodeficiency. A
significant difference in expression level(s) of TR21,
polynucleotides and/or polypeptides of the invention, and/or
agonists and/or antagonists thereof, between samples obtained from
the subject and the control suggests that the subject is afflicted
with an immunodeficiency.
[0339] In another embodiment, TR21 polynucleotides or polypeptides
(e.g., TR21 extracellular domain-Fc fusion polypeptides) or TR21
agonists (e.g., anti-TR21 antibodies) or antagonists (e.g.,
anti-TR21 antibodies) of the invention are used to treat, diagnose
and/or prognose an individual having common variable
immunodeficiency disease ("CVID"; also known as "acquired
agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease. According to this embodiment, an individual
having CVID or a subset of individuals having CVID expresses
aberrant levels of TR21 and/or Neutrokine-alpha or Neutrokine-alpha
SV on their B cells and/or monocytes, when compared to individuals
not having CVID. Any means described herein or otherwise known in
the art may be applied to detect TR21 polynucleotides or
polypeptides of the invention (e.g., FACS analysis or ELISA
detection of TR21 polypeptides of the invention and hybridization
or PCR detection of TR21 polynucleotides of the invention) and to
determine differentially the expression profile of TR21
polynucleotides or polypeptides of the invention in a sample
containing at least monocyte cells or some component thereof (e.g.,
RNA) as compared to a sample containing at least B cells or a
component thereof (e.g., RNA). In the instance where a sample
containing at least monocyte cells or some component thereof (e.g.,
RNA) is determined to reflect TR21 ligand (e.g., Neutrokine-alpha)
polynucleotide or polypeptide expression and a sample containing at
least B cells or a component thereof (e.g., RNA) is determined to
reflect less than normal levels of TR21 polynucleotide or
polypeptide expression, the samples may be correlated with the
occurrence of CVID (i.e., "acquired agammaglobulinemia" or
"acquired hypogammaglobulinemia").
[0340] A subset of persons afflicted with CVID may be characterized
by high levels of expression of Neutrokine-alpha, Neutrokine-alpha
SV, and/or TR21 polypeptides in peripheral or circulating B cells
when compared to that observed in individuals not having CVID. In
contrast, persons who are not afflicted with CVID are typically
characterized by low levels of Neutrokine-alpha expression and high
levels of TR21 expression in peripheral or circulating B cells.
Thus, TR21 polypeptides, polynucleotides and/or polypeptides of the
invention, and/or agonists or antagonists thereof, may be used
according to the methods of the invention in the differential
diagnosis of this subset of CVID. For example, a sample of
peripheral B cells obtained from a person suspected of being
afflicted with CVID ("the subject") may be analyzed for the
relative expression level(s) of Neutrokine-alpha, Neutrokine-alpha
SV, and/or TR21 polynucleotides and/or polypeptides of the
invention. The expression level(s) of one or more of these
molecules of the invention is (are) then compared to the expression
level(s) of the same molecules of the invention as expressed in a
person known not to be afflicted with CVID ("the control").
According to this example, a significant difference in expression
level(s) of Neutrokine-alpha, Neutrokine-alpha SV, and/or TR21
polynucleotides or polypeptides of the invention, and/or agonists
and/or antagonists thereof, between samples obtained from the
subject and the control suggests that the subject is afflicted with
this subset of CVID.
[0341] Cunningham-Rundles and Bodian followed 248 CVID patients
over a period of 1-25 years and discovered that a number of
associated diseases or conditions appear with increased frequency
in CVID patients (Cunningham-Rundles and Bodian, J. Clin. Immunol.,
92:34-48 (1999) which is herein incorporated by reference in its
entirety.) The most important clinical events include infections,
autoimmunity, inflammatory disorders, marked by gastrointestinal
and granulomatous disease, cancer and hepatitis. Most CVID patients
are at increased risk of recurrent infections particularly of the
respiratory tract. The types of acute and recurring bacterial
infections exhibited in most patients include pneumonia, bronchitis
and sinusitis. Children with CVID have a marked increased risk of
otitis media. Additionally, blood borne infections including
sepsis, meningitis, septic arthritis, and osteomyelitis are seen
with increased frequency in these patients.
[0342] In another specific embodiment, TR21 polynucleotides or
polypeptides, or agonists or antagonists thereof (e.g., anti-TR21
antibodies) are used to diagnose, prognose, treat, or prevent
conditions associated with CVID, including, but not limited to,
conditions associated with acute and recurring infections (e.g.,
pneumonia, bronchitis, sinusitis, otitis media, sepsis, meningitis,
septic arthritis, and osteomyelitis), chronic lung disease,
autoimmunity, granulomatous disease, lymphoma, cancers (e.g.,
cancers of the breast, stomach, colon, mouth, prostate, lung,
vagina, ovary, skin, and melanin forming cells (i.e. melanoma),
inflammatory bowel disease (e.g., Crohn's disease, ulcerative
colitis, and ulcerative proctitis), malabsorption, Hodgkin's
disease, and Waldenstrom's macroglobulinemia.
[0343] In a specific embodiment, TR21 polynucleotides or
polypeptides, or agonists or antagonists thereof (e.g., anti-TR21
antibodies) are used to diagnose, prognose, treat, or prevent a
disorder characterized by deficient serum immunoglobulin
production, recurrent infections, and/or immune system dysfunction.
Moreover, TR21 polynucleotides or polypeptides, or agonists or
antagonists thereof (e.g., anti-TR21 antibodies) may be used to
diagnose, prognose, treat, or prevent infections of the joints,
bones, skin, and/or parotid glands, blood-borne infections (e.g.,
sepsis, meningitis, septic arthritis, and/or osteomyelitis),
autoimmune diseases (e.g., those disclosed herein), inflammatory
disorders, and malignancies, and/or any disease or disorder or
condition associated with these infections, diseases, disorders
and/or malignancies) including, but not limited to, CVID, other
primary immune deficiencies, HIV disease, CLL, recurrent
bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,
hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),
and/or pneumocystis carnii.
[0344] Thus, the invention provides a diagnostic method useful
during diagnosis of a immune system disorder, including cancers of
this system, and immunodeficiencies and/or autoimmune diseases
which involves measuring the expression level of the gene encoding
TR21 polypeptide in immune system tissue or other cells or body
fluid from an individual and comparing the measured gene expression
level with a standard TR21 gene expression level, whereby an
increase or decrease in the gene expression level compared to the
standard is indicative of an immune system disorder.
[0345] Where a diagnosis of a disorder in the immune system,
including, but not limited to, diagnosis of a tumor, diagnosis of
an immunodeficiency, and/or diagnosis of an autoimmune disease, has
already been made according to conventional methods, the present
invention is useful as a prognostic indicator, whereby patients
exhibiting enhanced or depressed TR21 gene expression will
experience a worse clinical outcome relative to patients expressing
the gene at a level nearer the standard level.
[0346] By analyzing or determining the expression level of the gene
encoding the TR21 polypeptide is intended qualitatively or
quantitatively measuring or estimating the level of the TR21
polypeptide or the level of the mRNA encoding the TR21 polypeptide
in a first biological sample either directly (e.g., by determining
or estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the TR21 polypeptide level or mRNA level in
a second biological sample). Preferably, the TR21 polypeptide level
or mRNA level in the first biological sample is measured or
estimated and compared to a standard TR21 polypeptide level or mRNA
level, the standard being taken from a second biological sample
obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having a disorder of the immune system. As will be appreciated in
the art, once a standard TR21 polypeptide level or mRNA level is
known, it can be used repeatedly as a standard for comparison.
[0347] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains TR21 polypeptide or mRNA. As
indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain free
extracellular domains of the TR21 polypeptide, immune system
tissue, and other tissue sources found to express complete or free
extracellular domain of the TR21. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Where the biological sample is to include mRNA, a tissue biopsy is
the preferred source.
[0348] The compounds of the present invention are useful for
diagnosis, prognosis, or treatment of various immune system-related
disorders in mammals, preferably humans. Such disorders include,
but are not limited to tumors (e.g., B cell and monocytic cell
leukemias and lymphomas) and tumor metastasis, infections by
bacteria, viruses and other parasites, immunodeficiencies,
inflammatory diseases, lymphadenopathy, autoimmune diseases (e.g.,
rheumatoid arthritis, systemic lupus erythematosus, Sjogren's
syndrome, mixed connective tissue disease, and inflammatory
myopathies), and graft versus host disease.
[0349] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi Anal. Biochem. 162:156-159 (1987). Levels of
mRNA encoding the TR21 polypeptide are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0350] Assaying TR21 polypeptide levels in a biological sample can
occur using antibody-based techniques. For example, TR21
polypeptide expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al, J. Cell. Biol
101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting TR21 polypeptide gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115mIn, .sup.113mIn, .sup.112In,
.sup.111In), and technetium (.sup.99Tc, .sup.99mTc), thallium
(.sup.20Ti), gallium (.sup.68Ga, .sup.67Ga), palladium
(.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe), fluorine
(.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd, .sup.149Pm,
.sup.140La, .sup.175Yb, .sup.166 Ho, .sup.90Y, .sup.47Sc,
.sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh, .sup.97Ru;
luminescent labels, such as luminol; and fluorescent labels, such
as fluorescein and rhodamine, and biotin.
[0351] Techniques known in the art may be applied to label
polypeptides (including antibodies) of the invention. Such
techniques include, but are not limited to, the use of bifunctional
conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631;
5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139;
5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of
each of which are hereby incorporated by reference in its
entirety).
[0352] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the TR21
(such as, for example, cells of B cell lineage and the spleen). The
protein isolation methods employed herein may, for example, be such
as those described in Harlow and Lane (Harlow, E. and Lane, D.,
1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated
herein by reference in its entirety. The isolated cells can be
derived from cell culture or from a patient. The analysis of cells
taken from culture may be a necessary step in the assessment of
cells that could be used as part of a cell-based gene therapy
technique or, alternatively, to test the effect of compounds on the
expression of the TR21 gene.
[0353] For example, antibodies, or fragments of antibodies, such as
those described herein, may be used to quantitatively or
qualitatively detect the presence of TR21 gene products or
conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody coupled with light
microscopic, flow cytometric, or fluorimetric detection.
[0354] The antibodies (or fragments thereof) or TR21
polynucleotides or polypeptides, may additionally be employed
histologically, as in immunofluorescence, immunoelectron microscopy
or non-immunological assays, for in situ detection of TR21 gene
products or conserved variants or peptide fragments thereof, or for
Neutrokine-alpha and/or Neutrokine-alpha SV binding to TR21. In
situ detection may be accomplished by removing a histological
specimen from a patient, and applying thereto a labeled antibody or
TR21 polypeptide of the present invention. The antibody (or
fragment) or TR21 polypeptide is preferably applied by overlaying
the labeled antibody (or fragment) onto a biological sample.
Through the use of such a procedure, it is possible to determine
not only the presence of the TR21 gene product, or conserved
variants or peptide fragments, or TR21 polypeptide binding, but
also its distribution in the examined tissue. Using the present
invention, those of ordinary skill will readily perceive that any
of a wide variety of histological methods (such as staining
procedures) can be modified in order to achieve such in situ
detection.
[0355] Immunoassays and non-immunoassays for TR21 gene products or
conserved variants or peptide fragments thereof will typically
comprise incubating a sample, such as a biological fluid, a tissue
extract, freshly harvested cells, or lysates of cells which have
been incubated in cell culture, in the presence of a detectably
labeled antibody capable of identifying TR21 gene products or
conserved variants or peptide fragments thereof, and detecting the
bound antibody by any of a number of techniques well-known in the
art.
[0356] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled an anti-TR21 antibody or detectable
polypeptide. The solid phase support may then be washed with the
buffer a second time to remove unbound antibody or polypeptide.
Optionally the antibody is subsequently labeled. The amount of
bound label on solid support may then be detected by conventional
means.
[0357] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0358] The binding activity of a given lot of anti-TR21 antibody or
TR21 polypeptide may be determined according to well-known methods.
Those skilled in the art will be able to determine operative and
optimal assay conditions for each determination by employing
routine experimentation.
[0359] In addition to assaying TR21 polypeptide levels or
polynucleotide levels in a biological sample obtained from an
individual, TR21 polypeptides or polynucleotides can also be
detected in vivo by imaging. For example, in one embodiment of the
invention, TR21 polypeptide and/or anti-TR21 antibody is used to
image B cell lymphomas. In another embodiment, TR21 polypeptides
and/or anti-TR21 antibodies and/or TR21 polynucleotides of the
invention (e.g., polynucleotides complementary to all or a portion
of TR21 mRNA) is used to image lymphomas (e.g., monocyte and B cell
lymphomas).
[0360] Antibody labels or markers for in vivo imaging of TR21
polypeptide include those detectable by X-radiography, NMR, MRI,
CAT-scans or ESR. For X-radiography, suitable labels include
radioisotopes such as barium or cesium, which emit detectable
radiation but are not overtly harmful to the subject. Suitable
markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma. Where in vivo imaging is used to detect enhanced levels
of TR21 polypeptide for diagnosis in humans, it may be preferable
to use human antibodies or "humanized" chimeric monoclonal
antibodies. Such antibodies can be produced using techniques
described herein or otherwise known in the art. For example methods
for producing chimeric antibodies are known in the art. See, for
review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et
al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature
312:643 (1984); Neuberger et al., Nature 314:268 (1985).
[0361] Additionally, any TR21 polypeptide whose presence can be
detected can be administered. For example, TR21 polypeptides
labeled with a radio-opaque or other appropriate compound can be
administered and visualized in vivo, as discussed, above for
labeled antibodies. Further such TR21 polypeptides can be utilized
for in vitro diagnostic procedures.
[0362] A TR21 polypeptide-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging
moiety, such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc, (.sup.131I, .sup.125I, .sup.123I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.115mIn, .sup.113mIn, .sup.112In, .sup.111In), and technetium
(.sup.99Tc, .sup.99mTc), thallium (.sup.201Ti), gallium (.sup.68Ga,
.sup.67Ga), palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon
(.sup.133Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu,
.sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho,
.sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr,
.sup.105Rh, .sup.97Ru), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (for
example, parenterally, subcutaneously or intraperitoneally) into
the mammal to be examined for immune system disorder. It will be
understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to
produce diagnostic images. In the case of a radioisotope moiety,
for a human subject, the quantity of radioactivity injected will
normally range from about 5 to 20 millicuries of .sup.99mTc. The
labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain TR21 protein. In
vivo tumor imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
[0363] With respect to antibodies, one of the ways in which the
anti-TR21 antibody can be detectably labeled is by linking the same
to an enzyme and using the linked product in an enzyme immunoassay
(EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)",
1978, Diagnostic Horizons 2:1-7, Microbiological Associates
Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523
(1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca
Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme
Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is bound to the
antibody will react with an appropriate substrate, preferably a
chromogenic substrate, in such a manner as to produce a chemical
moiety which can be detected, for example, by spectrophotometric,
fluorimetric or by visual means. Enzymes which can be used to
detectably label the antibody include, but are not limited to,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. Additionally, the detection can be
accomplished by colorimetric methods which employ a chromogenic
substrate for the enzyme. Detection may also be accomplished by
visual comparison of the extent of enzymatic reaction of a
substrate in comparison with similarly prepared standards.
[0364] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect TR21
through the use of a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by means including, but not
limited to, a gamma counter, a scintillation counter, or
autoradiography.
[0365] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and
fluorescamine.
[0366] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0367] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0368] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling include, but are not limited to,
luciferin, luciferase and aequorin.
Treatment of Immune System-Related Disorders
[0369] As noted above, TR21 polynucleotides and polypeptides (e.g.,
TR21 extracellular domain-Fc fusion proteins), and anti-TR21
antibodies, are useful for diagnosis of conditions involving
abnormally high or low expression of TR21 activities. For example,
given the cells and tissues where TR21 is expressed as well as the
activities modulated by TR21, it is readily apparent that a
substantially altered (increased or decreased) level of expression
of TR21 in an individual compared to the standard or "normal" level
may produce pathological conditions related to the bodily system(s)
in which TR21 is expressed and/or is active.
[0370] It will also be appreciated by one of ordinary skill that,
since the TR21 polypeptides of the invention are members of the
TNFR family, the extracellular domains of the respective proteins
may be released in soluble form from the cells which express TR21
by proteolytic cleavage and therefore, when TR21 polypeptide
(particularly a soluble form of the respective extracellular
domains) is added from an exogenous source to cells, tissues or the
body of an individual, the polypeptide may inhibit the modulating
activities of its ligand (e.g., Neutrokine-alpha) on any of its
target cells of that individual. Also, cells expressing this type
III transmembrane protein may be added to cells, tissues or the
body of an individual whereby the added cells will bind to cells
expressing TR21 ligands (e.g., Neutrokine-alpha) whereby the cells
expressing the TR21 ligand (e.g., Neutrokine-alpha) can cause
actions (e.g., proliferation or cytotoxicity) on the ligand-bearing
target cells.
[0371] The present invention is further directed to TR21 based
therapies which involve administering TR21 based therapeutic
compounds of the invention to an animal, preferably a mammal, and
most preferably a human, patient for treating one or more of the
diseases, disorders, or conditions disclosed herein. Therapeutic
compounds of the invention include, but are not limited to, TR21
polypeptides (including fragments and variants of TR21
polypeptides), polynucleotides encoding these polypeptides,
antibodies that bind these polypeptides, and agonists and/or
antagonists of these polypeptides, polynucleotides and antibodies.
The TR21 polypeptides, polynucleotides, and antibodies of the
invention can be used to treat, ameliorate or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of Neutrokine-alpha, Neutrokine-alpha SV (See, e.g., U.S.
Pat. No. 5,969,102; and von Bulow et al., Science 278:138-141
(1997)), and/or TR21, including, but not limited to, any one or
more of the diseases, disorders, or conditions described herein.
The treatment and/or prevention of diseases, disorders, or
conditions associated with aberrant expression and/or activity of
Neutrokine-alpha, Neutrokine-alpha SV, and/or TR21, includes, but
is not limited to, alleviating symptoms associated with those
diseases, disorders or conditions. TR21 compositions of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0372] The TR21 polypeptides, polynucleotides, and antibodies of
the invention that function as agonists or antagonists of
Neutrokine alpha, Neutrokine-alpha SV and/or heteromultimeric
polypeptide complexes comprising one or more copies of
Neutrokine-alpha and/or Neutrokine-alpha SV, preferably of signal
transduction induced by Neutrokine alpha, Neutrokine-alpha SV
and/or heteromultimeric polypeptide complexes comprising one or
more copies of Neutrokine-alpha and/or Neutrokine-alpha SV, can be
administered to an animal to treat, prevent or ameliorate a disease
or disorder associated with aberrant expression of Neutrokine
alpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide
complexes comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV, lack of function of Neutrokine alpha,
Neutrokine-alpha SV and/or heteromultimeric polypeptide complexes
comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV, aberrant TR21 expression, aberrant TACI and/or
BCMA expression, lack of TR21 function and/or lack of TACI and/or
BCMA function. For example, TR21 polypeptides of the invention
which disrupt the interaction between TR21 and one or more of its
ligands may be administered to an animal to treat, prevent or
ameliorate a disease or disorder associated with aberrant
expression of Neutrokine alpha, Neutrokine-alpha SV and/or
heteromultimeric polypeptide complexes comprising one or more
copies of Neutrokine-alpha and/or Neutrokine-alpha SV, excessive
function of Neutrokine alpha, Neutrokine-alpha SV and/or
heteromultimeric polypeptide complexes comprising one or more
copies of Neutrokine-alpha and/or Neutrokine-alpha SV, aberrant
TR21 expression, aberrant TACI and/or BCMA expression, excessive
TR21 function or excessive TACI and/or BCMA function.
[0373] In a preferred embodiment, TR21 polypeptides of the
invention neutralize activity of Neutrokine alpha, Neutrokine-alpha
SV and/or heteromultimeric polypeptide complexes comprising one or
more copies of Neutrokine-alpha and/or Neutrokine-alpha SV. In
another preferred embodiment, TR21 polypeptides of the invention
inhibit B cell proliferation. In another preferred embodiment, TR21
polypeptides of the invention inhibit immunoglobulin production by
B cells.
[0374] In a preferred embodiment, TR21 polypeptides of the
invention (including TR21 fragments and variants, and anti-TR21
antibodies) inhibit or reduce binding of the soluble form of
Neutrokine alpha, Neutrokine-alpha SV and/or heteromultimeric
polypeptide complexes comprising one or more copies of
Neutrokine-alpha and/or Neutrokine-alpha SV to a Neutrokine alpha
receptor (e.g., TR21 and/or TACI and/or BCMA). In another preferred
embodiment TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) inhibit or reduce
B cell proliferation induced by the soluble form of Neutrokine
alpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide
complexes comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV. In another preferred embodiment TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) inhibit or reduce
immunoglobulin production induced by the soluble form of Neutrokine
alpha, Neutrokine-alpha SV and/or heteromultimeric polypeptide
complexes comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV. In another preferred embodiment TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) inhibit or reduce
immunoglobulin production in response to T cell dependent
immunogens. In another preferred embodiment TR21 polypeptides of
the invention (including TR21 fragments and variants, and anti-TR21
antibodies) inhibit or reduce immunoglobulin production in response
to T cell independent immunogens.
[0375] In another preferred embodiment TR21 polypeptides of the
invention (including TR21 fragments and variants, and anti-TR21
antibodies) promote or enhance B cell proliferation induced by the
soluble form of Neutrokine alpha, Neutrokine-alpha SV and/or
heteromultimeric polypeptide complexes comprising one or more
copies of Neutrokine-alpha and/or Neutrokine-alpha SV. In another
preferred embodiment TR21 polypeptides of the invention (including
TR21 fragments and variants, and anti-TR21 antibodies) increase or
enhance immunoglobulin production induced by the soluble form of
Neutrokine alpha, Neutrokine-alpha SV and/or heteromultimeric
polypeptide complexes comprising one or more copies of
Neutrokine-alpha and/or Neutrokine-alpha SV. In another preferred
embodiment TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) increase or
enhance immunoglobulin production in response to T cell dependent
immunogens. In another preferred embodiment TR21 polypeptides of
the invention (including TR21 fragments and variants, and anti-TR21
antibodies) increase or enhance immunoglobulin production in
response to T cell independent immunogens.
[0376] In one embodiment, the invention provides a method of
delivering radiolabeled TR21 (including TR21 fragments and
variants, and anti-TR21 antibodies) polypeptide conjugates of the
invention to targeted cells, such as, for example, monocytic cells
expressing membrane-bound forms of Neutrokine alpha,
Neutrokine-alpha SV and/or heteromultimeric polypeptide complexes
comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV.
[0377] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing immunoglobulin production
(e.g. IgM, IgG, and/or IgA production), comprising, or
alternatively consisting of, contacting an effective amount of TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) with Neutrokine alpha,
Neutrokine-alpha SV and/or heteromultimeric polypeptide complexes
comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV, wherein the effective amount of TR21
polypeptide inhibits or reduces immunoglobulin production mediated
by Neutrokine alpha, Neutrokine-alpha SV and/or heteromultimeric
polypeptide complexes comprising one or more copies of
Neutrokine-alpha and/or Neutrokine-alpha SV. In another embodiment,
the invention provides methods and compositions for inhibiting or
reducing immunoglobulin production (e.g. IgM, IgG, and/or IgA
production), comprising, or alternatively consisting of,
administering to an animal in which such inhibition or reduction is
desired, TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) in an amount
effective to inhibit or reduce immunoglobulin production.
[0378] In another embodiment, the invention provides methods and
compositions for stimulating immunoglobulin production (e.g. IgM,
IgG, and/or IgA production), comprising, or alternatively
consisting of, contacting an effective amount of TR21 polypeptides
of the invention (including TR21 fragments and variants, and
anti-TR21 antibodies) with Neutrokine alpha, Neutrokine-alpha SV
and/or heteromultimeric polypeptide complexes comprising one or
more copies of Neutrokine-alpha and/or Neutrokine-alpha SV, wherein
the effective amount of the TR21 polypeptide stimulates
immunoglobulin production mediated by Neutrokine alpha,
Neutrokine-alpha SV and/or heteromultimeric polypeptide complexes
comprising one or more copies of Neutrokine-alpha and/or
Neutrokine-alpha SV. In another embodiment, the invention provides
methods and compositions for stimulating immunoglobulin production
(e.g. IgM, IgG, and/or IgA production) comprising, or alternatively
consisting of, administering to an animal in which such stimulation
is desired, a TR21 polypeptide of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) in an amount
effective to stimulate immunoglobulin production. Determination of
immunoglobulin levels are most often performed by comparing the
level of immunoglobulin in a sample to a standard containing a
known amount of immunoglobulin using ELISA assays. Determination of
immunoglobulin levels in a given sample can readily be determined
using ELISA or other method known in the art.
[0379] Investigation of Neutrokine-alpha induced signaling in human
tonsillar B cells co-stimulated with Staphylococcus aureus Cowan
consistently revealed that mRNA for ERK-1 and PLK were upregulated
by treatment with Neutrokine-alpha combined with SAC (data not
shown). Polo like kinases (PLK) belong to a sub family of
serine/threonine kinases related to the Saccharomyces cerevisiae
cell cycle protein CDC5. The expression of PLK is induced during G2
and S phase of the cell cycle. PLK is reported to play a role in
cell proliferation (See e.g., Lee et al., Proc. Natl. Acad. Sci.
95:9301-9306). The role of extracellular-signal related kinases 1
and 2 (ERK1 and 2) in cell survival and proliferation in response
to stimulation by growth factors and other agonists has been
extensively studied. The induced expression of PLK and ERK-1 is
consistent with the survival and proliferative effects of
Neutrokine-alpha on B cells.
[0380] Additionally, in some samples of human tonsillar B cells
stimulated with Neutrokine-alpha and SAC, mRNA for CD25 (IL-2R
alpha) was upregulated. Nuclear extracts from human tonsillar B
cells treated with Neutrokine-alpha and from IM-9 cells treated
with Neutrokine-alpha were able to shift probes from the CD25
promoter region containing sites for NF-kappaB, SRF, ELF-1 and
HMGI/Y in an electromobility shift assay. ELF-1 for example, is a
transcription factor that is part of the ETS family of proteins and
whose expression appears to be restricted to T and B cells. Binding
sites for ELF-1 have been described in the promoters of a number of
proteins that are important in the regulation of the immune
response.
[0381] Thus Neutrokine-alpha induced signaling has been shown to be
consistent with the activation of cellular activation and cellular
proliferation pathways as well as with cellular signaling pathways
that regulate B cell lifespan. Further, Neutrokine-alpha treatment
of B cells induces cellular proliferation and immunoglobulin
secretion, a characteristic of activation of B cells (Moore et al.,
Science 285:260-263, 1999). TR21 polypeptides complexed with
Neutrokine-alpha may inhibit, stimulate, or not significantly alter
these Neutrokine-alpha mediated activities.
[0382] In one embodiment, the invention provides methods and
compositions for inhibiting or reducing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha, wherein the effective amount of TR21 polypeptide
inhibits or reduces Neutrokine-alpha mediated B cell
proliferation.
[0383] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha SV, wherein the effective amount of TR21
polypeptide inhibits or reduces Neutrokine-alpha SV mediated B cell
proliferation.
[0384] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount of TR21 polypeptide inhibits or reduces B cell
proliferation mediated by said hetermiultimeric polypeptide
complexes.
[0385] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing B cell proliferation
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a TR21
polypeptide in an amount effective to inhibit or reduce B cell
proliferation.
[0386] In a further embodiment, the invention provides methods and
compositions for stimulating or increasing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha, wherein the effective amount of TR21 polypeptide
stimulates or increases Neutrokine-alpha mediated B cell
proliferation.
[0387] In another embodiment, the invention provides methods and
compositions for stimulating or increasing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha SV, wherein the effective amount of TR21
polypeptide stimulates or increases Neutrokine-alpha SV mediated B
cell proliferation.
[0388] In another embodiment, the invention provides methods and
compositions for stimulating or increasing B cell proliferation,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount of TR21 polypeptide stimulates or increases B
cell proliferation mediated by said hetermiultimeric polypeptide
complexes.
[0389] In another embodiment, the invention provides methods and
compositions for stimulating or increasing B cell proliferation
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a TR21
polypeptide in an amount effective to stimulate or increase B cell
proliferation.
[0390] In yet another embodiment, the invention provides methods
and compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha, wherein the effective amount TR21 polypeptides
inhibits or reduces Neutrokine-alpha mediated B cell
activation.
[0391] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha SV, wherein the effective amount TR21 polypeptides
inhibits or reduces Neutrokine-alpha SV mediated B cell
activation.
[0392] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount TR21 polypeptides inhibits or reduces B cell
activation mediated by said heteromultimeric polypeptide
complexes.
[0393] In another embodiment, the invention provides methods and
compositions for inhibiting or reducing activation of B cells,
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a TR21
polypeptide in an amount effective to inhibit or reduce B cell
activation.
[0394] In yet another embodiment, the invention provides methods
and compositions for stimulating or increasing activation of B
cells, comprising, or alternatively consisting of, contacting an
effective amount of TR21 polypeptides of the invention (including
TR21 fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha, wherein the effective amount TR21 polypeptides
stimulates or increases Neutrokine-alpha mediated B cell
activation.
[0395] In another embodiment, the invention provides methods and
compositions for stimulating or increasing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha SV, wherein the effective amount TR21 polypeptides
stimulates or increases Neutrokine-alpha SV mediated B cell
activation.
[0396] In another embodiment, the invention provides methods and
compositions for stimulating or increasing activation of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount TR21 polypeptides stimulates or increases B
cell activation mediated by said heteromultimeric polypeptide
complexes.
[0397] In another embodiment, the invention provides methods and
compositions for stimulating or increasing activation of B cells,
comprising, or alternatively consisting of, administering to an
animal in which such inhibition or reduction is desired, a TR21
polypeptide in an amount effective to stimulate or increase B cell
activation.
[0398] B cell activation can measured in a variety of ways, such as
FACS analysis of activation markers expressed on B cells. B cells
activation markers include, but are not limited to, CD26, CD28,
CD30, CD38, CD39, CD69, CD70, CD71, CD 77, CD 83, CD126, CDw130,
and B220. Additionally, B cell activation may be measured by
analysis of the activation of signaling molecules involved in B
cell activation. By way of non-limiting example, such analysis may
take the form of analyzing mRNA levels of signaling molecules by
Northern analysis or real time PCR. One can also measure, for
example, the phosphorylation of signaling molecules using
anti-phosphotyrosine antibodies in a Western blot. B cell
activation may also be measured by measuring the calcium levels in
B cells. These and other methods of determining B cell activation
are commonly known in the art and could be routinely adapted for
the use of determining the effect of TR21 polypeptides of the
invention on B cell activation.
[0399] In yet another embodiment, the invention provides methods
and compositions for reducing or decreasing lifespan of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha, wherein the effective amount of TR21 polypeptide
reduces or decreases Neutrokine-alpha regulated lifespan of B
cells.
[0400] In another embodiment, the invention provides methods and
compositions for reducing or decreasing lifespan of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with
Neutrokine-alpha SV, wherein the effective amount of TR21
polypeptide reduces or decreases Neutrokine-alpha SV regulated
lifespan of B cells.
[0401] In another embodiment, the invention provides methods and
compositions for reducing or decreasing lifespan of B cells,
comprising, or alternatively consisting of, contacting an effective
amount of TR21 polypeptides of the invention (including TR21
fragments and variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount of TR21 polypeptide reduces or decreases
lifespan of B cells regulated by said heteromultimeric polypeptide
complexes.
[0402] In another embodiment, the invention provides methods and
compositions for reducing or decreasing lifespan of B cells,
comprising, or alternatively consisting of, administering to an
animal in which such decrease is desired, a TR21 polypeptide of the
invention in an amount effective to reduce or decrease B cell
lifespan.
[0403] In a further embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) with Neutrokine-alpha, wherein
the effective amount of TR21 polypeptides increases
Neutrokine-alpha regulated lifespan of B cells.
[0404] In another embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) with Neutrokine-alpha SV,
wherein the effective amount of TR21 polypeptides increases
Neutrokine-alpha SV regulated lifespan of B cells.
[0405] In another embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, contacting an effective amount of TR21
polypeptides of the invention (including TR21 fragments and
variants, and anti-TR21 antibodies) with one or more
heteromultimeric polypeptide complexes comprising one or more
Neutrokine-alpha and/or Neutrokine-alpha SV polypeptides, wherein
the effective amount of TR21 polypeptide increases lifespan of B
cells regulated by said heteromultimeric polypeptide complexes.
[0406] In one embodiment, the invention provides methods and
compositions for increasing lifespan of B cells, comprising, or
alternatively consisting of, administering to an animal in which
such increase is desired, a TR21 polypeptide of the invention
(e.g., anti-TR21 antibody) in an amount effective to increase
lifespan of B cells.
[0407] B cell lifespan in vivo may be measured by
5-bromo-2'-deoxyuridine (BrdU) labeling experiments which are well
known to one skilled in the art. BrdU is a thymidine analogue that
gets incorporated into the DNA of dividing cells. Cells containing
BrdU in their DNA can be detected using, for example fluorescently
labeled anti-BrdU antibody and flow cytometry. Briefly, an animal
is injected with BrdU in an amount sufficient to label developing B
cells. Then, a sample of B cells is withdrawn from the animal, for
example, from peripheral blood, and analyzed for the percentage of
cells that contain BrdU. Such an analysis performed at several time
points can be used to calculate the halflife of B cells.
Alternatively, B cell survival may be measured in vitro. For
example, B cells may be cultured under conditions where
proliferation does not occur, (for example the media should contain
no reagents that crosslink the immunoglobulin receptor, such as
anti-IgM antibodies) for a period of time (usually 2-4 days). At
the end of this time, the percent of surviving cells is determined,
using for instance, the vital dye Trypan Blue, or by staining cells
with propidium iodide or any other agent designed to specifically
stain apoptotic cells and analyzing the percentage of cells stained
using flow cytometry. One could perform this experiment under
several conditions, such as B cells treated with Neutrokine-alpha,
B cells treated with Neutrokine-alpha SV, B cells treated with
Neutrokine-alpha/TR21 complexes, B cells treated with
Neutrokine-alpha SV/TR21 complexes, and untreated B cells in order
to determine the effects of Neutrokine-alpha, Neutrokine-alpha SV
and TR21 polypeptides on B cell survival. These and other methods
for determining B cell lifespan are commonly known in the art and
could routinely be adapted to determining the effect of TR21
polypeptides on Neutrokine-alpha and/or Neutrokine-alpha SV
regulation of B cell lifespan.
[0408] In one embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing TR21 polypeptides or
anti-TR21 antibodies associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs) to targeted cells,
such as, for example, monocytic cells expressing TR21 ligand (e.g.,
Neutrokine-alpha and/or Neutrokine-alpha SV), or B cells expressing
TR21. TR21 polypeptides (e.g., soluble TR21 extracellular domain or
fragments thereof) or anti-TR21 antibodies of the invention may be
associated with heterologous polypeptides, heterologous nucleic
acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic
and/or covalent interactions.
[0409] In one embodiment, the invention provides a method for the
specific delivery of compositions of the invention to cells by
administering polypeptides of the invention (e.g., TR21
polypeptides or anti-TR21 antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the
invention provides a method for delivering a therapeutic protein
into the targeted cell. In another example, the invention provides
a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA
that can integrate into the cell's genome or replicate episomally
and that can be transcribed) into the targeted cell.
[0410] In another embodiment, the invention provides a method for
the specific destruction of cells (e.g., the destruction of tumor
cells) by administering polypeptides of the invention (e.g., TR21
polypeptides or anti-TR21 antibodies) in association with toxins or
cytotoxic prodrugs.
[0411] In a specific embodiment, the invention provides a method
for the specific destruction of cells of monocytic lineage (e.g.,
monocytic cell related leukemias or lymphomas, such as, for example
acute myelogenous leukemia) by administering TR21 polypeptides
(e.g., a soluble fragment of the TR21 extracellular domain) and/or
anti-TR21 antibodies) in association with toxins or cytotoxic
prodrugs.
[0412] In another specific embodiment, the invention provides a
method for the specific destruction of cells of B cell lineage
(e.g., B cell related leukemias or lymphomas such as chronic
lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, and
Hodgkin's disease) by administering anti-TR21 antibodies in
association with toxins or cytotoxic prodrugs.
[0413] By "toxin" is meant compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins,
modified toxins, catalytic subunits of toxins, cytotoxins
(cytotoxic agents), or any molecules or enzymes not normally
present in or on the surface of a cell that under defined
conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not
limited to, radioisotopes known in the art, compounds such as, for
example, antibodies (or complement fixing containing portions
thereof) that bind an inherent or induced endogenous cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein,
alpha-sarcin and cholera toxin. "Toxin" also includes a cytostatic
or cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha-emitters such as, for example, .sup.213Bi, or other
radioisotopes such as, for example, .sup.103Pd, .sup.133Xe,
.sup.131I, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.35S, .sup.90Y, .sup.153Sm, .sup.153Gd, .sup.169Yb, .sup.51Cr,
.sup.54Mn, .sup.75Se, .sup.113Sn, .sup.90Yttrium, .sup.117Tin,
.sup.186Rhenium, .sup.166Holmium, and .sup.188Rhenium; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin.
[0414] Techniques known in the art may be applied to label
antibodies of the invention. Such techniques include, but are not
limited to, the use of bifunctional conjugating agents (see e.g.,
U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361;
5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003; the contents of each of which are hereby
incorporated by reference in its entirety). A cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0415] By "cytotoxic prodrug" is meant a non-toxic compound that is
converted by an enzyme, normally present in the cell, into a
cytotoxic compound. Cytotoxic prodrugs that may be used according
to the methods of the invention include, but are not limited to,
glutamyl derivatives of benzoic acid mustard alkylating agent,
phosphate derivatives of etoposide or mitomycin C, cytosine
arabinoside, daunorubisin, and phenoxyacetamide derivatives of
doxorubicin.
[0416] In specific embodiments, TR21 and/or anti-TR21 polypeptides
in association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of autoimmune diseases. In
preferred embodiments, TR21 and/or anti-TR21 polypeptides in
association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of systemic lupus
erythematosus. In further preferred embodiments, TR21 and/or
anti-TR21 polypeptides in association with radioisotopes, toxins or
cytotoxic prodrugs are used to treat or ameliorate the symptoms of
rheumatoid arthritis, including advanced rheumatoid arthritis. In
preferred embodiments, TR21 and/or anti-TR21 polypeptides in
association with radioisotopes, toxins or cytotoxic prodrugs are
used to treat or ameliorate the symptoms of idiopathic
thrombocytopenic purpura (ITP).
[0417] In other preferred embodiments TR21 and/or anti-TR21
polypeptides in association with radioisotopes, toxins or cytotoxic
prodrugs are used to treat or ameliorate the symptoms of Sjogren's
syndrome. In other preferred embodiments, TR21 and/or anti-TR21
polypeptides in association with radioisotopes, toxins or cytotoxic
prodrugs are used to treat or ameliorate the symptoms of IgA
nephropathy. In other preferred embodiments, TR21 and/or anti-TR21
polypeptides in association with radioisotopes, toxins or cytotoxic
prodrugs are used to treat or ameliorate the symptoms of Myasthenia
gravis. In preferred embodiments, TR21 and/or anti-TR21
polypeptides in association with radioisotopes, toxins or cytotoxic
prodrugs are used to treat or ameliorate the symptoms of multiple
sclerosis.
[0418] It will be appreciated that conditions caused by a decrease
in the standard or normal level of TR21 activity or TR21 ligand
(e.g., Neutrokine-alpha) activity in an individual, particularly
disorders of the immune system (e.g., an immunodeficiency), can be
treated by administration of TR21 polypeptide (e.g., in the form of
soluble extracellular domain or fragments thereof, or cells
expressing the complete protein) or agonist. Thus, the invention
also provides a method of treatment of an individual in need of an
increased level of TR21 activity or TR21 ligand activity comprising
administering to such an individual a pharmaceutical composition
comprising an amount of an isolated TR21 polypeptide of the
invention, or agonist thereof, effective to increase the TR21
activity level in such an individual.
[0419] It will also be appreciated that conditions caused by a
increase in the standard or normal level of TR21 activity or TR21
ligand (e.g., Neutrokine-alpha) activity in an individual,
particularly disorders of the immune system (e.g., autoimmune
diseases, such as, for example, lupus, rheumatoid arthritis, and
myasthenia gravis), can be treated by administration of TR21
polypeptides (e.g., in the form of soluble extracellular domain or
fragments thereof, or cells expressing the complete protein) or
antagonist (e.g., an anti-TR21 antibody). Thus, the invention also
provides a method of treatment of an individual in need of a
decreased level of TR21 activity or TR21 ligand activity comprising
administering to such an individual a pharmaceutical composition
comprising an amount of an isolated TR21 polypeptide of the
invention, or antagonist thereof, effective to decrease the TR21
activity level in such an individual.
[0420] Autoantibody production is common to several autoimmune
diseases and contributes to tissue destruction and exacerbation of
disease. Autoantibodies can also lead to the occurrence of immune
complex deposition complications and lead to many symptoms of
systemic lupus erythematosis, including kidney failure, neuralgic
symptoms and death. Modulating antibody production independent of
cellular response would also be beneficial in many disease states.
B cells have also been shown to play a role in the secretion of
arthritogenic immunoglobulins in rheumatoid arthritis, (Korganow et
al., Immunity 10:451-61, 1999). As such, inhibition of
Neutrokine-alpha mediated antibody production would be beneficial
in treatment of autoimmune diseases such as myasthenia gravis and
rheumatoid arthritis. Compounds of the invention that selectively
block or neutralize the action of B-lymphocytes would be useful for
such purposes. To verify these capabilities in compositions of the
present invention, such compositions are evaluated using assays
known in the art and described herein.
[0421] The invention provides methods employing compositions of the
invention (e.g., TR21 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof) for
selectively blocking or neutralizing the actions of B-cells in
association with end stage renal diseases, which may or may not be
associated with autoimmune diseases. Such methods would also be
useful for treating immunologic renal diseases. Such methods would
be would be useful for treating glomerulonephritis associated with
diseases such as membranous nephropathy, IgA nephropathy or
Berger's Disease, IgM nephropathy, Goodpasture's Disease,
post-infectious glomerulonephritis, mesangioproliferative disease,
minimal-change nephrotic syndrome. Such methods would also serve as
therapeutic applications for treating secondary glomerulonephritis
or vasculitis associated with such diseases as lupus,
polyarteritis, Henloch-Scoenlein, Scleroderma, HIV-related
diseases, amyloidosis or hemolytic uremic syndrome. The methods of
the present invention would also be useful as part of a therapeutic
application for treating interstitial nephritis or pyelonephritis
associated with chronic pyelonephritis, analgesic abuse,
nephrocalcinosis, nephropathy caused by other agents,
nephrolithiasis, or chronic or acute interstitial nephritis.
[0422] The methods of the present invention also include use of
compositions of the invention in the treatment of hypertensive or
large vessel diseases, including renal artery stenosis or occlusion
and cholesterol emboli or renal emboli.
[0423] The present invention also provides methods for diagnosis
and treatment of renal or urological neoplasms, multiple mylelomas,
lymphomas, light chain neuropathy or amyloidosis.
[0424] The invention also provides methods for blocking or
inhibiting activated B cells using compositions of the invention
for the treatment of asthma and other chronic airway diseases such
as bronchitis and emphysema.
[0425] TR21 polynucleotides or polypeptides of the invention, or
agonists of TR21 (e.g., anti-TR21 agonistic antibodies), can be
used in the treatment of infectious agents. For example, by
increasing the immune response, particularly increasing the
proliferation and differentiation of B cells, infectious diseases
may be treated. The immune response may be increased by either
enhancing an existing immune response, or by initiating a new
immune response. Alternatively TR21 polynucleotides or polypeptides
of the invention, or agonists of TR21 (e.g., anti-TR21 agonistic
antibodies), may also directly inhibit the infectious agent,
without necessarily eliciting an immune response.
[0426] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated, prevented, and/or
diagnosed by TR21 polynucleotides or polypeptides of the invention,
or agonists of TR21 (e.g., anti-TR21 agonistic antibodies).
Examples of viruses, that can be treated, prevented, and/or
diagnosed with the compositions of the invention include, but are
not limited to one or more of the following DNA and RNA viruses and
viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae
(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes
Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,
Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,
Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,
Parvoviridae, Picomaviridae, Poxviridae (such as Smallpox or
Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I,
HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses
falling within these families can cause a variety of diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A,
B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin,
Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's
Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,
sexually transmitted diseases, skin diseases (e.g., Kaposi's,
warts), and viremia. TR21 polynucleotides or polypeptides, or
agonists or antagonists of TR21, can be used to treat, prevent,
diagnose, and/or detect any of these symptoms or diseases. In
specific embodiments, TR21 polynucleotides or polypeptides, or
agonists of TR21 are used to treat, prevent, and/or diagnose:
meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In
an additional specific embodiment TR21 polynucleotides,
polypeptides, or agonists are used to treat patients nonresponsive
to one or more other commercially available hepatitis vaccines. In
a further specific embodiment, TR21 polynucleotides, polypeptides,
or agonists are used to treat, prevent, and/or diagnose AIDS. In an
additional specific embodiment TR21 polynucleotides, polypeptides,
agonists, and/or antagonists are used to treat, prevent, and/or
diagnose patients with cryptosporidiosis.
[0427] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated, prevented, and/or diagnosed by
TR21 polynucleotides or polypeptides, or agonists or antagonists of
TR21, include, but not limited to, one or more of the following
Gram-Negative and Gram-positive bacteria and bacterial families and
fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,
Nocardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae
(e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,
Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis,
Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and
Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella,
Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi),
Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis,
Leptospirosis, Listeria (e.g., Listeria monocytogenes),
Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Neisseria meningitidis, Pasteurellacea Infections (e.g.,
Actinobacillus, Haemophilus (e.g., Haemophilus influenza type B),
Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,
Shigella spp., Staphylococcal, Meningococcal, Pneumococcal and
Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the
following diseases or symptoms, including, but not limited to:
bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g.,
AIDS related infections), paronychia, prosthesis-related
infections, Reiter's Disease, respiratory tract infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid,
pneumonia, Gonorrhea, meningitis (e.g., meningitis types A and B),
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. TR21 polynucleotides or polypeptides,
or agonists or antagonists of TR21, can be used to treat, prevent,
diagnose, and/or detect any of these symptoms or diseases. In
specific embodiments, TR21 polynucleotides, polypeptides, or
agonists thereof are used to treat, prevent, and/or diagnose:
tetanus, Diphtheria, botulism, and/or meningitis type B.
[0428] Moreover, parasitic agents causing disease or symptoms that
can be treated, prevented, and/or diagnosed by TR21 polynucleotides
or polypeptides, or agonists or antagonists of TR21, include, but
not limited to, a member of one or more of the following families
or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and
Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium
falciparium, Plasmodium malariae and Plasmodium ovale). These
parasites can cause a variety of diseases or symptoms, including,
but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal disease (e.g., dysentery, giardiasis), liver disease,
lung disease, opportunistic infections (e.g., AIDS related),
malaria, pregnancy complications, and toxoplasmosis. TR21
polynucleotides or polypeptides, or agonists or antagonists of
TR21, can be used to treat, prevent, diagnose, and/or detect any of
these symptoms or diseases. In specific embodiments, TR21
polynucleotides, polypeptides, or agonists thereof are used to
treat, prevent, and/or diagnose malaria.
[0429] In another embodiment, TR21 polynucleotides or polypeptides
of the invention and/or agonists and/or antagonists thereof, are
used to treat, prevent, and/or diagnose inner ear infection (such
as, for example, otitis media), as well as other infections
characterized by infection with Streptococcus pneumoniae and other
pathogenic organisms.
[0430] In a specific embodiment, TR21 polynucleotides or
polypeptides, or agonists or antagonists thereof (e.g., anti-TR21
antibodies) are used to treat or prevent a disorder characterized
by deficient serum immunoglobulin production, recurrent infections,
and/or immune system dysfunction. Moreover, TR21 polynucleotides or
polypeptides, or agonists or antagonists thereof (e.g., anti-TR21
antibodies) may be used to treat or prevent infections of the
joints, bones, skin, and/or parotid glands, blood-borne infections
(e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),
autoimmune diseases (e.g., those disclosed herein), inflammatory
disorders, and malignancies, and/or any disease or disorder or
condition associated with these infections, diseases, disorders
and/or malignancies) including, but not limited to, CVID, other
primary immune deficiencies, HIV disease, CLL, multiple myeloma,
recurrent bronchitis, sinusitis, otitis media, conjunctivitis,
pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe
herpes zoster), and/or pneumocystis carnii.
[0431] TR21 polynucleotides or polypeptides of the invention, or
agonists or antagonists thereof, may be used to diagnose, prognose,
treat or prevent one or more of the following diseases or
disorders, or conditions associated therewith: primary
immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki
syndrome, bone marrow transplant (e.g., recent bone marrow
transplant in adults or children), chronic B-cell lymphocytic
leukemia, HIV infection (e.g., adult or pediatric HIV infection),
chronic inflammatory demyelinating polyneuropathy, and
post-transfusion purpura.
[0432] Additionally, TR21 polynucleotides or polypeptides of the
invention, or agonists or antagonists thereof, may be used to
diagnose, prognose, treat or prevent one or more of the following
diseases, disorders, or conditions associated therewith,
Guillain-Barre syndrome, anemia (e.g., anemia associated with
parvovirus B19, stable mutliple myeloma with high risk for
infection (e.g., recurrent infection), autoimmune hemolytic anemia
(e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia
(e.g., neonatal thrombocytopenia), and immune-mediated
neutropenia), infection through transplantation (e.g.,
cytomegalovirus (CMV)-negative recipients of CMV-positive organs),
hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with
risk factor for infection or morbidity), epilepsy (e.g.,
intractable epilepsy), systemic vasculitic syndromes, myasthenia
gravis (e.g., decompensation in myasthenia gravis),
dermatomyositis, and polymyositis.
[0433] Additional preferred embodiments of the invention include,
but are not limited to, the use of TR21 polypeptides, TR21
polynucleotides, and functional agonists or antagonists thereof,
(e.g., example an anti-TR21 agonistic antibody) in the following
applications:
[0434] Administration to an animal (e.g., mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse,
cow, sheep, dog, cat, non-human primate, and human, most preferably
human) to boost the immune system to produce increased quantities
of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE),
and/or to increase an immune response. In a specific nonexclusive
embodiment, TR21 polypeptides of the invention, and/or agonists
thereof, are administered to boost the immune system to produce
increased quantities of IgG. In another specific nonexclusive
embodiment, TR21 polypeptides of the invention and/or agonists
thereof, are administered to boost the immune system to produce
increased quantities of IgA. In another specific nonexclusive
embodiment, TR21 polypeptides of the invention and/or agonists
thereof, are administered to boost the immune system to produce
increased quantities of IgM.
[0435] Administration to an animal (including, but not limited to,
those listed above, and also including transgenic animals)
incapable of producing functional endogenous antibody molecules or
having an otherwise compromised endogenous immune system, but which
is capable of producing human immunoglobulin molecules by means of
a reconstituted or partially reconstituted immune system from
another animal (see, e.g., published PCT Application Nos.
WO98/24893, WO/9634096, WO/9633735, and WO/9110741).
[0436] A vaccine adjuvant that enhances immune responsiveness to
specific antigen. In a specific embodiment, the vaccine is a TR21
polypeptide described herein. In another specific embodiment, the
vaccine adjuvant is a polynucleotide described herein (e.g., a TR21
polynucleotide genetic vaccine adjuvant). As discussed herein, TR21
polynucleotides may be administered using techniques known in the
art, including but not limited to, liposomal delivery, recombinant
vector delivery, injection of naked DNA, and gene gun delivery.
[0437] An adjuvant to enhance tumor-specific immune responses.
[0438] An adjuvant to enhance anti-viral immune responses.
Anti-viral immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include, but are not
limited to, virus and virus associated diseases or symptoms
described herein or otherwise known in the art. In specific
embodiments, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: AIDS, meningitis,
Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific
embodiment, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or
symptom selected from the group consisting of: HIV/AIDS,
Respiratory syncytial virus, Dengue, Rotavirus, Japanese B
encephalitis, Influenza A and B, Parainfluenza, Measles,
Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever,
Herpes simplex, and yellow fever. In another specific embodiment,
the compositions of the invention are used as an adjuvant to
enhance an immune response to the HIV gp120 antigen.
[0439] An adjuvant to enhance anti-bacterial or anti-fungal immune
responses. Anti-bacterial or anti-fungal immune responses that may
be enhanced using the compositions of the invention as an adjuvant,
include bacteria or fungus and bacteria or fungus associated
diseases or symptoms described herein or otherwise known in the
art. In specific embodiments, the compositions of the invention are
used as an adjuvant to enhance an immune response to a bacterium or
fungus, disease, or symptom selected from the group consisting of:
tetanus, Diphtheria, botulism, and meningitis type B. In another
specific embodiment, the compositions of the invention are used as
an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or symptom selected from the group consisting of: Vibrio
cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella
paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group
B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium
(malaria).
[0440] An adjuvant to enhance anti-parasitic immune responses.
Anti-parasitic immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include parasite and
parasite associated diseases or symptoms described herein or
otherwise known in the art. In specific embodiments, the
compositions of the invention are used as an adjuvant to enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the invention are used as an adjuvant to enhance an
immune response to Plasmodium falciparum (malaria).
[0441] As a stimulator of B cell responsiveness to pathogens.
[0442] As an agent that elevates the immune status of an individual
prior to their receipt of immunosuppressive therapies.
[0443] As an agent to induce higher affinity antibodies.
[0444] As an agent to increase serum immunoglobulin
concentrations.
[0445] As an agent to accelerate recovery of immunocompromised
individuals.
[0446] As an agent to boost immunoresponsiveness among aged
populations.
[0447] As an immune system enhancer prior to, during, or after bone
marrow transplant and/or other transplants (e.g., allogenic or
xenogenic organ transplantation). With respect to transplantation,
compositions of the invention may be administered prior to,
concomitant with, and/or after transplantation. In a specific
embodiment, compositions of the invention are administered after
transplantation, prior to the beginning of recovery of T-cell
populations. In another specific embodiment, compositions of the
invention are first administered after transplantation after the
beginning of recovery of T cell populations, but prior to full
recovery of B cell populations.
[0448] As an agent to boost immunoresponsiveness among B cell
immunodeficient individuals, such as, for example, an individual
who has undergone a partial or complete splenectomy. B cell
immunodeficiencies that may be ameliorated or treated by
administering the TR21 polypeptides or polynucleotides of the
invention, or agonists thereof, include, but are not limited to,
severe combined immunodeficiency (SCID)-X linked, SCID-autosomal,
adenosine deaminase deficiency (ADA deficiency), X-linked
agammaglobulinemia (XLA), Bruton's disease, congenital
agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
transient hypogammaglobulinemia of infancy, unspecified
hypogammaglobulinemia, agammaglobulinemia, common variable
immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),
X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, selective IgA deficiency, IgG
subclass deficiency (with or without IgA deficiency), antibody
deficiency with normal or elevated Igs, immunodeficiency with
thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell
lymphoproliferative disorder (BLPD), selective IgM
immunodeficiency, recessive agammaglobulinemia (Swiss type),
reticular dysgenesis, neonatal neutropenia, severe congenital
leukopenia, thymic alymphoplasia-aplasia or dysplasia with
immunodeficiency, ataxia-telangiectasia, short limbed dwarfism,
X-linked lymphoproliferative syndrome (XLP), Nezelof
syndrome-combined immunodeficiency with Igs, purine nucleoside
phosphorylase deficiency (PNP), MHC Class II deficiency (Bare
Lymphocyte Syndrome) and severe combined immunodeficiency.
[0449] In a specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate selective IgA deficiency.
[0450] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate ataxia-telangiectasia.
[0451] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate common variable
immunodeficiency.
[0452] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate X-linked
agammaglobulinemia.
[0453] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate severe combined
immunodeficiency (SCID).
[0454] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate Wiskott-Aldrich syndrome.
[0455] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat or ameliorate X-linked Ig deficiency with
hyper IgM.
[0456] As an agent to boost immunoresponsiveness among individuals
having an acquired loss of B cell function. Conditions resulting in
an acquired loss of B cell function that may be ameliorated or
treated by administering TR21 polypeptides or polynucleotides of
the invention, or agonists thereof include, but are not limited to,
HIV Infection, AIDS, bone marrow transplant, multiple myeloma and B
cell chronic lymphocytic leukemia (CLL).
[0457] Patients with CLL and myeloma are at risk for increased
infections. Thus, one aspect of the present invention provides for
the use of TR21 and/or anti-TR21 polynucleotides and/or
polypeptides as an agent to boost immunoresponsiveness in CLL and
myeloma patients.
[0458] As an agent to boost immunoresponsiveness among individuals
having a temporary immune deficiency or compromised immune system.
Conditions resulting in a temporary immune deficiency/compromised
immune system that may be ameliorated or treated by administering
TR21 polypeptides or polynucleotides of the invention, or agonists
or antagonists thereof, include, but are not limited to, recovery
from viral infections (e.g., influenza), conditions associated with
malnutrition, recovery from infectious mononucleosis, or conditions
associated with stress, recovery from measles, recovery from blood
transfusion, recovery from surgery and recovery from burns.
[0459] As a regulator of antigen presentation by monocytes,
dendritic cells, T cells and/or B-cells. In one embodiment, TR21
polypeptides (in soluble, membrane-bound or transmembrane forms) or
polynucleotides enhance antigen presentation or antagonize antigen
presentation in vitro or in vivo. Moreover, in related embodiments,
this enhancement or antagonism of antigen presentation may be
useful in anti-tumor treatment or to modulate the immune
system.
[0460] As a mediator of mucosal immune responses. The expression of
Neutrokine-alpha on monocytes, the expression of TR21 on B cells,
and the responsiveness of B cells to Neutrokine-alpha suggests that
it may be involved in exchange of signals between B cells and
monocytes or their differentiated progeny. This activity is in many
ways analogous to the CD40-CD154 signaling between B cells and T
cells. TR21 may therefore be an important regulator of T cell
independent immune responses to environmental pathogens. In
particular, the unconventional B cell populations (CD5+) that are
associated with mucosal sites and responsible for much of the
innate immunity in humans may respond to TR21 thereby enhancing an
individual's protective immune status.
[0461] As an agent to direct an individual's immune system towards
development of a humoral response (i.e. TH2) as opposed to a TH1
cellular response.
[0462] As a means to induce tumor proliferation and thus make it
more susceptible to anti-neoplastic agents. For example, multiple
myeloma is a slowly dividing disease and is thus refractory to
virtually all anti-neoplastic regimens. If these cells were forced
to proliferate more rapidly their susceptibility profile would
likely change.
[0463] As a monocyte cell specific binding protein to which
specific activators or inhibitors of cell growth may be attached.
The result would be to focus the activity of such activators or
inhibitors onto normal, diseased, or neoplastic B cell
populations.
[0464] As a B cell specific binding protein to which specific
activators or inhibitors of cell growth may be attached. The result
would be to focus the activity of such activators or inhibitors
onto normal, diseased, or neoplastic B cell populations.
[0465] As a means of detecting B-lineage cells by virtue of its
specificity. This application may require labeling the protein with
biotin or other agents (e.g., as described herein) to afford a
means of detection.
[0466] As a stimulator of B cell production in pathologies such as
AIDS, chronic lymphocyte disorder and/or Common Variable
Immunodeficiency.
[0467] As part of a B cell selection device the function of which
is to isolate B cells from a heterogeneous mixture of cell types.
TR21 could be coupled to a solid support to which B cells would
then specifically bind. Unbound cells would be washed out and the
bound cells subsequently eluted. A nonlimiting use of this
selection would be to allow purging of tumor cells from, for
example, bone marrow or peripheral blood prior to transplant.
[0468] As a therapy for generation and/or regeneration of lymphoid
tissues following surgery, trauma or genetic defect.
[0469] As a gene-based therapy for genetically inherited disorders
resulting in immuno-incompetence such as observed among SCID
patients.
[0470] As an antigen for the generation of antibodies to inhibit or
enhance TR21 mediated responses.
[0471] As a means of activating monocytes/macrophages to defend
against parasitic diseases that effect monocytes such as
Leishmaniasis.
[0472] As pretreatment of bone marrow samples prior to transplant.
Such treatment would increase B cell representation and thus
accelerate recovery.
[0473] As a means of regulating secreted cytokines that are
elicited by TR21. For example, as a means of regulating secreted
cytokines that are elicited by TR21.
[0474] TR21 polypeptides or polynucleotides of the invention, or
agonists may be used to modulate IgE concentrations in vitro or in
vivo.
[0475] Additionally, TR21 polypeptides or polynucleotides of the
invention, or agonists thereof, may be used to treat, prevent,
and/or diagnose IgE-mediated allergic reactions. Such allergic
reactions include, but are not limited to, asthma, rhinitis, and
eczema.
[0476] In a specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
selective IgA deficiency.
[0477] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
ataxia-telangiectasia.
[0478] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate common
variable immunodeficiency.
[0479] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
X-linked agammaglobulinemia.
[0480] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate severe
combined immunodeficiency (SCID).
[0481] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
Wiskott-Aldrich syndrome.
[0482] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
X-linked Ig deficiency with hyper IgM. In a specific embodiment,
TR21 polypeptides or polynucleotides of the invention, or agonists
thereof, is administered to treat, prevent, diagnose, and/or
ameliorate X-linked Ig deficiency with hyper IgM.
[0483] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists or antagonists (e.g.,
anti-TR21 antibodies) thereof, is administered to treat, prevent,
and/or diagnose chronic myelogenous leukemia, acute myelogenous
leukemia, leukemia, hystiocytic leukemia, monocytic leukemia (e.g.,
acute monocytic leukemia), leukemic reticulosis, Shilling Type
monocytic leukemia, and/or other leukemias derived from monocytes
and/or monocytic cells and/or tissues.
[0484] In another specific embodiment, TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
monocytic leukemoid reaction, as seen, for example, with
tuberculosis.
[0485] In another specific embodiment TR21 polypeptides or
polynucleotides of the invention, or agonists thereof, is
administered to treat, prevent, diagnose, and/or ameliorate
monocytic leukocytosis, monocytic leukopenia, monocytopenia, and/or
monocytosis.
[0486] In a specific embodiment, TR21 polynucleotides or
polypeptides of the invention, and/or anti-TR21 antibodies, and/or
agonists or antagonists thereof, are used to treat, prevent,
detect, and/or diagnose primary B lymphocyte disorders and/or
diseases, and/or conditions associated therewith. In one
embodiment, such primary B lymphocyte disorders, diseases, and/or
conditions are characterized by a complete or partial loss of
humoral immunity. Primary B lymphocyte disorders, diseases, and/or
conditions associated therewith that are characterized by a
complete or partial loss of humoral immunity and that may be
prevented, treated, detected and/or diagnosed with compositions of
the invention include, but are not limited to, X-Linked
Agammaglobulinemia (XLA), severe combined immunodeficiency disease
(SCID), and selective IgA deficiency.
[0487] In a preferred embodiment, TR21 polynucleotides,
polypeptides, and/or agonists and/or antagonists thereof are used
to treat, prevent, and/or diagnose diseases or disorders affecting
or conditions associated with any one or more of the various mucous
membranes of the body. Such diseases or disorders include, but are
not limited to, for example, mucositis, mucoclasis, mucocolitis,
mucocutaneous leishmaniasis (such as, for example, American
leishmaniasis, leishmaniasis americana, nasopharyngeal
leishmaniasis, and New World leishmaniasis), mucocutaneous lymph
node syndrome (for example, Kawasaki disease), mucoenteritis,
mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial
dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid
degeneration; myxomatous degeneration; myxomatosis, mucoid medial
degeneration (for example, cystic medial necrosis), mucolipidosis
(including, for example, mucolipidosis I, mucolipidosis II,
mucolipidosis III, and mucolipidosis IV), mucolysis disorders,
mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis
(such as, for example, type I mucopolysaccharidosis (i.e., Hurler's
syndrome), type IS mucopolysaccharidosis (i.e., Scheie's syndrome
or type V mucopolysaccharidosis), type II mucopolysaccharidosis
(i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e.,
Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e.,
Morquio's syndrome), type VI mucopolysaccharidosis (i.e.,
Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e,
mucopolysaccharidosis due to beta-glucuronidase deficiency), and
mucosulfatidosis), mucopolysacchariduria, mucopurulent
conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal
disease (i.e., bovine virus diarrhea), mucous colitis (such as, for
example, mucocolitis and myxomembranous colitis), and
mucoviscidosis (such as, for example, cystic fibrosis, cystic
fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic
disease of the pancreas, mucoviscidosis, and viscidosis). In a
highly preferred embodiment, TR21 polynucleotides, polypeptides,
and/or agonists and/or antagonists thereof are used to treat,
prevent, and/or diagnose mucositis, especially as associated with
chemotherapy.
[0488] In a preferred embodiment, TR21 polynucleotides,
polypeptides, and/or agonists and/or antagonists thereof are used
to treat, prevent, and/or diagnose diseases or disorders affecting
or conditions associated with sinusitis.
[0489] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by TR21 polynucleotides or
polypeptides, or agonists of TR21 (e.g., anti-TR21 agonistic
antibodies), is osteomyelitis.
[0490] An additional condition, disease or symptom that can be
treated, prevented, and/or diagnosed by TR21 polynucleotides or
polypeptides, or agonists of TR21 (e.g., anti-TR21 agonistic
antibodies), is endocarditis.
[0491] All of the above described applications as they may apply to
veterinary medicine.
[0492] Antagonists of TR21 include binding and/or inhibitory
antibodies (e.g., anti-TR21 antagonistic antibodies), antisense
nucleic acids, ribozymes, and TR21 polypeptides of the invention.
These would be expected to reverse many of the activities of the
ligand described above as well as find clinical or practical
application as:
[0493] A means of blocking various aspects of immune responses to
foreign agents or self. Examples include autoimmune disorders such
as lupus, and arthritis, as well as immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and pathogens.
Although there appears to be a clear potential role of TR21 in B
cell and monocyte related pathologies, it remains possible that
other cell types may gain expression or responsiveness to TR21.
Thus, TR21 may, like CD40 and its ligand, be regulated by the
status of the immune system and the microenvironment in which the
cell is located.
[0494] A therapy for preventing the B cell proliferation and Ig
secretion associated with autoimmune diseases such as idiopathic
thrombocytopenic purpura, systemic lupus erythematosus and MS.
[0495] An inhibitor of graft versus host disease or transplant
rejection.
[0496] A therapy for B cell malignancies such as ALL, Hodgkin's
disease, non-Hodgkin's lymphoma, Chronic lymphocyte leukemia,
plasmacytomas, multiple myeloma, Burkitt's lymphoma, and
EBV-transformed diseases.
[0497] A therapy for chronic hypergammaglobulinemeia evident in
such diseases as monoclonalgammopathy of undetermined significance
(MGUS), Waldenstrom's disease, related idiopathic
monoclonalgammopathies, and plasmacytomas.
[0498] A therapy for decreasing cellular proliferation of Large
B-cell Lymphomas.
[0499] A means of decreasing the involvement of B cells and Ig
associated with Chronic Myelogenous Leukemia.
[0500] As a B cell specific binding protein to which specific
activators or inhibitors of cell growth may be attached. The result
would be to focus the activity of such activators or inhibitors
onto normal, diseased, or neoplastic B cell populations.
[0501] As part of a B cell selection device the function of which
is to isolate B cells from a heterogeneous mixture of cell types.
Anti-TR21 antibody or TNF ligands that bind TR21 could be coupled
to a solid support to which B cells would then specifically bind.
Unbound cells would be washed out and the bound cells subsequently
eluted. This technique would allow purging of tumor cells from, for
example, bone marrow or peripheral blood prior to transplant.
[0502] An immunosuppressive agent(s).
[0503] TR21 polypeptides or polynucleotides of the invention, or
antagonists may be used to modulate IgE concentrations in vitro or
in vivo.
[0504] In another embodiment, administration of TR21 polypeptides
or polynucleotides of the invention, or antagonists thereof, may be
used to treat, prevent, and/or diagnose IgE-mediated allergic
reactions including, but not limited to, asthma, rhinitis, and
eczema.
[0505] An inhibitor of signaling pathways involving ERK1, COX2 and
Cyclin D2.
[0506] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
[0507] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described herein.
[0508] The antagonists may be employed, for instance, to inhibit
TR21-mediated chemotaxis and activation of macrophages and their
precursors, and of neutrophils, basophils, B lymphocytes and some
T-cell subsets, e.g., activated and CD8 cytotoxic T cells and
natural killer cells, in certain auto-immune and chronic
inflammatory and infective diseases. Examples of auto-immune
diseases include multiple sclerosis, and insulin-dependent
diabetes. The antagonists may also be employed to treat, prevent,
and/or diagnose infectious diseases including silicosis,
sarcoidosis, idiopathic pulmonary fibrosis by preventing the
recruitment and activation of mononuclear phagocytes. They may also
be employed to treat, prevent, and/or diagnose idiopathic
hyper-eosinophilic syndrome by preventing eosinophil production and
migration. Endotoxic shock may also be treated by the antagonists
by preventing the migration of macrophages and their production of
the TR21 polypeptides of the present invention. The antagonists may
also be employed for treating atherosclerosis, by preventing
monocyte infiltration in the artery wall. The antagonists may also
be employed to treat, prevent, and/or diagnose histamine-mediated
allergic reactions and immunological disorders including late phase
allergic reactions, chronic urticaria, and atopic dermatitis by
inhibiting chemokine-induced mast cell and basophil degranulation
and release of histamine. IgE-mediated allergic reactions such as
allergic asthma, rhinitis, and eczema may also be treated. The
antagonists may also be employed to treat, prevent, and/or diagnose
chronic and acute inflammation by preventing the attraction of
monocytes to a wound area. They may also be employed to regulate
normal pulmonary macrophage populations, since chronic and acute
inflammatory pulmonary diseases are associated with sequestration
of mononuclear phagocytes in the lung. Antagonists may also be
employed to treat, prevent, and/or diagnose rheumatoid arthritis by
preventing the attraction of monocytes into synovial fluid in the
joints of patients. Monocyte influx and activation plays a
significant role in the pathogenesis of both degenerative and
inflammatory arthropathies. The antagonists may be employed to
interfere with the deleterious cascades attributed primarily to
IL-1 and TNF, which prevents the biosynthesis of other inflammatory
cytokines. In this way, the antagonists may be employed to prevent
inflammation. The antagonists may also be employed to inhibit
prostaglandin-independent fever induced by TR21. The antagonists
may also be employed to treat, prevent, and/or diagnose cases of
bone marrow failure, for example, aplastic anemia and
myelodysplastic syndrome. The antagonists may also be employed to
treat, prevent, and/or diagnose asthma and allergy by preventing
eosinophil accumulation in the lung. The antagonists may also be
employed to treat, prevent, and/or diagnose subepithelial basement
membrane fibrosis which is a prominent feature of the asthmatic
lung. The antagonists may also be employed to treat, prevent,
and/or diagnose lymphomas (e.g., one or more of the extensive, but
not limiting, list of lymphomas provided herein).
[0509] All of the above described applications as they may apply to
veterinary medicine. Moreover, all applications described herein
may also apply to veterinary medicine.
[0510] TR21 polynucleotides or polypeptides of the invention and/or
agonists and/or antagonists thereof, may be used to treat, prevent,
and/or diagnose various immune system-related disorders and/or
conditions associated with these disorders, in mammals, preferably
humans. Many autoimmune disorders result from inappropriate
recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to
the destruction of the host tissue. Therefore, the administration
of TR21 polynucleotides or polypeptides of the invention and/or
agonists and/or antagonists thereof that can inhibit an immune
response, particularly the proliferation of B cells and/or the
production of immunoglobulins, may be an effective therapy in
treating and/or preventing autoimmune disorders. Thus, in preferred
embodiments, TR21 polypeptides and/or TR21 antagonists of the
invention (e.g., polypeptide fragments of TR21 and anti-TR21
antibodies) are used to treat, prevent, and/or diagnose an
autoimmune disorder.
[0511] Autoimmune disorders and conditions associated with these
disorders that may be treated, prevented, and/or diagnosed with the
TR21 polynucleotides, polypeptides, and/or antagonist (e.g.,
anti-TR21 antibodies) of the invention, include, but are not
limited to, autoimmune hemolytic anemia, autoimmune neonatal
thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmune
neutropenia, autoimmunocytopenia, hemolytic anemia,
antiphospholipid syndrome, dermatitis, gluten-sensitive
enteropathy, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, glomerulonephritis (e.g.,
IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,
Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),
Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary
Inflammation, myocarditis, IgA glomerulonephritis, dense deposit
disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin
dependent diabetes mellitis, and autoimmune inflammatory eye
disease.
[0512] Additional autoimmune disorders (that are highly probable)
that may be treated, prevented, and/or diagnosed with the
compositions of the invention (e.g., TR21 polynucleotides,
polypeptides, and/or antagonists (e.g., anti-TR21 antibodies))
include, but are not limited to, autoimmune thyroiditis,
hypothyroidism (i.e., Hashimoto's thyroiditis) (often
characterized, e.g., by cell-mediated and humoral thyroid
cytotoxicity), systemic lupus erhythematosus (often characterized,
e.g., by circulating and locally generated immune complexes),
discoid lupus, Goodpasture's syndrome (often characterized, e.g.,
by anti-basement membrane antibodies), Pemphigus (often
characterized, e.g., by epidermal acantholytic antibodies),
Receptor autoimmunities such as, for example, (a) Graves' Disease
(often characterized, e.g., by TSH receptor antibodies), (b)
Myasthenia Gravis (often characterized, e.g., by acetylcholine
receptor antibodies), and (c) insulin resistance (often
characterized, e.g., by insulin receptor antibodies), autoimmune
hemolytic anemia (often characterized, e.g., by phagocytosis of
antibody-sensitized RBCs), autoimmune thrombocytopenic purpura
(often characterized, e.g., by phagocytosis of antibody-sensitized
platelets.
[0513] Additional autoimmune disorders (that are probable) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention (e.g., TR21 polynucleotides, polypeptides, and/or
antagonists (e.g., anti-TR21 antibodies)) include, but are not
limited to, rheumatoid arthritis (often characterized, e.g., by
immune complexes in joints), scleroderma with anti-collagen
antibodies (often characterized, e.g., by nucleolar and other
nuclear antibodies), mixed connective tissue disease (often
characterized, e.g., by antibodies to extractable nuclear antigens
(e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often
characterized, e.g., by nonhistone ANA), pernicious anemia (often
characterized, e.g., by antiparietal cell, microsomes, and
intrinsic factor antibodies), idiopathic Addison's disease (often
characterized, e.g., by humoral and cell-mediated adrenal
cytotoxicity, infertility (often characterized, e.g., by
antispermatozoal antibodies), glomerulonephritis (often
characterized, e.g., by glomerular basement membrane antibodies or
immune complexes) such as primary glomerulonephritis and IgA
nephropathy, bullous pemphigoid (often characterized, e.g., by IgG
and complement in basement membrane), Sjogren's syndrome (often
characterized, e.g., by multiple tissue antibodies, and/or a
specific nonhistone ANA (SS-B)), diabetes mellitus (often
characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and adrenergic drug resistance (including adrenergic
drug resistance with asthma or cystic fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
[0514] Additional autoimmune disorders (that are possible) that may
be treated, prevented, and/or diagnosed with the compositions of
the invention (e.g., TR21 polynucleotides, polypeptides, and/or
antagonists (e.g., anti-TR21 antibodies)) include, but are not
limited to, chronic active hepatitis (often characterized, e.g., by
smooth muscle antibodies), primary biliary cirrhosis (often
characterized, e.g., by mitchondrial antibodies), other endocrine
gland failure (often characterized, e.g., by specific tissue
antibodies in some cases), vitiligo (often characterized, e.g., by
melanocyte antibodies), vasculitis (often characterized, e.g., by
Ig and complement in vessel walls and/or low serum complement),
post-MI (often characterized, e.g., by myocardial antibodies),
cardiotomy syndrome (often characterized, e.g., by myocardial
antibodies), urticaria (often characterized, e.g., by IgG and IgM
antibodies to IgE), atopic dermatitis (often characterized, e.g.,
by IgG and IgM antibodies to IgE), asthma (often characterized,
e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies,
and many other inflammatory, granulamatous, degenerative, and
atrophic disorders.
[0515] In a preferred embodiment, the autoimmune diseases and
disorders and/or conditions associated with the diseases and
disorders recited above are treated, prevented, and/or diagnosed
using anti-TR21 antibodies.
[0516] In a specific preferred embodiment, multiple sclerosis is
treated, prevented, and/or diagnosed anti-TR21 antibodies and/or
other TR21 antagonist of the invention.
[0517] In a specific preferred embodiment, rheumatoid arthritis is
treated, prevented, and/or diagnosed anti-TR21 antibodies and/or
other TR21 antagonist of the invention.
[0518] In a specific preferred embodiment, lupus is treated,
prevented, and/or diagnosed using anti-TR21 antibodies and/or other
TR21 antagonist of the invention.
[0519] In a specific preferred embodiment, nephritis associated
with lupus is treated, prevented, and/or diagnosed anti-TR21
antibodies and/or other TR21 antagonist of the invention.
[0520] In a specific embodiment, TR21 polynucleotides or
polypeptides, or antagonists thereof (e.g., anti-TR21 antibodies)
are used to treat or prevent systemic lupus erythematosus and/or
diseases, disorders or conditions associated therewith.
Lupus-associated diseases, disorders, or conditions that may be
treated or prevented with TR21 polynucleotides or polypeptides, or
antagonists of the invention, include, but are not limited to,
hematologic disorders (e.g., hemolytic anemia, leukopenia,
lymphopenia, and thrombocytopenia), immunologic disorders (e.g.,
anti-DNA antibodies, and anti-Sm antibodies), rashes,
photosensitivity, oral ulcers, arthritis, fever, fatigue, weight
loss, serositis (e.g., pleuritus (pleuricy)), renal disorders
(e.g., nephritis), neurological disorders (e.g., seizures,
peripheral neuropathy, CNS related disorders), gastroinstestinal
disorders, Raynaud phenomenon, and pericarditis. In a preferred
embodiment, the TR21 polynucleotides or polypeptides, or
antagonists thereof (e.g., anti-TR21 antibodies) are used to treat
or prevent renal disorders associated with systemic lupus
erythematosus. In a most preferred embodiment, TR21 polynucleotides
or polypeptides, or antagonists thereof (e.g., anti-TR21
antibodies) are used to treat or prevent nephritis associated with
systemic lupus erythematosus.
[0521] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by TR21 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof. Moreover,
these molecules can be used to treat, prevent, and/or diagnose
anaphylaxis, hypersensitivity to an antigenic molecule, or blood
group incompatibility.
[0522] TR21 polynucleotides or polypeptides of the invention and/or
antagonists thereof (e.g., antagonistic anti-TR21 antibodies), may
also be used to modulate blood clotting and to treat or prevent
blood-clotting disorders, such as, for example, antibody-mediated
thrombosis (i.e., antiphospholipid antibody syndrome (APS)). For
example, TR21 polynucleotides or polypeptides of the invention
and/or antagonists thereof, may inhibit the proliferation and
differentiation of cells involved in producing anticardiolipin
antibodies. These compositions of the invention can be used to
treat, prevent, and/or diagnose, thrombotic related events
including, but not limited to, stroke (and recurrent stroke), heart
attack, deep vein thrombosis, pulmonary embolism, myocardial
infarction, coronary artery disease (e.g., antibody-mediated
coronary artery disease), thrombosis, graft reocclusion following
cardiovascular surgery (e.g., coronary arterial bypass grafts,
recurrent fetal loss, and recurrent cardiovascular thromboembolic
events.
[0523] TR21 polynucleotides or polypeptides of the invention and/or
agonists and/or antagonists thereof may also be used to treat,
prevent, and/or diagnose organ rejection or graft-versus-host
disease (GVHD) and/or conditions associated therewith. Organ
rejection occurs by host immune cell destruction of the
transplanted tissue through an immune response. Similarly, an
immune response is also involved in GVHD, but, in this case, the
foreign transplanted immune cells destroy the host tissues. The
administration of TR21 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, that inhibits
an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD.
[0524] Similarly, TR21 polynucleotides or polypeptides of the
invention and/or agonists and/or antagonists thereof, may also be
used to modulate inflammation. For example, TR21 polynucleotides or
polypeptides of the invention and/or agonists and/or antagonists
thereof, may inhibit the proliferation and differentiation of cells
involved in an inflammatory response. These molecules can be used
to treat, prevent, and/or diagnose inflammatory conditions, both
chronic and acute conditions, including chronic prostatitis,
granulomatous prostatitis and malacoplakia, inflammation associated
with infection (e.g., septic shock, sepsis, or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion
injury, endotoxin lethality, arthritis, complement-mediated
hyperacute rejection, nephritis, cytokine or chemokine induced lung
injury, inflammatory bowel disease, Crohn's disease, or due to
overproduction of cytokines (e.g., TNF or IL-1).
[0525] In a specific embodiment, anti-TR21 antibodies of the
invention are used to treat, prevent, modulate, detect, and/or
diagnose inflammation.
[0526] In a specific embodiment, anti-TR21 antibodies of the
invention are used to treat, prevent, modulate, detect, and/or
diagnose inflammatory disorders.
[0527] In another specific embodiment, anti-TR21 antibodies of the
invention are used to treat, prevent, modulate, detect, and/or
diagnose allergy and/or hypersensitivity.
[0528] Antibodies against TR21 may be employed to bind to and
inhibit TR21 activity to treat, prevent, and/or diagnose ARDS, by
preventing infiltration of neutrophils into the lung after injury.
The agonists and antagonists of the instant may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described hereinafter.
[0529] TR21 polynucleotides or polypeptides of the invention and/or
agonists and/or antagonists thereof (e.g., antagonistic anti-TR21
antibodies), may be used to treat, prevent, and/or diagnose
diseases and disorders of the pulmonary system (e.g., bronchi such
as, for example, sinopulmonary and bronchial infections and
conditions associated with such diseases and disorders and other
respiratory diseases and disorders. In specific embodiments, such
diseases and disorders include, but are not limited to, bronchial
adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial
pneumonia, bronchopneumonia, and tuberculous bronchopneumonia),
chronic obstructive pulmonary disease (COPD), bronchial polyps,
bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical
bronchiectasis, and saccular bronchiectasis), bronchiolar
adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as,
e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans,
and proliferative bronchiolitis), bronchiolo-alveolar carcinoma,
bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis,
Castellani's bronchitis, chronic bronchitis, croupous bronchitis,
fibrinous bronchitis, hemorrhagic bronchitis, infectious avian
bronchitis, obliterative bronchitis, plastic bronchitis,
pseudomembranous bronchitis, putrid bronchitis, and verminous
bronchitis), bronchocentric granulomatosis, bronchoedema,
bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic
cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as,
e.g., bronchopulmonary aspergillosis), bronchopulmonary
spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm,
bronchostaxis, bronchostenosis, Biot's respiration, bronchial
respiration, Kussmaul respiration, Kussmaul-Kien respiration,
respiratory acidosis, respiratory alkalosis, respiratory distress
syndrome of the newborn, respiratory insufficiency, respiratory
scleroma, respiratory syncytial virus, and the like.
[0530] In a specific embodiment, TR21 polynucleotides or
polypeptides of the invention and/or agonists thereof (e.g.,
agonistic anti-TR21 antibodies), are used to treat, prevent, and/or
diagnose chronic obstructive pulmonary disease (COPD).
[0531] In another embodiment, TR21 polynucleotides or polypeptides
of the invention and/or agonists and/or antagonists thereof, are
used to treat, prevent, and/or diagnose fibroses and conditions
associated with fibroses, including, but not limited to, cystic
fibrosis (including such fibroses as cystic fibrosis of the
pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the
pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis,
idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis,
mediastinal fibrosis, nodular subepidermal fibrosis, pericentral
fibrosis, perimuscular fibrosis, pipestem fibrosis, replacement
fibrosis, subadventitial fibrosis, and Symmers' clay pipestem
fibrosis.
[0532] The TNF family ligands are known to be among the most
pleiotropic cytokines, inducing a large number of cellular
responses, including cytotoxicity, anti-viral activity,
immunoregulatory activities, and the transcriptional regulation of
several genes (D. V. Goeddel et al., "Tumor Necrosis Factors: Gene
Structure and Biological Activities," Symp. Quant. Biol. 51:597-609
(1986), Cold Spring Harbor; B. Beutler and A. Cerami, Annu. Rev.
Biochem. 57:505-518 (1988); L. J. Old, Sci. Am. 258:59-75 (1988);
W. Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands,
including the TR21 ligands (e.g., Neutrokine-alpha), induce such
various cellular responses by binding to TNF-family receptors,
including TR21. TNF-ligand (e.g., Neutrokine-alpha)/TR21
interactions may elicit a potent cellular response including any
genotypic, phenotypic, and/or morphologic change to the cell, cell
line, tissue, tissue culture or patient. As indicated, such
cellular responses include not only normal physiological responses
to TNF-family ligands, but such responses may lead to diseases
associated with dysregulation of these physiological responses,
such as, for example, diseases associated with increased apoptosis
or the inhibition of apoptosis. Apoptosis-programmed cell death-is
a physiological mechanism involved in the deletion of peripheral B
and/or T lymphocytes of the immune system, and its dysregulation
can lead to a number of different pathogenic processes (J. C.
Ameisen, AIDS 8:1197-1213 (1994); P. H. Krammer et al., Curr. Opin.
Immunol. 6:279-289 (1994)).
[0533] TR21 polynucleotides, polypeptides, agonists and/or
antagonists (e.g., agonistic antibodies) of the invention may be
administered to a patient (e.g., mammal, preferably human)
afflicted with any disease or disorder mediated (directly or
indirectly) by defective, or deficient levels of, TR21 or TR21
ligand (e.g., Neutrokine-alpha). Alternatively, a gene therapy
approach may be applied to treat such diseases or disorders. In one
embodiment of the invention, TR21 polynucleotide sequences are used
to detect mutein TR21 genes, including defective genes. Mutein
genes may be identified in in vitro diagnostic assays, and by
comparison of the TR21 nucleotide sequence disclosed herein with
that of a TR21 gene obtained from a patient suspected of harboring
a defect in this gene. Defective genes may be replaced with normal
TR21-encoding genes using techniques known to one skilled in the
art.
[0534] In another embodiment, the TR21 polypeptides,
polynucleotides, agonists and/or antagonists of the present
invention are used as research tools for studying the phenotypic
effects that result from inhibiting TR21/TR21 ligand interactions
on various cell types. TR21 polypeptides and antagonists (e.g.
monoclonal antibodies to TR21) also may be used in in vitro assays
for detecting TR21, TR21 ligands, or the interactions thereof.
[0535] Diseases associated with increased cell survival, or the
inhibition of apoptosis that may be diagnosed, treated, or
prevented with the TR21 polynucleotides or polypeptides (including
anti-TR21 antibodies) of the invention, and agonists and
antagonists thereof, include cancers (such as follicular lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to, colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma,
adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders (such as systemic lupus
erythematosus and immune-related glomerulonephritis rheumatoid
arthritis); viral infections (such as herpes viruses, pox viruses
and adenoviruses); inflammation; graft vs. host disease; acute
graft rejection and chronic graft rejection. Thus, in preferred
embodiments TR21 polynucleotides or polypeptides of the invention
and/or agonists or antagonists thereof, are used to treat, prevent,
and/or diagnose autoimmune diseases and/or inhibit the growth,
progression, and/or metastasis of cancers, including, but not
limited to, those cancers disclosed herein, such as, for example,
lymphocytic leukemias (including, for example, MLL and chronic
lymphocytic leukemia (CLL)) and follicular lymphomas. In another
embodiment TR21 polynucleotides or polypeptides of the invention
are used to activate, differentiate or proliferate cancerous cells
or tissue (e.g., B cell lineage related cancers (e.g., CLL and
MLL), lymphocytic leukemia, or lymphoma) and thereby render the
cells more vulnerable to cancer therapy (e.g., chemotherapy or
radiation therapy).
[0536] Moreover, in other embodiments, TR21 polynucleotides or
polypeptides of the invention (including anti-TR21 antibodies) or
agonists or antagonists thereof, may be used to inhibit the growth,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)), acute myelogenous leukemia, and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,
Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid
tumors including, but not limited to, sarcomas and carcinomas such
as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma. In specific embodiments, TR21 polynucleotides or
polypeptides (e.g., TR21 extracellular domain-Fc fusion proteins)
of the invention or agonists or antagonists thereof (e.g.,
anti-TR21 antibodies) are used to diagnose, treat, or prevent acute
myelogenous leukemia. In further specific embodiments, TR21
polypeptides or agonists or antagonists (e.g., anti-TR21
antibodies) are used to diagnose, treat, or prevent chronic
lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma,
and/or Hodgkin's disease.
[0537] Diseases associated with increased apoptosis and that may be
treated or prevented by the polynucleotides, polypeptides and/or
agonists or antagonists of the invention include, but are not
limited to, AIDS; neurodegenerative disorders (such as Alzheimer's
disease, Parkinson's disease, Amyotrophic lateral sclerosis,
Retinitis pigmentosa, Cerebellar degeneration and brain tumor or
prior associated disease); autoimmune disorders (such as, multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis); myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (such as hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia. In preferred
embodiments, TR21 polynucleotides, polypeptides and/or agonists are
used to treat the diseases and disorders listed above.
[0538] In preferred embodiments, TR21 polypeptides of the invention
and/or agonists or antagonists thereof (e.g., anti-TR21 antibodies)
inhibit the growth of human histiocytic lymphoma U-937 cells in a
dose-dependent manner. In additional preferred embodiments, TR21
polypeptides of the invention and/or agonists or antagonists
thereof (e.g., anti-TR21 antibodies) inhibit the growth of PC-3
cells, HT-29 cells, HeLa cells, MCF-7 cells, and A293 cells. In
highly preferred embodiments, TR21 polynucleotides or polypeptides
of the invention and/or agonists or antagonists thereof (e.g.,
anti-TR21 antibodies) are used to inhibit growth, progression,
and/or metastasis of prostate cancer, colon cancer, cervical
carcinoma, and breast carcinoma.
[0539] Thus, in additional preferred embodiments, the present
invention is directed to a method for enhancing apoptosis induced
by a TNF-family ligand, which involves administering to a cell
which expresses TR21 an effective amount of TR21 ligand (e.g.,
Neutrokine-alpha), or an agonist or antagonist of TR21, capable of
increasing or decreasing TR21 mediated signaling. Preferably, TR21
mediated signaling is increased or decreased to treat, prevent,
and/or diagnose a disease wherein decreased apoptosis or decreased
cytokine and adhesion molecule expression is exhibited. An agonist
or antagonist can include soluble forms of TR21 and monoclonal
antibodies directed against the TR21 polypeptide.
[0540] Many of the pathologies associated with HIV are mediated by
apoptosis, including HIV-induced nephropathy and HIV encephalitis.
Thus, in additional preferred embodiments, TR21 polynucleotides,
polypeptides, and/or TR21 agonists (e.g., anti-TR21 antibodies) or
antagonists of the invention are used to treat AIDS and pathologies
associated with AIDS.
[0541] The state of immunodeficiency that defines AIDS is secondary
to a decrease in the number and function of CD4.sup.+
T-lymphocytes. Recent reports estimate the daily loss of CD4.sup.+
T cells to be between 3.5.times.10.sup.7 and 2.times.10.sup.9 cells
(Wei et al., Nature 373:117-122 (1995)). One cause of CD4.sup.+ T
cell depletion in the setting of HIV infection is believed to be
HIV-induced apoptosis (see, for example, Meyaard et al., Science
257:217-219, 1992; Groux et al., J Exp. Med., 175:331, 1992; and
Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection,
Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114).
Indeed, HIV-induced apoptotic cell death has been demonstrated not
only in vitro but also, more importantly, in infected individuals
(J. C. Ameisen, AIDS 8:1197-1213 (1994); T. H. Finkel and N. K.
Banda, Curr. Opin. Immunol. 6:605-615(1995); C. A. Muro-Cacho et
al., J. Immunol. 154:5555-5566 (1995)). Furthermore, apoptosis and
CD4.sup.+ T-lymphocyte depletion is tightly correlated in different
animal models of AIDS (T. Brunner et al., Nature 373:441-444
(1995); M. L. Gougeon et al., AIDS Res. Hum. Retroviruses 9:553-563
(1993)) and, apoptosis is not observed in those animal models in
which viral replication does not result in AIDS. Id. Further data
indicates that uninfected but primed or activated T lymphocytes
from HIV-infected individuals undergo apoptosis after encountering
the TNF-family ligand FasL. Using monocytic cell lines that result
in death following HIV infection, it has been demonstrated that
infection of U937 cells with HIV results in the de novo expression
of FasL and that FasL mediates HIV-induced apoptosis (A. D. Badley
et al., J. Virol. 70:199-206 (1996)). Further, the TNF-family
ligand was detectable in uninfected macrophages and its expression
was upregulated following HIV infection resulting in selective
killing of uninfected CD4 T-lymphocytes. Id. Thus, by the
invention, a method for treating HIV.sup.+ individuals is provided
which involves administering TR21 and/or TR21 agonists (e.g.,
anti-TR21 antibodies) or antagonists of the present invention to
reduce selective killing of CD4.sup.+ T-lymphocytes. Modes of
administration and dosages are discussed in detail below.
[0542] Activated human T cells are induced to undergo programmed
cell death (apoptosis) upon triggering through the CD3/T cell
receptor complex, a process termed activated-induced cell death
(AICD). AICD of CD4.sup.+ T cells isolated from HIV-Infected
asymptomatic individuals has been reported (Groux et al., supra).
Thus, AICD may play a role in the depletion of CD4.sup.+ T cells
and the progression to AIDS in HIV-infected individuals. Thus, the
present invention provides a method of inhibiting TNF
ligand-mediated T cell death in HIV patients, comprising
administering a TR21 polypeptide of the invention (preferably, a
soluble TR21 polypeptide) to the patients. In one embodiment, the
patient is asymptomatic when treatment with TR21 commences. If
desired, prior to treatment, peripheral blood T cells may be
extracted from an HIV patient, and tested for susceptibility to TNF
ligand-mediated cell death by procedures known in the art. In one
embodiment, a patient's blood or plasma is contacted with TR21 ex
vivo. The TR21 may be bound to a suitable chromatography matrix by
procedures known in the art. The patient's blood or plasma flows
through a chromatography column containing TR21 bound to the
matrix, before being returned to the patient. The immobilized TR21
binds TNF ligand, thus removing TNF ligand protein from the
patient's blood.
[0543] In additional embodiments a TR21 polypeptide of the
invention is administered in combination with other inhibitors of T
cell apoptosis. For example, Fas-mediated apoptosis and
TRAIL-mediated apoptosis have also has been implicated in loss of T
cells in HIV individuals (See, e.g., Katsikis et al., J. Exp. Med.
181:2029-2036 (1995)). Thus, a patient susceptible to Fas ligand
mediated and/or TRAIL mediated T cell death may be treated with an
agent that blocks Fas-ligand/Fas receptor interactions and/or an
agent that blocks TRAIL/TRAIL interactions.
[0544] Suitable agents for blocking binding of Fas-ligand to Fas
that may be administered with the TR21 polynucleotides or
polypeptides of the invention (including TR21 agonists (e.g.,
agonistic antibodies) and/or antagonists) include, but are not
limited to, soluble Fas polypeptides; multimeric forms of soluble
Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies
that bind Fas without transducing the biological signal that
results in apoptosis; anti-Fas-ligand antibodies that block binding
of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fas but
do not transduce the biological signal that results in apoptosis.
Preferably, the antibodies employed according to this method are
monoclonal antibodies. Examples of suitable agents for blocking
Fas-ligand/Fas interactions, including blocking anti-Fas monoclonal
antibodies, are described in International application publication
number WO 95/10540, hereby incorporated by reference.
[0545] Suitable agents, which also block binding of TRAIL to a
TRAIL receptor that may be administered with the polynucleotides
and/or polypeptides of the present invention include, but are not
limited to, soluble TRAIL receptor polypeptides (e.g., a soluble
form of OPG, DR4 (International application publication number WO
98/32856); TR5 (International application publication number WO
98/30693); and DR5 (International application publication number WO
98/41629)); multimeric forms of soluble TRAIL receptor
polypeptides; and TRAIL receptor antibodies that bind the TRAIL
receptor without transducing the biological signal that results in
apoptosis, anti-TRAIL antibodies that block binding of TRAIL to one
or more TRAIL receptors, and muteins of TRAIL that bind TRAIL
receptors but do not transduce the biological signal that results
in apoptosis. Preferably, the antibodies employed according to this
method are monoclonal antibodies.
[0546] In a further aspect, the present invention is directed to a
method for inhibiting apoptosis induced by a TNF-family ligand,
which involves administering to a cell which expresses the TR21 an
effective amount of an agonist or antagonist capable of increasing
or decreasing signaling mediated by the TR21. Preferably, TR21
mediated signaling is increased or decreased to treat, prevent,
and/or diagnose a disease wherein increased apoptosis or NF-kappaB
expression is exhibited. An agonist or antagonist can include
soluble forms of TR21 and monoclonal antibodies directed against
the TR21 polypeptide.
[0547] TR21 polypeptides or polynucleotides encoding TR21 and
anti-TR21 antibodies of the invention of the invention may be used
to treat cardiovascular disorders, including peripheral artery
disease, such as limb ischemia.
[0548] Cardiovascular disorders include cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous
fistula, cerebral arteriovenous malformations, congenital heart
defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects include aortic coarctation, cor triatriatum, coronary
vessel anomalies, crisscross heart, dextrocardia, patent ductus
arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic
left heart syndrome, levocardia, tetralogy of fallot, transposition
of great vessels, double outlet right ventricle, tricuspid atresia,
persistent truncus arteriosus, and heart septal defects, such as
aortopulmonary septal defect, endocardial cushion defects,
Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal
defects, and conditions characterized by clotting of small blood
vessels.
[0549] Cardiovascular disorders also include heart disease, such as
arrhythmias, carcinoid heart disease, high cardiac output, low
cardiac output, cardiac tamponade, endocarditis (including
bacterial), heart aneurysm, cardiac arrest, congestive heart
failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left
ventricular hypertrophy, right ventricular hypertrophy,
post-infarction heart rupture, ventricular septal rupture, heart
valve diseases, myocardial diseases, myocardial ischemia,
pericardial effusion, pericarditis (including constrictive and
tuberculous), pneumopericardium, postpericardiotomy syndrome,
pulmonary heart disease, rheumatic heart disease, ventricular
dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[0550] Arrhythmias include sinus arrhythmia, atrial fibrillation,
atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome,
bundle-branch block, sinoatrial block, long QT syndrome,
parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type
pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia, Torsades de Pointes, and ventricular tachycardia.
[0551] Heart valve diseases include aortic valve insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral
valve prolapse, tricuspid valve prolapse, mitral valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[0552] Myocardial diseases include alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic
subvalvular stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[0553] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0554] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, thrombotic microangiopathies (e.g., thrombotic
thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome
(HUS)), and venous insufficiency.
[0555] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0556] Arterial occlusive diseases include arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular
dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[0557] Cerebrovascular disorders include carotid artery diseases,
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral arteriosclerosis, cerebral arteriovenous malformation,
cerebral artery diseases, cerebral embolism and thrombosis, carotid
artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma,
subarachnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar insufficiency.
[0558] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[0559] Ischemia includes cerebral ischemia, ischemic colitis,
compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis,
Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's granulomatosis.
[0560] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. See e.g., Rastinejad et al.,
Cell 56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions,
such as wound healing, organ regeneration, embryonic development,
and female reproductive processes, angiogenesis is stringently
regulated and spatially and temporally delimited. Under conditions
of pathological angiogenesis such as that characterizing solid
tumor growth, these regulatory controls fail. Unregulated
angiogenesis becomes pathologic and sustains progression of many
neoplastic and non-neoplastic diseases. A number of serious
diseases are dominated by abnormal neovascularization including
solid tumor growth and metastases, arthritis, some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al.,
Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein
and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz,
Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science
221:719-725 (1983). In a number of pathological conditions, the
process of angiogenesis contributes to the disease state. For
example, significant data have accumulated which suggest that the
growth of solid tumors is dependent on angiogenesis. Folkman and
Klagsbrun, Science 235:442-447 (1987).
[0561] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
the TR21 polynucleotides and/or polypeptides of the invention
(including TR21 agonists (e.g., agonistic antibodies) and/or
antagonists). Malignant and metastatic conditions which can be
treated with the polynucleotides and polypeptides of the invention
include, but are not limited to those malignancies, solid tumors,
and cancers described herein and otherwise known in the art (for a
review of such disorders, see Fishman et al., Medicine, 2d Ed., J.
B. Lippincott Co., Philadelphia (1985)).
[0562] Additionally, ocular disorders associated with
neovascularization which can be treated with the TR21
polynucleotides and polypeptides of the present invention
(including TR21 agonists and TR21 antagonists) include, but are not
limited to: neovascular glaucoma, diabetic retinopathy,
retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of
prematurity macular degeneration, corneal graft neovascularization,
as well as other eye inflammatory diseases, ocular tumors and
diseases associated with choroidal or iris neovascularization. See,
e.g., reviews by Waltman et al., Am. J. Ophthal 85:704-710 (1978)
and Gartner et al., Surv. Ophthal. 22:291-312 (1978).
[0563] Additionally, disorders which can be treated with the TR21
polynucleotides and polypeptides of the present invention
(including TR21 agonists and TR21 antagonists) include, but are not
limited to, hemangioma, arthritis, psoriasis, angiofibroma,
atherosclerotic plaques, delayed wound healing, granulations,
hemophilic joints, hypertrophic scars, nonunion fractures,
Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma,
and vascular adhesions.
[0564] The polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof, can also be employed to
inhibit the proliferation and differentiation of hematopoietic
cells and therefore may be employed to protect bone marrow stem
cells from chemotherapeutic agents during chemotherapy. This
antiproliferative effect may allow administration of higher doses
of chemotherapeutic agents and, therefore, more effective
chemotherapeutic treatment.
[0565] The polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof, may also be employed
for the expansion of immature hematopoietic progenitor cells, for
example, granulocytes, macrophages or monocytes (e.g., C-kit+,
Sca-1+), by temporarily preventing their differentiation. These
bone marrow cells may be cultured in vitro. Thus, TR21 may be
useful as a modulator of hematopoietic stem cells in vitro for the
purpose of bone marrow transplantation and/or gene therapy. Since
stem cells are rare and are most useful for introducing genes into
for gene therapy, TR21 can be used to isolate enriched populations
of stem cells. Stem cells can be enriched by culturing cells in the
presence of cytotoxins, such as 5-Fu, which kills rapidly dividing
cells, where as the stem cells will be protected by TR21. These
stem cells can be returned to a bone marrow transplant patient or
can then be used for transfection of the desired gene for gene
therapy. In addition, TR21 can be injected into animals which
results in the release of stem cells from the bone marrow of the
animal into the peripheral blood. These stem cells can be isolated
for the purpose of autologous bone marrow transplantation or
manipulation for gene therapy. After the patient has finished
chemotherapy or radiation treatment, the isolated stem cells can be
returned to the patient.
[0566] In a specific embodiment, polynucleotides and/or
polypeptides of the invention and/or agonists and/or antagonists
thereof may be used to increase the concentration of blood cells in
individuals in need of such increase (i.e., in hematopoietic
therapy). Conditions that may be ameliorated by administering the
compositions of the invention include, but are not limited to,
neutropenia, anemia, and thrombocytopenia.
[0567] In a specific embodiment, the polynucleotides and/or
polypeptides of the invention (and/or agonists or antagonists
thereof) are used in erythropoietin therapy, which is directed
toward supplementing the oxygen carrying capacity of blood.
Polynucleotides and/or polypeptides of the invention (and/or
agonists or antagonists thereof) may be used to treat or prevent
diseases or conditions in patients generally requiring blood
transfusions, such as, for example, trauma victims, surgical
patients, dialysis patients, and patients with a variety of blood
composition-affecting disorders, such as, for example, hemophilia,
cystic fibrosis, pregnancy, menstrual disorders, early anemia of
prematurity, spinal cord injury, aging, various neoplastic disease
states, and the like. Examples of patient conditions that require
supplementation of the oxygen carrying capacity of blood and which
are within the scope of this invention, include, but are not
limited to: treatment of blood disorders characterized by low or
defective red blood cell production, anemia associated with chronic
renal failure, stimulation of reticulocyte response, development of
ferrokinetic effects (such as plasma iron turnover effects and
marrow transit time effects), erythrocyte mass changes, stimulation
of hemoglobin C synthesis, and increasing levels of hematocrit in
vertebrates. The invention also provides for treatment to enhance
the oxygen-carrying capacity of an individual, such as for example,
an individual encountering hypoxic environmental conditions.
[0568] TR21 polynucleotides, polypeptides and/or agonists or
antagonists may also be employed to regulate hematopoiesis, by
regulating the activation and differentiation of various
hematopoietic progenitor cells, for example, to release mature
leukocytes from the bone marrow following chemotherapy, i.e., in
stem cell mobilization. TR21 polynucleotides, polypeptides and/or
agonists or antagonists may also be employed to treat sepsis.
[0569] TR21 polynucleotides, polypeptides and/or agonists or
antagonists may also be employed to inhibit T-cell proliferation by
the inhibition of IL-2 biosynthesis for the treatment of T-cell
mediated auto-immune diseases and lymphocytic leukemias (including,
for example, chronic lymphocytic leukemia (CLL)).
[0570] TR21 polynucleotides, polypeptides and/or agonists or
antagonists may also be employed to stimulate wound healing, both
via the recruitment of debris clearing and connective tissue
promoting inflammatory cells. In this same manner, TR21
polynucleotides, polypeptides and/or agonists or antagonists may
also be employed to treat other fibrotic disorders, including liver
cirrhosis, osteoarthritis and pulmonary fibrosis.
[0571] TR21 polynucleotides, polypeptides and/or agonists or
antagonists may also be employed to enhance host defenses against
resistant chronic and acute infections, for example, myobacterial
infections via the attraction and activation of microbicidal
leukocytes.
[0572] TR21 polynucleotides, polypeptides and/or agonists or
antagonists also increases the presence of eosinophils which have
the distinctive function of killing the larvae of parasites that
invade tissues, as in schistosomiasis, trichinosis and
ascariasis.
[0573] Because TR21 belongs to the TNFR superfamily, the
polypeptides should also modulate angiogenesis. In addition, since
TR21 inhibits immune cell functions, the polypeptides will have a
wide range of anti-inflammatory activities. TR21 may be employed as
an anti-neovascularizing agent to treat, prevent, and/or diagnose
solid tumors by stimulating the invasion and activation of host
defense cells, e.g., cytotoxic T cells and macrophages and by
inhibiting the angiogenesis of tumors. Those of skill in the art
will recognize other non-cancer indications where blood vessel
proliferation is not wanted. They may also be employed to enhance
host defenses against resistant chronic and acute infections, for
example, myobacterial infections via the attraction and activation
of microbicidal leukocytes. TR21 may also be employed to inhibit
T-cell proliferation by the inhibition of IL-2 biosynthesis for the
treatment of T-cell mediated auto-immune diseases and lymphocytic
leukemias (including, for example, chronic lymphocytic leukemia
(CLL)). TR21 may also be employed to stimulate wound healing, both
via the recruitment of debris clearing and connective tissue
promoting inflammatory cells. In this same manner, TR21 may also be
employed to treat, prevent, and/or diagnose other fibrotic
disorders, including liver cirrhosis, osteoarthritis and pulmonary
fibrosis. TR21 also increases the presence of eosinophils that have
the distinctive function of killing the larvae of parasites that
invade tissues, as in schistosomiasis, trichinosis and ascariasis.
It may also be employed to regulate hematopoiesis, by regulating
the activation and differentiation of various hematopoietic
progenitor cells, for example, to release mature leukocytes from
the bone marrow following chemotherapy, i.e., in stem cell
mobilization. TR21 may also be employed to treat, prevent, and/or
diagnose sepsis.
[0574] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in the diagnosis and
treatment or prevention of a wide range of diseases and/or
conditions. Such diseases and conditions include, but are not
limited to, cancer (e.g., immune cell related cancers, breast
cancer, prostate cancer, ovarian cancer, follicular lymphoma,
cancer associated with mutation or alteration of p53, brain tumor,
bladder cancer, uterocervical cancer, colon cancer, colorectal
cancer, non-small cell carcinoma of the lung, small cell carcinoma
of the lung, stomach cancer, etc.), lymphoproliferative disorders
(e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.)
infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus
infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV,
HHV-6, HHV-7, EBV), adenovirus infection, poxvirus infection, human
papilloma virus infection, hepatitis infection (e.g., HAV, HBV,
HCV, etc.), Helicobacter pylori infection, invasive Staphylococci,
etc.), parasitic infection, nephritis, bone disease (e.g.,
osteoporosis), atherosclerosis, pain, cardiovascular disorders
(e.g., neovascularization, hypovascularization or reduced
circulation (e.g., ischemic disease (e.g., myocardial infarction,
stroke, etc.)), AIDS, allergy, inflammation, neurodegenerative
disease (e.g., Alzheimer's disease, Parkinson's disease,
amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar
degeneration, etc.), graft rejection (acute and chronic), graft vs.
host disease, diseases due to osteomyelodysplasia (e.g., aplastic
anemia, etc.), joint tissue destruction in rheumatism, liver
disease (e.g., acute and chronic hepatitis, liver injury, and
cirrhosis), autoimmune disease (e.g., multiple sclerosis,
rheumatoid arthritis, systemic lupus erythematosus, immune complex
glomerulonephritis, autoimmune diabetes, autoimmune
thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis,
etc.), cardiomyopathy (e.g., dilated cardiomyopathy), diabetes,
diabetic complications (e.g., diabetic nephropathy, diabetic
neuropathy, diabetic retinopathy), influenza, asthma, psoriasis,
glomerulonephritis, septic shock, and ulcerative colitis.
[0575] Polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in promoting
angiogenesis, wound healing (e.g., wounds, burns, and bone
fractures). Polynucleotides and/or polypeptides of the invention
and/or agonists and/or antagonists thereof are also useful as an
adjuvant to enhance immune responsiveness to specific antigen,
anti-viral immune responses.
[0576] More generally, polynucleotides and/or polypeptides of the
invention and/or agonists and/or antagonists thereof are useful in
regulating (i.e., elevating or reducing) immune response. For
example, polynucleotides and/or polypeptides of the invention may
be useful in preparation or recovery from surgery, trauma,
radiation therapy, chemotherapy, and transplantation, or may be
used to boost immune response and/or recovery in the elderly and
immunocompromised individuals. Alternatively, polynucleotides
and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful as immunosuppressive agents, for
example in the treatment or prevention of autoimmune disorders. In
specific embodiments, polynucleotides and/or polypeptides of the
invention are used to treat or prevent chronic inflammatory,
allergic or autoimmune conditions, such as those described herein
or are otherwise known in the art.
[0577] In one aspect, the present invention is directed to a method
for enhancing TR21 mediated signaling by a TNF-family ligand, which
involves administering to a cell which expresses the TR21
polypeptide an effective amount of TR21 ligand, analog or an
agonist capable of increasing TR21 mediated signaling. Preferably,
TR21 mediated signaling is increased to treat a disease wherein
increased apoptosis, decreased cytokine and adhesion molecule
expression, or decreased cell proliferation is exhibited. An
agonist can include soluble forms of TR21 and monoclonal antibodies
directed against the TR21 polypeptide.
[0578] In a further aspect, the present invention is directed to a
method for inhibiting TR21 mediated signaling induced by a
TNF-family ligand, which involved administering to a cell which
expresses the TR21 polypeptide an effective amount of an antagonist
capable of decreasing TR21 mediated signaling. Preferably, TR21
mediated signaling is decreased to treat a disease wherein
decreased apoptosis or NF-kappaB expression, or increased cell
proliferation, is exhibited. An antagonist can include soluble
forms of TR21 and monoclonal antibodies directed against the TR21
polypeptide.
[0579] Preferably, treatment using TR21 polynucleotides or
polypeptides, and/or agonists or antagonists of TR21 (e.g.,
anti-TR21 antibodies), could either be by administering an
effective amount of TR21 polypeptide of the invention, or agonist
or antagonist thereof, to the patient, or by removing cells from
the patient, supplying the cells with TR21 polynucleotide, and
returning the engineered cells to the patient (ex vivo therapy).
Moreover, as further discussed herein, the TR21 polypeptide or
polynucleotide can be used as an adjuvant in a vaccine to raise an
immune response against infectious disease.
Formulations and Administration
[0580] The TR21 polypeptide composition (preferably containing
anti-TR21 antibody or a polypeptide which is a soluble form of the
TR21 extracellular domain) will be formulated and dosed in a
fashion consistent with good medical practice, taking into account
the clinical condition of the individual patient (especially the
side effects of treatment with TR21 polypeptide alone), the site of
delivery of the TR21 polypeptide composition, the method of
administration, the scheduling of administration, and other factors
known to practitioners. The "effective amount" of TR21 polypeptide
for purposes herein is thus determined by such considerations.
[0581] As a general proposition, the total pharmaceutically
effective amount of TR21 polypeptide administered parenterally per
dose will be in the range of about 1 microgram/kg/day to 10
mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day.
[0582] In another embodiment, the TR21 polypeptide of the invention
is administered to a human at a dose between 0.0001 and 0.045
mg/kg/day, preferably, at a dose between 0.0045 and 0.045
mg/kg/day, and more preferably, at a dose of about 45
microgram/kg/day in humans; and at a dose of about 3 mg/kg/day in
mice.
[0583] If given continuously, the TR21 polypeptide is typically
administered at a dose rate of about 1 microgram/kg/hour to about
50 micrograms/kg/hour, either by 1-4 injections per day or by
continuous subcutaneous infusions, for example, using a mini-pump.
An intravenous bag solution may also be employed.
[0584] The length of treatment needed to observe changes and the
interval following treatment for responses to occur appears to vary
depending on the desired effect.
[0585] In a specific embodiment, the total pharmaceutically
effective amount of TR21 polypeptide administered parenterally per
dose will be in the range of about 0.1 microgram/kg/day to 45
micrograms/kg/day of patient body weight, although, as noted above,
this will be subject to therapeutic discretion. More preferably,
this dose is at least 0.1 microgram/kg/day, and most preferably for
humans between about 0.01 and 50 micrograms/kg/day for the protein.
TR21 polypeptides of the invention may be administered as a
continuous infusion, multiple discrete injections per day (e.g.,
three or more times daily, or twice daily), single injection per
day, or as discreet injections given intermittently (e.g., twice
daily, once daily, every other day, twice weekly, weekly, biweekly,
monthly, bimonthly, and quarterly). If given continuously, the TR21
polypeptide is typically administered at a dose rate of about 0.001
to 10 microgram/kg/hour to about 50 micrograms/kg/hour, either by
1-4 injections per day or by continuous subcutaneous infusions, for
example, using a mini-pump.
[0586] Effective dosages of the compositions of the present
invention to be administered may be determined through procedures
well known to those in the art which address such parameters as
biological half-life, bioavailability, and toxicity. Such
determination is well within the capability of those skilled in the
art, especially in light of the detailed disclosure provided
herein.
[0587] Bioexposure of an organism to TR21 polypeptide during
therapy may also play an important role in determining a
therapeutically and/or pharmacologically effective dosing regime.
Variations of dosing such as repeated administrations of a
relatively low dose of TR21 polypeptide for a relatively long
period of time may have an effect which is therapeutically and/or
pharmacologically distinguishable from that achieved with repeated
administrations of a relatively high dose of TR21 for a relatively
short period of time.
[0588] Using the equivalent surface area dosage conversion factors
supplied by Freireich, E. J., et al. (Cancer Chemotherapy Reports
50(4):219-44 (1966)), one of ordinary skill in the art is able to
conveniently convert data obtained from the use of TR21 in a given
experimental system into an accurate estimation of a
pharmaceutically effective amount of TR21 polypeptide to be
administered per dose in another experimental system. Experimental
data obtained through the administration of TR21 in mice may
converted through the conversion factors supplied by Freireich, et
al., to accurate estimates of pharmaceutically effective doses of
TR21 in rat, monkey, dog, and human. The following conversion table
(Table III) is a summary of the data provided by Freireich, et al.
Table III gives approximate factors for converting doses expressed
in terms of mg/kg from one species to an equivalent surface area
dose expressed as mg/kg in another species tabulated.
TABLE-US-00003 TABLE III Equivalent Surface Area Dosage Conversion
Factors. --TO-- Mouse Rat Monkey Dog Human --FROM-- (20 g) (150 g)
(3.5 kg) (8 kg) (60 kg) Mouse 1 1/2 1/4 1/6 1/12 Rat 2 1 1/2 1/4
1/7 Monkey 4 2 1 3/5 1/3 Dog 6 4 5/3 1 1/2 Human 12 7 3 2 1
[0589] Thus, for example, using the conversion factors provided in
Table III, a dose of 50 mg/kg in the mouse converts to an
appropriate dose of 12.5 mg/kg in the monkey because (50
mg/kg).times.(1/4)=12.5 mg/kg. As an additional example, doses of
0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate to effect doses
of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7 micrograms/kg,
166.7 micrograms/kg, and 0.667 mg/kg, respectively, in the
human.
[0590] Pharmaceutical compositions containing TR21 polypeptides of
the invention may be administered orally, rectally, parenterally,
subcutaneously, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, ointments, drops or
transdermal patch), bucally, or as an oral or nasal spray (e.g.,
via inhalation of a vapor or powder). In one embodiment,
"pharmaceutically acceptable carrier" means a non-toxic solid,
semisolid or liquid filler, diluent, encapsulating material or
formulation auxiliary of any type. In a specific embodiment,
"pharmaceutically acceptable" means approved by a regulatory agency
of the federal or a state government or listed in the U.S.
Pharmacopoeia or other generally recognized pharmacopoeia for use
in animals, and more particularly humans. Nonlimiting examples of
suitable pharmaceutical carriers according to this embodiment are
provided in "Remington's Pharmaceutical Sciences" by E. W. Martin,
and include sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as
peanut oil, soybean oil, mineral oil, sesame oil and the like.
Water is a preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can be employed as liquid carriers,
particularly for injectable solutions. The composition, if desired,
can also contain minor amounts of wetting or emulsifying agents, or
pH buffering agents. These compositions can take the form of
solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like.
[0591] The term "parenteral" as used herein refers to modes of
administration which include intravenous, intramuscular,
intraperitoneal, intracisternal, subcutaneous and intraarticular
injection and infusion.
[0592] In a preferred embodiment, TR21 compositions of the
invention (including polypeptides, polynucleotides, and antibodies,
and agonists and/or antagonists thereof) are administered
subcutaneously.
[0593] In another preferred embodiment, TR21 compositions of the
invention (including polypeptides, polynucleotides, and antibodies,
and agonists and/or antagonists thereof) are administered
intravenously.
[0594] For parenteral administration, in one embodiment, the TR21
polypeptide is formulated generally by mixing it at the desired
degree of purity, in a unit dosage injectable form (solution,
suspension, or emulsion), with a pharmaceutically acceptable
carrier, i.e., one that is non-toxic to recipients at the dosages
and concentrations employed and is compatible with other
ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0595] Generally, the formulations are prepared by contacting the
TR21 polypeptide uniformly and intimately with liquid carriers or
finely divided solid carriers or both. Then, if necessary, the
product is shaped into the desired formulation. Preferably the
carrier is a parenteral carrier, more preferably a solution that is
isotonic with the blood of the recipient. Examples of such carrier
vehicles include water, saline, Ringer's solution, and dextrose
solution. Non-aqueous vehicles such as fixed oils and ethyl oleate
are also useful herein, as well as liposomes.
[0596] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, sucrose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; preservatives,
such as cresol, phenol, chlorobutanol, benzyl alcohol and parabens,
and/or nonionic surfactants such as polysorbates, poloxamers, or
PEG.
[0597] The TR21 polypeptide is typically formulated in such
vehicles at a concentration of about 0.001 mg/ml to 100 mg/ml, or
0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml or 1-10 mg/ml, at a
pH of about 3 to 10, or 3 to 8, more preferably 5-8, most
preferably 6-7. It will be understood that the use of certain of
the foregoing excipients, carriers, or stabilizers will result in
the formation of TR21 polypeptide salts.
[0598] TR21 polypeptide to be used for therapeutic administration
must be sterile. Sterility is readily accomplished by filtration
through sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic TR21 polypeptide compositions generally are placed into
a container having a sterile access port, for example, an
intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
[0599] TR21 polypeptide ordinarily will be stored in unit or
multi-dose containers, for example, sealed ampoules or vials, as an
aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
TR21 polypeptide solution, and the resulting mixture is
lyophilized. The infusion solution is prepared by reconstituting
the lyophilized TR21 polypeptide using bacteriostatic
Water-for-Injection.
[0600] Alternatively, TR21 polypeptide is stored in single dose
containers in lyophilized form. The infusion selection is
reconstituted using a sterile carrier for injection.
[0601] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally, associated with such container(s) is a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In addition, the polypeptides of the
present invention may be employed in conjunction with other
therapeutic compounds.
[0602] Pharmaceutical compositions of the present invention for
parenteral injection can comprise pharmaceutically acceptable
sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions as well as sterile powders for reconstitution into
sterile injectable solutions or dispersions just prior to use. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like.
[0603] In addition to soluble TR21 polypeptides, TR21 polypeptides
containing the transmembrane region can also be used when
appropriately solubilized by including detergents, such as CHAPS or
NP-40, with buffer.
[0604] TR21 compositions of the invention are also suitably
administered by sustained-release systems. Suitable examples of
sustained-release compositions include suitable polymeric materials
(such as, for example, semi-permeable polymer matrices in the form
of shaped articles, e.g., films, or microcapsules), suitable
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, and sparingly soluble derivatives
(such as, for example, a sparingly soluble salt).
[0605] Sustained-release matrices include polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and
gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.)
or poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0606] Sustained-release compositions also include liposomally
entrapped compositions of the invention (see generally, Langer,
Science 249:1527-1533 (1990); Treat et al., in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)).
Liposomes containing TR21 polypeptide may be prepared by methods
known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
(USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.
(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP
143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.
4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes
are of the small (about 200-800 Angstroms) unilamellar type in
which the lipid content is greater than about 30 mol. percent
cholesterol, the selected proportion being adjusted for the optimal
a polypeptide therapy.
[0607] In another embodiment sustained release compositions of the
invention include crystal formulations known in the art.
[0608] In yet an additional embodiment, the compositions of the
invention are delivered by way of a pump (see Langer, supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574
(1989)).
[0609] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0610] The compositions of the invention may be administered alone
or in combination with other adjuvants. Adjuvants that may be
administered with the compositions of the invention include, but
are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE
(Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a
specific embodiment, compositions of the invention are administered
in combination with alum. In another specific embodiment,
compositions of the invention are administered in combination with
QS-21. Further adjuvants that may be administered with the
compositions of the invention include, but are not limited to,
Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,
CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.
Vaccines that may be administered with the compositions of the
invention include, but are not limited to, vaccines directed toward
protection against MMR (measles, mumps, rubella), polio, varicella,
tetanus/diphtheria, hepatitis A, hepatitis B, haemophilus
influenzae B, whooping cough, pneumonia, influenza, Lyme disease,
rotavirus, cholera, yellow fever, Japanese encephalitis,
poliomyelitis, rabies, typhoid fever, and pertussis, and/or
PNEUMOVAX-23.TM.. Combinations may be administered either
concomitantly, e.g., as an admixture, separately but simultaneously
or concurrently; or sequentially. This includes presentations in
which the combined agents are administered together as a
therapeutic mixture, and also procedures in which the combined
agents are administered separately but simultaneously, e.g., as
through separate intravenous lines into the same individual.
Administration "in combination" further includes the separate
administration of one of the compounds or agents given first,
followed by the second.
[0611] In a specific embodiment, compositions of the invention may
be administered to patients as vaccine adjuvants. In a further
specific embodiment, compositions of the invention may be
administered as vaccine adjuvants to patients suffering from an
immune-deficiency. In a further specific embodiment, compositions
of the invention may be administered as vaccine adjuvants to
patients suffering from HIV.
[0612] In a specific embodiment, compositions of the invention may
be used to increase or enhance antigen-specific antibody responses
to standard and experimental vaccines. In a specific embodiment,
compositions of the invention may be used to enhance seroconversion
in patients treated with standard and experimental vaccines. In
another specific embodiment, compositions of the invention may be
used to increase the repertoire of antibodies recognizing unique
epitopes in response to standard and experimental vaccination.
[0613] In a preferred embodiment, TR21 polypeptides of the
invention (including TR21 fragments and variants, and anti-TR21
antibodies) increase or enhance antigen-specific antibody responses
to standard and experimental vaccines by regulating binding of the
soluble form of a TR21 ligand (e.g., Neutrokine-alpha,
Neutroline-alpha SV and/or one or more heteromultimeric polypeptide
complexes comprising one or more Neutrokine-alpha and/or
Neutrokine-alpha SV polypeptides), to TR21 and/or another
Neutrokine-alpha receptor (e.g., TACI and/or BCMA).
[0614] In a preferred embodiment, TR21 polypeptides of the
invention (including TR21 fragments and variants, and anti-TR21
antibodies) increase or enhance seroconversion in patients treated
with standard and experimental vaccines by regulating binding of
the soluble form of a TR21 ligand (e.g., Neutrokine-alpha,
Neutroline-alpha SV and/or one or more heteromultimeric polypeptide
complexes comprising one or more Neutrokine-alpha and/or
Neutrokine-alpha SV polypeptides), to TR21 and/or another
Neutrokine alpha receptor (e.g., TACI and/or BCMA).
[0615] In a preferred embodiment, TR21 polypeptides of the
invention (including TR21 fragments and variants, and anti-TR21
antibodies) increase or enhance the repertoire of antibodies
recognizing unique epitopes in response to standard and
experimental vaccination by regulating binding of the soluble form
of a TR21 ligand (e.g., Neutrokine-alpha, Neutroline-alpha SV
and/or one or more heteromultimeric polypeptide complexes
comprising one or more Neutrokine-alpha and/or Neutrokine-alpha SV
polypeptides), to TR21 and/or another Neutrokine alpha receptor
(e.g., TACI and/or BCMA).
[0616] In another specific embodiment, compositions of the
invention are used in combination with PNEUMOVAX-23.TM. to treat,
prevent, and/or diagnose infection and/or any disease, disorder,
and/or condition associated therewith. In one embodiment,
compositions of the invention are used in combination with
PNEUMOVAX-23.TM. to treat, prevent, and/or diagnose any
Gram-positive bacterial infection and/or any disease, disorder,
and/or condition associated therewith. In another embodiment,
compositions of the invention are used in combination with
PNEUMOVAX-23.TM. to treat, prevent, and/or diagnose infection
and/or any disease, disorder, and/or condition associated with one
or more members of the genus Enterococcus and/or the genus
Streptococcus. In another embodiment, compositions of the invention
are used in any combination with PNEUMOVAX-23.TM. to treat,
prevent, and/or diagnose infection and/or any disease, disorder,
and/or condition associated with one or more members of the Group B
streptococci. In another embodiment, compositions of the invention
are used in combination with PNEUMOVAX-23.TM. to treat, prevent,
and/or diagnose infection and/or any disease, disorder, and/or
condition associated with Streptococcus pneumoniae.
[0617] The compositions of the invention may be administered alone
or in combination with other therapeutic agents, including but not
limited to, chemotherapeutic agents, antibiotics, antivirals,
steroidal and non-steroidal anti-inflammatories, conventional
immunotherapeutic agents and cytokines. Combinations may be
administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially.
This includes presentations in which the combined agents are
administered together as a therapeutic mixture, and also procedures
in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into
the same individual. Administration "in combination" further
includes the separate administration of one of the compounds or
agents given first, followed by the second.
[0618] In one embodiment, the compositions of the invention are
administered in combination with other members of the TNF family.
TNF, TNF-related or TNF-like molecules that may be administered
with the compositions of the invention include, but are not limited
to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328),
TRAIL, AIM-II (International Publication No. WO 97/34911), APRIL
(International Publication Number WO 97/33902; J. Exp. Med.
188(6):1185-1190) (1998)), endokine-alpha (International
Publication No. WO 98/07880), Neutrokine-alpha and/or
Neutrokine-alpha SV (International Application Publication No. WO
98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble
forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International
Publication No. WO 96/34095), DR3 (International Publication No. WO
97/33904), DR4 (International Publication No. WO 98/32856), TR5
(International Publication No. WO 98/30693), TR6 (International
Publication No. WO 98/30694), TR7 (International Publication No. WO
98/41629), TRANK, TR9 (International Publication No. WO 98/56892),
312C2 (International Publication No. WO 98/06842), TR12, TACI (See,
e.g., U.S. Pat. No. 5,969,102; and von Bulow et al., Science
278:138-141 (1997)), CD 154, CD70, and CD153.
[0619] In a preferred embodiment, the compositions of the invention
are administered in combination with CD40 ligand (CD40L), a soluble
form of CD40L (e.g., AVREND.TM.), biologically active fragments,
variants, or derivatives of CD40L, anti-CD40L antibodies (e.g.,
agonistic or antagonistic antibodies), and/or anti-CD40 antibodies
(e.g., agonistic or antagonistic antibodies).
[0620] In a preferred embodiment, the compositions of the invention
are administered in combination with TACI (See e.g., U.S. Pat. No.
5,969,102; and von Bulow et al., Science 278:138-141 (1997)), a
soluble form of TACI, biologically active fragments, variants, or
derivatives of TACI (e.g., TACI-Fc), and/or anti-TACI antibodies
(e.g., agonistic or antagonistic antibodies).
[0621] In a preferred embodiment, the compositions of the invention
are administered in combination with Neutrokine-alpha and/or
Neutrokine-alpha SV (International Publication No. WO 98/18921), a
soluble form of Neutrokine alpha and/or Neutrokine-alpha SV,
biologically active fragments, variants, or derivatives of
Neutrokine-alpha and/or Neutrokine-alpha SV, and/or
anti-Neutrokine-alpha and/or anti-Neutrokine-alpha SV antibodies
(e.g., agonistic or antagonistic antibodies).
[0622] In a preferred embodiment, the compositions of the invention
are administered in combination with APRIL (International
Publication Number WO 97/33902; J. Exp. Med. 188(6):1185-1190
(1998)), a soluble form of APRIL, biologically active fragments,
variants, or derivatives of APRIL, and/or anti-APRIL antibodies
(e.g., agonistic or antagonistic antibodies).
[0623] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-angiogenic agent(s). Anti-angiogenic agents that may be
administered with the compositions of the invention include, but
are not limited to, Angiostatin (Entremed, Rockville, Md.),
Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel (Taxol),
Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor
of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1,
Plasminogen Activator Inhibitor-2, and various forms of the lighter
"d group" transition metals.
[0624] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
[0625] Representative examples of vanadium complexes include oxo
vanadium complexes such as vanadate and vanadyl complexes. Suitable
vanadate complexes include metavanadate and orthovanadate complexes
such as, for example, ammonium metavanadate, sodium metavanadate,
and sodium orthovanadate. Suitable vanadyl complexes include, for
example, vanadyl acetylacetonate and vanadyl sulfate including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
[0626] Representative examples of tungsten and molybdenum complexes
also include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[0627] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include, but are not limited to, platelet
factor 4; protamine sulphate; sulphated chitin derivatives
(prepared from queen crab shells), (Murata et al., Cancer Res.
51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex
(SP-PG) (the function of this compound may be enhanced by the
presence of steroids such as estrogen, and tamoxifen citrate);
Staurosporine; modulators of matrix metabolism, including for
example, proline analogs, cishydroxyproline,
d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl,
aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomikinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin
Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et
al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987);
anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol.
Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer
Institute); Lobenzarit disodium
(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or "CCA";
(Takeuchi et al., Agents Actions 36:312-316, 1992); and
metalloproteinase inhibitors such as BB94.
[0628] Additional anti-angiogenic factors that may also be utilized
within the context of the present invention include Thalidomide,
(CELGENE.TM., Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem
and J. Folkman, J Pediatr. Surg. 28:445-51 (1993)); an integrin
alpha v beta 3 antagonist (C. Storgard et al., J Clin. Invest.
103:47-54 (1999)); carboxyaminolmidazole; Carboxyamidotriazole
(CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin
A-4 (CA4P) (OXiGENE.TM., Boston, Mass.); Squalamine (Magainin
Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (TAP
PHARMACEUTICALS.TM., Deerfield, Ill.); ZD-0101 ASTRAZENECA.TM.
(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555);
CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA;
Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839);
Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint;
PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644);
Tamoxifen (NOLVADEX.TM.); Tazarotene; Tetrathiomolybdate;
XELODA.TM. (Capecitabine); and 5-Fluorouracil.
[0629] Anti-angiogenic agents that may be administered in
combination with the compounds of the invention may work through a
variety of mechanisms including, but not limited to, inhibiting
proteolysis of the extracellular matrix, blocking the function of
endothelial cell-extracellular matrix adhesion molecules, by
antagonizing the function of angiogenesis inducers such as growth
factors, and inhibiting integrin receptors expressed on
proliferating endothelial cells. Examples of anti-angiogenic
inhibitors that interfere with extracellular matrix proteolysis and
which may be administered in combination with the compositions of
the invention include, but are not limited to, AG-3340
(AGOURON.TM., La Jolla, Calif.), BAY-12-9566 (BAYER.TM., West
Haven, Conn.), BMS-275291 (BRISTOL MYERS SQUIBB.TM., Princeton,
N.J.), CGS-27032A (NOVARTIS.TM., East Hanover, N.J.),
MARIMASTAT.TM. (BRITISH BIOTECH.TM., Oxford, UK), and METASTAT.TM.
(AETERNA.TM., St-Foy, Quebec). Examples of anti-angiogenic
inhibitors that act by blocking the function of endothelial
cell-extracellular matrix adhesion molecules and which may be
administered in combination with the compositions of the invention
include, but are not limited to, EMD-121974 (MERCK.TM. KcgaA
Darmstadt, Germany) and VITAXIN.TM. (IXSYS.TM., La Jolla,
Calif./MEDIMMUNE.TM., Gaithersburg, Md.). Examples of
anti-angiogenic agents that act by directly antagonizing or
inhibiting angiogenesis inducers and which may be administered in
combination with the compositions of the invention include, but are
not limited to, ANGIOZYME.TM. (RIBOZYME.TM., Boulder, Colo.),
Anti-VEGF antibody (GENENTECH.TM., S. San Francisco, Calif.),
PTK-787/ZK-225846 (NOVARTIS.TM., Basel, Switzerland), SU-101
(SUGEN.TM., S. San Francisco, Calif), SU-5416
(SUGEN.TM./PHARMACIA.TM. Upjohn, Bridgewater, N.J.), and SU-6668
(SUGEN.TM.). Other anti-angiogenic agents act to indirectly inhibit
angiogenesis. Examples of indirect inhibitors of angiogenesis which
may be administered in combination with the compositions of the
invention include, but are not limited to, IM-862 (CYTRAN.TM.,
Kirkland, Wash.), Interferon-alpha, IL-12 (ROCHE.TM., Nutley,
N.J.), and Pentosan polysulfate (Georgetown University, Washington,
D.C.).
[0630] In particular embodiments, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
an autoimmune disease, such as for example, an autoimmune disease
described herein.
[0631] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use of compositions
of the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
rheumatoid arthritis or conditions associated therewith.
[0632] In a particular embodiment, the use of compositions of the
invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
arthritis. In a more particular embodiment, the use of compositions
of the invention in combination with anti-angiogenic agents is
contemplated for the treatment, prevention, and/or amelioration of
systemic lupus erythematosus or conditions associated
therewith.
[0633] In another embodiment, compositions of the invention are
administered in combination with an anticoagulant. Anticoagulants
that may be administered with the compositions of the invention
include, but are not limited to, heparin, warfarin, and aspirin. In
a specific embodiment, compositions of the invention are
administered in combination with heparin and/or warfarin. In
another specific embodiment, compositions of the invention are
administered in combination with warfarin. In another specific
embodiment, compositions of the invention are administered in
combination with warfarin and aspirin. In another specific
embodiment, compositions of the invention are administered in
combination with heparin. In another specific embodiment,
compositions of the invention are administered in combination with
heparin and aspirin.
[0634] In another embodiment, compositions of the invention are
administered in combination with an agent that suppresses the
production of anticardiolipin antibodies. In specific embodiments,
the polynucleotides of the invention are administered in
combination with an agent that blocks and/or reduces the ability of
anticardiolipin antibodies to bind phospholipid-binding plasma
protein beta 2-glycoprotein I (b2GPI).
[0635] In another embodiment, therapeutic or pharmaceutical
compositions of the invention are administered to an animal to
treat, prevent or ameliorate ischemia and arteriosclerosis.
Examples of such disorders include, but are not limited to,
reperfusion damage (e.g., in the heart and/or brain) and cardiac
hypertrophy.
[0636] Therapeutic or pharmaceutical compositions of the invention
may also be administered to modulate blood clotting and to treat or
prevent blood-clotting disorders, such as, for example,
antibody-mediated thrombosis (i.e., antiphospholipid antibody
syndrome (APS)). For example, therapeutic or pharmaceutical
compositions of the invention may inhibit the proliferation and
differentiation of cells involved in producing anticardiolipin
antibodies. These compositions of the invention can be used to
treat, prevent, ameliorate, diagnose, and/or prognose thrombotic
related events including, but not limited to, stroke (and recurrent
stroke), heart attack, deep vein thrombosis, pulmonary embolism,
myocardial infarction, coronary artery disease (e.g.,
antibody-mediated coronary artery disease), thrombosis, graft
reocclusion following cardiovascular surgery (e.g., coronary
arterial bypass grafts, recurrent fetal loss, and recurrent
cardiovascular thromboembolic events.
[0637] In certain embodiments, compositions of the invention are
administered in combination with antiretroviral agents, nucleoside
reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors. Nucleoside
reverse transcriptase inhibitors that may be administered in
combination with the compositions of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HIVID.TM. (zalcitabine/ddC), ZERIT.TM.
(stavudine/d4T), EPIVIR.TM. (lamivudine/3TC), and COMBIVIR.TM.
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase
inhibitors that may be administered in combination with the
compositions of the invention, include, but are not limited to,
VIRAMUNE.TM. (nevirapine), RESCRIPTOR.TM. (delavirdine), and
SUSTIVA.TM. (efavirenz). Protease inhibitors that may be
administered in combination with the compositions of the invention,
include, but are not limited to, CRIXIVAN.TM. (indinavir),
NORVIR.TM. (ritonavir), INVIRASE.TM. (saquinavir), and VIRACEPT.TM.
(nelfinavir). In a specific embodiment, antiretroviral agents,
nucleoside reverse transcriptase inhibitors, non-nucleoside reverse
transcriptase inhibitors, and/or protease inhibitors may be used in
any combination with compositions of the invention to treat,
prevent, and/or diagnose AIDS and/or to treat, prevent, and/or
diagnose HIV infection.
[0638] Additional NRTIs include LODENOSINE.TM. (F-ddA; an
acid-stable adenosine NRTI; Triangle/Abbott; COVIRACIL.TM.
(emtricitabine/FTC; structurally related to lamivudine (3TC) but
with 3- to 10-fold greater activity in vitro; Triangle/Abbott);
dOTC (BCH-10652, also structurally related to lamivudine but
retains activity against a substantial proportion of
lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused
approval for anti-HIV therapy by FDA; Gilead Sciences);
PREVEON.RTM. (Adefovir Dipivoxil, the active prodrug of adefovir;
its active form is PMEA-pp); TENOFOVIR.TM. (bis-POC PMPA, a PMPA
prodrug; Gilead); DAPD/DXG (active metabolite of DAPD;
Triangle/Abbott); D-D4FC (related to 3TC, with activity against
AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN.TM.
(abacavir/159U89; Glaxo Wellcome Inc.); CS-87
(3'azido-2',3'-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl
(SATE)-bearing prodrug forms of .beta.-L-FD4C and .beta.-L-FddC (WO
98/17281).
[0639] Additional NNRTIs include COACTINON.TM. (Emivirine/MKC-442,
potent NNRTI of the HEPT class; Triangle/Abbott); CAPRAVIRINE.TM.
(AG-1549/S-1153, a next generation NNRTI with activity against
viruses containing the K103N mutation; AGOURON.TM.); PNU-142721
(has 20- to 50-fold greater activity than its predecessor
delavirdine and is active against K103N mutants; PHARMACIA.TM.
& Upjohn); DPC-961 and DPC-963 (second-generation derivatives
of efavirenz, designed to be active against viruses with the K103N
mutation; DuPont); GW-420867X (has 25-fold greater activity than
HBY097 and is active against K103N mutants; Glaxo Wellcome);
CALANOLIDE A (naturally occurring agent from the latex tree; active
against viruses containing either or both the Y181C and K103N
mutations); and Propolis (WO 99/49830).
[0640] Additional protease inhibitors include LOPINAVIR.TM.
(ABT378/r; Abbott Laboratories); BMS-232632 (an azapeptide;
Bristol-Myers Squibb); TIPRANAVIR.TM. (PNU-140690, a non-peptic
dihydropyrone; PHARMACIA.TM. & Upjohn); PD-178390 (a
nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide;
Bristol-Myers Squibb); L-756,423 (an indinavir analog; Merck);
DMP-450 (a cyclic urea compound; Avid & DuPont); AG-1776 (a
peptidomimetic with in vitro activity against protease
inhibitor-resistant viruses; AGOURON.TM.); VX-175/GW-433908
(phosphate prodrug of amprenavir; Vertex & Glaxo Welcome);
CGP61755 (Ciba); and AGENERASE.TM. (amprenavir; Glaxo Wellcome
Inc.).
[0641] Additional antiretroviral agents include fusion
inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include
T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane
protein ectodomain which binds to gp41 in its resting state and
prevents transformation to the fusogenic state; Trimeris) and
T-1249 (a second-generation fusion inhibitor; Trimeris).
[0642] Additional antiretroviral agents include fusion
inhibitors/chemokine receptor antagonists. Fusion
inhibitors/chemokine receptor antagonists include CXCR4 antagonists
such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C
(a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and
the T22 analogs T134 and T140; CCR5 antagonists such as RANTES
(9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4
antagonists such as NSC 651016 (a distamycin analog). Also included
are CCR2B, CCR3, and CCR6 antagonists. Chemokine receptor agonists
such as RANTES, SDF-1, MIP-1.alpha., MIP-1.beta., etc., may also
inhibit fusion.
[0643] Additional antiretroviral agents include integrase
inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA)
acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid);
quinalizarin (QLC) and related anthraquinones; ZINTEVIR.TM. (AR
177, an oligonucleotide that probably acts at cell surface rather
than being a true integrase inhibitor; Arondex); and naphthols such
as those disclosed in WO 98/50347.
[0644] Additional antiretroviral agents include hydroxyurea-like
compounds such as BCX-34 (a purine nucleoside phosphorylase
inhibitor; Biocryst); ribonucleotide reductase inhibitors such as
DIDOX.TM. (Molecules for Health); inosine monophosphate
dehydrogenase (IMPDH) inhibitors such as VX-497 (Vertex); and
myvopholic acids such as CellCept (mycophenolate mofetil;
ROCHE.TM.).
[0645] Additional antiretroviral agents include inhibitors of viral
integrase, inhibitors of viral genome nuclear translocation such as
arylene bis(methylketone) compounds; inhibitors of HIV entry such
as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble
complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100;
nucleocapsid zinc finger inhibitors such as dithiane compounds;
targets of HIV Tat and Rev; and pharmacoenhancers such as
ABT-378.
[0646] Other antiretroviral therapies and adjunct therapies include
cytokines and lymphokines such as MIP-1.alpha., MIP-1.beta.,
SDF-1.alpha., IL-2, PROLEUKIN.TM. (aldesleukin/L2-7001;
CHIRON.TM.), IL-4, IL-10, IL-12, and IL-13; interferons such as
IFN-.alpha.2a; antagonists of TNFs, NF.kappa.B, GM-CSF, M-CSF, and
IL-10; agents that modulate immune activation such as cyclosporin
and prednisone; vaccines such as Remune.TM. (HIV Immunogen), APL
400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E)
recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2
rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched
synthetic peptide derived from discontinuous gp120 C3/C4 domain,
fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines;
gene-based therapies such as genetic suppressor elements (GSEs; WO
98/54366), and intrakines (genetically modified CC chemokines
targeted to the ER to block surface expression of newly synthesized
CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med.
3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5,
the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and
PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3
antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D,
268-D and 50.1, anti-Tat antibodies, anti-TNF-.alpha. antibodies,
and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor
agonists and antagonists such as TCDD,
3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and
.alpha.-naphthoflavone (WO 98/30213); and antioxidants such as
.gamma.-L-glutamyl-L-cysteine ethyl ester (.gamma.-GCE; WO
99/56764).
[0647] In other embodiments, compositions of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the compositions of the invention, include, but
are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM.,
PENTAMIDINE.TM., ATOVAQUONE.TM., ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., ETHAMBUTOL.TM., RIFABUTIN.TM.,
CLARITHROMYCIN.TM., AZITHROMYCIN.TM., GANCICLOVIR.TM.,
FOSCARNET.TM., CIDOFOVIR.TM., FLUCONAZOLE.TM., ITRACONAZOLE.TM.,
KETOCONAZOLE.TM., ACYCLOVIR.TM., FAMCICOLVIR.TM.,
PYRIMETHAMINE.TM., LEUCOVORIN.TM., NEUPOGEN.TM. (filgrastim/G-CSF),
and LEUKINE.TM. (sargramostim/GM-CSF). In a specific embodiment,
compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat, prevent, and/or
diagnose an opportunistic Pneumocystis carnii pneumonia infection.
In another specific embodiment, compositions of the invention are
used in any combination with ISONIAZID.TM., RIFAMPIN.TM.,
PYRAZINAMIDE.TM., and/or ETHAMBUTOL.TM. to prophylactically treat,
prevent, and/or diagnose an opportunistic Mycobacterium avium
complex infection. In another specific embodiment, compositions of
the invention are used in any combination with RIFABUTIN.TM.,
CLARITHROMYCIN.TM., and/or AZITHROMYCIN.TM. to prophylactically
treat, prevent, and/or diagnose an opportunistic Mycobacterium
tuberculosis infection. In another specific embodiment,
compositions of the invention are used in any combination with
GANCICLOVIR.TM., FOSCARNET.TM., and/or CIDOFOVIR.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
cytomegalovirus infection. In another specific embodiment,
compositions of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
fungal infection. In another specific embodiment, compositions of
the invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic herpes simplex virus type I and/or type II
infection. In another specific embodiment, compositions of the
invention are used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat, prevent, and/or diagnose
an opportunistic Toxoplasma gondii infection. In another specific
embodiment, compositions of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat, prevent, and/or diagnose an opportunistic
bacterial infection.
[0648] In a further embodiment, the compositions of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the compositions of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[0649] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases,
Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin,
ciprofloxacin, erythromycin, fluoroquinolones, macrolides,
metronidazole, penicillins, quinolones, rifampin, streptomycin,
sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamethoxazole, and vancomycin.
[0650] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs cyclophosphamide, cyclophosphamide IV,
methylprednisolone, prednisolone, azathioprine, FK-506,
15-deoxyspergualin, and other immunosuppressive agents that act by
suppressing the function of responding T cells. Other
immunosuppressive agents, that may be administered in combination
with the compositions of the invention include, but are not limited
to, prednisolone, methotrexate, thalidomide, methoxsalen,
rapamycin, leflunomide, mizoribine (BREDINN.TM.), brequinar,
deoxyspergualin, and azaspirane (SKF 105685).
[0651] In specific embodiments, compositions of the invention are
administered in combination with immunosuppressants.
Immunosuppressants preparations that may be administered with the
compositions of the invention include, but are not limited to,
ORTHOCLONE.TM. (OKT3), (muromonab-CD3), SANDIMMUNE.TM., NEORAL.TM.,
SANGDYA.TM. (cyclosporine), PROGRAF.RTM. (FK506, tacrolimus),
CELLCEPT.RTM. (mycophenolate motefil, of which the active
metabolite is mycophenolic acid), IMURAN.TM. (azathioprine),
corticosteroids, adrenocortical steroids such as DELTASONE.TM.
(prednisone) and HYDELTRASOL.TM. (prednisolone), FOLEX.TM. and
MEXATE.TM. (methotrexate), OXSORALEN-ULTRA.TM. (methoxsalen) and
RAPAMUNE.TM. (sirolimus). In a specific embodiment,
immunosuppressants may be used to prevent rejection of organ or
bone marrow transplantation.
[0652] In a preferred embodiment, the compositions of the invention
are administered in combination with steroid therapy. Steroids that
may be administered in combination with the compositions of the
invention, include, but are not limited to, oral corticosteroids,
prednisone, and methylprednisolone (e.g., IV methylprednisolone).
In a specific embodiment, compositions of the invention are
administered in combination with prednisone. In a further specific
embodiment, the compositions of the invention are administered in
combination with prednisone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and prednisone are those described
herein, and include, but are not limited to, azathioprine,
cyclophosphamide, and cyclophosphamide IV. In another specific
embodiment, compositions of the invention are administered in
combination with methylprednisolone. In a further specific
embodiment, the compositions of the invention are administered in
combination with methylprednisolone and an immunosuppressive agent.
Immunosuppressive agents that may be administered with the
compositions of the invention and methylprednisolone are those
described herein, and include, but are not limited to,
azathioprine, cyclophosphamide, and cyclophosphamide IV.
[0653] In a preferred embodiment, the compositions of the invention
are administered in combination with an antimalarial. Antimalarials
that may be administered with the compositions of the invention
include, but are not limited to, hydroxychloroquine, chloroquine,
and/or quinacrine.
[0654] In a preferred embodiment, the compositions of the invention
are administered in combination with an NSAID.
[0655] In a nonexclusive embodiment, the compositions of the
invention are administered in combination with one, two, three,
four, five, ten, or more of the following drugs: NRD-101 (HOECHST
MARION ROUSSEL.TM.), diclofenac (Dimethaid), oxaprozin potassium
(MONSANTO.TM.), mecasermin (CHIRON.TM.), T-614 (TOYAMA.TM.),
pemetrexed disodium (ELI LILLY.TM.), atreleuton (ABBOTT.TM.),
valdecoxib (MONSANTO.TM.), eltenac (Byk Gulden), CAMPATH.TM.,
AGM-1470 (TAKEDA.TM.), CDP-571 (CELLTECH CHIROSCIENCE.TM.), CM-101
(CarboMed), ML-3000 (Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS
Biomedix), IL-1Ra gene therapy (VALENTIS.TM.), JTE-522 (JAPAN
TOBACCO.TM.), paclitaxel (ANGIOTECH.TM.), DW-166HC (Dong Wha),
darbufelone mesylate (WARNER-LAMBERT.TM.), soluble TNF receptor 1
(SYNERGEN.TM.; AMGEN.TM.), IPR-6001 (Institute for Pharmaceutical
Research), trocade (HOFFMAN-LA ROCHE.TM.), EF-5 (SCOTIA
PHARMACEUTICALS.TM.), BIIL-284 (BOEHRINGER INGELHEIM.TM.),
BIIF-1149 (BOEHRINGER INGELHEIM.TM.), LEUKOVAX.TM.
(INFLAMMATICS.TM.), MK-663 (MERCK.TM.), ST-1482 (Sigma-Tau), and
butixocort propionate (WARNER-LAMBERT.TM.).
[0656] In one embodiment, the compositions of the invention are
administered in combination with one or more of the following
drugs: infliximab (also known as REMICADE.TM. Centocor, Inc.),
Trocade (ROCHE.TM., RO-32-3555), Leflunomide (also known as
ARAVA.TM. from HOECHST MARION ROUSSEL.TM.), KINERET.TM. (an IL-1
Receptor antagonist also known as Anakinra from Amgen, Inc.),
SCIO-469 (p38 kinase inhibitor from Scios, Inc), and/or ASLERA.TM.
(prasterone, dehydroepiandrosterone, GL701) from Genelabs
Technologies Inc.
[0657] In a preferred embodiment, the compositions of the invention
are administered in combination with one, two, three, four, five or
more of the following drugs: methotrexate, sulfasalazine, sodium
aurothiomalate, auranofin, cyclosporine, penicillamine,
azathioprine, an antimalarial drug (e.g., as described herein),
cyclophosphamide, chlorambucil, gold, ENBREL.TM. (Etanercept),
anti-TNF antibody, LJP 394 (La Jolla Pharmaceutical Company, San
Diego, Calif.) and prednisolone.
[0658] In a more preferred embodiment, the compositions of the
invention are administered in combination with an antimalarial,
methotrexate, anti-TNF antibody, ENBREL.TM. and/or suflasalazine.
In one embodiment, the compositions of the invention are
administered in combination with methotrexate. In another
embodiment, the compositions of the invention are administered in
combination with anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
methotrexate and anti-TNF antibody. In another embodiment, the
compositions of the invention are administered in combination with
suflasalazine. In another specific embodiment, the compositions of
the invention are administered in combination with methotrexate,
anti-TNF antibody, and suflasalazine. In another embodiment, the
compositions of the invention are administered in combination
ENBREL.TM.. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM. and
methotrexate. In another embodiment, the compositions of the
invention are administered in combination with ENBREL.TM.,
methotrexate and suflasalazine. In another embodiment, the
compositions of the invention are administered in combination with
ENBREL.TM., methotrexate and suflasalazine. In other embodiments,
one or more antimalarials are combined with one of the
above-recited combinations. In a specific embodiment, the
compositions of the invention are administered in combination with
an antimalarial (e.g., hydroxychloroquine), ENBREL.TM.,
methotrexate and suflasalazine. In another specific embodiment, the
compositions of the invention are administered in combination with
an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNF
antibody, and methotrexate.
[0659] In an additional embodiment, compositions of the invention
are administered alone or in combination with one or more
intravenous immune globulin preparations. Intravenous immune
globulin preparations that may be administered with the
compositions of the invention include, but not limited to,
GAMMAR.TM., IVEEGAM.TM., SANDOGLOBULIN.TM., GAMMAGARD S/D.TM., and
GAMIMUNE.TM.. In a specific embodiment, compositions of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0660] In an additional embodiment, the compositions of the
invention are administered in combination with CD40 ligand (CD40L),
a soluble form of CD40L (e.g., AVREND.TM.), biologically active
fragments, variants, or derivatives of CD40L, anti-CD40L antibodies
(e.g., agonistic or antagonistic antibodies), and/or anti-CD40
antibodies (e.g., agonistic or antagonistic antibodies).
[0661] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0662] In another embodiment, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
methoxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0663] In a specific embodiment, compositions of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or any combination of the
components of CHOP. In one embodiment, the compositions of the
invention are administered in combination with anti-CD20
antibodies, human monoclonal anti-CD20 antibodies. In another
embodiment, the compositions of the invention are administered in
combination with anti-CD20 antibodies and CHOP, or anti-CD20
antibodies and any combination of one or more of the components of
CHOP, particularly cyclophosphamide and/or prednisone. In a
specific embodiment, compositions of the invention are administered
in combination with Rituximab. In a further embodiment,
compositions of the invention are administered with Rituximab and
CHOP, or Rituximab and any combination of one or more of the
components of CHOP, particularly cyclophosphamide and/or
prednisone. In a specific embodiment, compositions of the invention
are administered in combination with tositumomab (anti-CD20
antibody from Coulter Pharmaceuticals, San Francisco, Calif.). In a
further embodiment, compositions of the invention are administered
with tositumomab and CHOP, or tositumomab and any combination of
one or more of the components of CHOP, particularly
cyclophosphamide and/or prednisone. Tositumomab may optionally be
associated with .sup.131I. The anti-CD20 antibodies may optionally
be associated with radioisotopes, toxins or cytotoxic prodrugs.
[0664] In another specific embodiment, the compositions of the
invention are administered in combination Zevalin.TM.. In a further
embodiment, compositions of the invention are administered with
Zevalin.TM. and CHOP, or Zevalin.TM. and any combination of one or
more of the components of CHOP, particularly cyclophosphamide
and/or prednisone. Zevalin.TM. may be associated with one or more
radioisotopes. Particularly preferred isotopes are .sup.90Y and
.sup.111In.
[0665] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5,
IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-alpha,
IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another
embodiment, compositions of the invention may be administered with
any interleukin, including, but not limited to, IL-1alpha,
IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, IL-21, and IL-22. In preferred embodiments, the compositions
of the invention are administered in combination with IL4 and
IL10.
[0666] In one embodiment, the compositions of the invention are
administered in combination with one or more chemokines. In
specific embodiments, the compositions of the invention are
administered in combination with an .alpha.(C.times.C) chemokine
selected from the group consisting of gamma-interferon inducible
protein-10 (.gamma.IP-10), interleukin-8 (IL-8), platelet factor-4
(PF4), neutrophil activating protein (NAP-2), GRO-.alpha.,
GRO-.beta., GRO-.gamma., neutrophil-activating peptide (ENA-78),
granulocyte chemoattractant protein-2 (GCP-2), and stromal
cell-derived factor-1 (SDF-1, or pre-B cell stimulatory factor
(PBSF)); and/or a .beta.(CC) chemokine selected from the group
consisting of: RANTES (regulated on activation, normal T expressed
and secreted), macrophage inflammatory protein-1 alpha
(MIP-1.alpha.), macrophage inflammatory protein-1 beta
(MIP-1.beta.), monocyte chemotactic protein-1 (MCP-1), monocyte
chemotactic protein-2 (MCP-2), monocyte chemotactic protein-3
(MCP-3), monocyte chemotactic protein-4 (MCP-4) macrophage
inflammatory protein-1 gamma (MIP-1.gamma.), macrophage
inflammatory protein-3 alpha (MIP-3.alpha.), macrophage
inflammatory protein-3 beta (MIP-3.beta.), macrophage inflammatory
protein-4 (MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and I-309; and/or
the .gamma.(C) chemokine, lymphotactin.
[0667] In another embodiment, the compositions of the invention are
administered with chemokine beta-8, chemokine beta-1, and/or
macrophage inflammatory protein-4. In a preferred embodiment, the
compositions of the invention are administered with chemokine
beta-8.
[0668] In an additional embodiment, the compositions of the
invention are administered in combination with an IL-4 antagonist.
IL-4 antagonists that may be administered with the compositions of
the invention include, but are not limited to: soluble IL-4
receptor polypeptides, multimeric forms of soluble IL-4 receptor
polypeptides; anti-IL-4 receptor antibodies that bind the IL-4
receptor without transducing the biological signal elicited by
IL-4, anti-IL4 antibodies that block binding of IL-4 to one or more
IL-4 receptors, and muteins of IL-4 that bind IL-4 receptors but do
not transduce the biological signal elicited by IL-4. Preferably,
the antibodies employed according to this method are monoclonal
antibodies (including antibody fragments, such as, for example,
those described herein).
[0669] In an additional embodiment, the compositions of the
invention are administered in combination with an IL-13 antagonist.
IL-13 antagonists that may be administered with the compositions of
the invention include, but are not limited to: soluble IL-13
receptor polypeptides, multimeric forms of soluble IL-13 receptor
polypeptides; anti-IL-13 receptor antibodies that bind the IL-13
receptor without transducing the biological signal elicited by
IL-13, anti-IL-13 antibodies that block binding of IL-13 to one or
more IL-13 receptors, and muteins of IL-13 that bind IL-13
receptors but do not transduce the biological signal elicited by
IL-13. Preferably, the antibodies employed according to this method
are monoclonal antibodies (including antibody fragments, such as,
for example, those described herein).
[0670] The invention also encompasses combining the polynucleotides
and/or polypeptides of the invention (and/or agonists or
antagonists thereof) with other proposed or conventional
hematopoietic therapies. Thus, for example, the polynucleotides
and/or polypeptides of the invention (and/or agonists or
antagonists thereof) can be combined with compounds that singly
exhibit erythropoietic stimulatory effects, such as erythropoietin,
testosterone, progenitor cell stimulators, insulin-like growth
factor, prostaglandins, serotonin, cyclic AMP, prolactin, and
triiodothyzonine. Also encompassed are combinations of the
compositions of the invention with compounds generally used to
treat aplastic anemia, such as, for example, methenolene,
stanozolol, and nandrolone; to treat iron-deficiency anemia, such
as, for example, iron preparations; to treat malignant anemia, such
as, for example, vitamin B.sub.12 and/or folic acid; and to treat
hemolytic anemia, such as, for example, adrenocortical steroids,
e.g., corticoids. See e.g., Resegotti et al., Panminerva Medica,
23:243-248 (1981); Kurtz, FEBS Letters, 14a:105-108 (1982);
McGonigle et al., Kidney Int., 25:437-444 (1984); and
Pavlovic-Kantera, Expt. Hematol., 8(supp. 8) 283-291 (1980), the
contents of each of which are hereby incorporated by reference in
their entireties.
[0671] Compounds that enhance the effects of or synergize with
erythropoietin are also useful as adjuvants herein, and include but
are not limited to, adrenergic agonists, thyroid hormones,
androgens, hepatic erythropoietic factors, erythrotropins, and
erythrogenins, See for e.g., Dunn, "Current Concepts in
Erythropoiesis", John Wiley and Sons (Chichester, England, 1983);
Kalmani, Kidney Int., 22:383-391 (1982); Shahidi, New Eng. J. Med.,
289:72-80 (1973); Urabe et al., J. Exp. Med., 149:1314-1325 (1979);
Billat et al., Expt. Hematol., 10:133-140 (1982); Naughton et al.,
Acta Haemat, 69:171-179 (1983); Cognote et al. in abstract 364,
Proceedings 7th Intl. Cong. of Endocrinology (Quebec City, Quebec,
Jul. 1-7, 1984); and Rothman et al., 1982, J. Surg. Oncol.,
20:105-108 (1982). Methods for stimulating hematopoiesis comprise
administering a hematopoietically effective amount (i.e., an amount
which effects the formation of blood cells) of a pharmaceutical
composition containing polynucleotides and/or polypeptides of the
invention (and/or agonists or antagonists thereof) to a patient.
The polynucleotides and/or polypeptides of the invention and/or
agonists or antagonists thereof is administered to the patient by
any suitable technique, including but not limited to, parenteral,
sublingual, topical, intrapulmonary and intranasal, and those
techniques further discussed herein. The pharmaceutical composition
optionally contains one or more members of the group consisting of
erythropoietin, testosterone, progenitor cell stimulators,
insulin-like growth factor, prostaglandins, serotonin, cyclic AMP,
prolactin, triiodothyzonine, methenolene, stanozolol, and
nandrolone, iron preparations, vitamin B.sub.12, folic acid and/or
adrenocortical steroids.
[0672] In an additional embodiment, the compositions of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the compositions of the invention include, but are not limited to,
LEUKINE.TM. (SARGRAMOSTIM.TM.) and NEUPOGEN.TM.
(FILGRASTIM.TM.).
[0673] In an additional embodiment, the compositions of the
invention are administered in combination with fibroblast growth
factors. Fibroblast growth factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0674] Additionally, the compositions of the invention may be
administered alone or in combination with other therapeutic
regimens, including but not limited to, radiation therapy. Such
combinatorial therapy may be administered sequentially and/or
concomitantly.
Agonists and Antagonists--Assays and Molecules
[0675] The invention also provides a method of screening compounds
to identify those compounds which enhance or block the action of
TR21 polypeptide on cells, such as its interaction with TR21
binding molecules such as ligand molecules. An agonist is a
compound which increases the natural biological functions of TR21
or which functions in a manner similar to TR21 while antagonists
decrease or eliminate such functions.
[0676] In another embodiment, the invention provides a method for
identifying a ligand protein or other ligand-binding protein which
binds specifically to TR21 polypeptide. For example, a cellular
compartment, such as a membrane or a preparation thereof, may be
prepared from a cell that expresses a molecule that binds
Neutrokine-alpha. The preparation is incubated with labeled
Neutrokine-alpha and complexes of Neutrokine-alpha bound to TR21 or
other binding protein are isolated and characterized according to
routine methods known in the art. Alternatively, the TR21 ligand
polypeptide may be bound to a solid support so that binding
molecules solubilized from cells are bound to the column and then
eluted and characterized according to routine methods.
[0677] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
TR21 such as a molecule of a signaling or regulatory pathway
modulated by TR21. The preparation is incubated with labeled TR21
in the absence or the presence of a candidate molecule which may be
a TR21 agonist or antagonist. The ability of the candidate molecule
to bind the binding molecule is reflected in decreased binding of
the labeled ligand. Molecules which bind gratuitously, i.e.,
without inducing the effects of TR21 on binding the TR21 binding
molecule, are most likely to be good antagonists. Molecules that
bind well and elicit effects that are the same as or closely
related to TR21 are agonists.
[0678] By "agonist" is intended naturally occurring and synthetic
compounds capable of enhancing or potentiating TR21 mediated
signaling. By "antagonist" is intended naturally occurring and
synthetic compounds capable of inhibiting apoptosis. Whether any
candidate "agonist" or "antagonist" of the present invention can
enhance or inhibit TR21 mediated signaling can be determined using
art-known TNF-family ligand/receptor cellular response assays,
including those described in more detail below.
[0679] One such screening procedure involves the use of
melanophores which are transfected to express the receptor of the
present invention. Such a screening technique is described in PCT
WO 92/01810. Such an assay may be employed, for example, for
screening for a compound which inhibits (or enhances) activation of
the receptor polypeptide of the present invention by contacting the
melanophore cells which encode the receptor with both a TNF-family
ligand and the candidate antagonist (or agonist). Inhibition or
enhancement of the signal generated by the ligand indicates that
the compound is an antagonist or agonist of the ligand/receptor
signaling pathway.
[0680] Other screening techniques include the use of cells which
express the receptor (for example, transfected CHO cells) in a
system which measures extracellular pH changes caused by receptor
activation. For example, compounds may be contacted with a cell
which expresses the receptor polypeptide of the present invention
and a second messenger response, e.g., signal transduction or pH
changes, may be measured to determine whether the potential
compound activates or inhibits the receptor.
[0681] Another such screening technique involves introducing RNA
encoding the receptor into Xenopus oocytes to transiently express
the receptor. The receptor oocytes may then be contacted with the
receptor ligand and a compound to be screened, followed by
detection of inhibition or activation of a calcium signal in the
case of screening for compounds which are thought to inhibit
activation of the receptor.
[0682] Another screening technique well known in the art involves
expressing in cells a construct wherein the receptor is linked to a
phospholipase C or D. Exemplary cells include endothelial cells,
smooth muscle cells, embryonic kidney cells, etc. The screening may
be accomplished as hereinabove described by detecting activation of
the receptor or inhibition of activation of the receptor from the
phospholipase signal.
[0683] Another method involves screening for compounds which
inhibit activation of the receptor polypeptide of the present
invention antagonists by determining inhibition of binding of
labeled ligand to cells which have the receptor on the surface
thereof. Such a method involves transfecting a eukaryotic cell with
DNA encoding the receptor such that the cell expresses the receptor
on its surface and contacting the cell with a compound in the
presence of a labeled form of a known ligand. The ligand can be
labeled, e.g., by radioactivity. The amount of labeled ligand bound
to the receptors is measured, e.g., by measuring radioactivity of
the receptors. If the compound binds to the receptor as determined
by a reduction of labeled ligand which binds to the receptors, the
binding of labeled ligand to the receptor is inhibited.
[0684] Further screening assays for agonists and antagonists of the
present invention are described in L. A. Tartaglia and D. V.
Goeddel, J. Biol. Chem. 267:4304-4307(1992).
[0685] Thus, in a further aspect, a screening method is provided
for determining whether a candidate agonist or antagonist is
capable of enhancing or inhibiting a cellular response to a
TNF-family ligand. The method involves contacting cells which
express the TR21 polypeptide with a candidate compound and a
TNF-family ligand, assaying a cellular response, and comparing the
cellular response to a standard cellular response, the standard
being assayed when contact is made with the ligand in absence of
the candidate compound, whereby an increased cellular response over
the standard indicates that the candidate compound is an agonist of
the ligand/receptor signaling pathway and a decreased cellular
response compared to the standard indicates that the candidate
compound is an antagonist of the ligand/receptor signaling pathway.
By "assaying a cellular response" is intended qualitatively or
quantitatively measuring a cellular response to a candidate
compound and/or a TNF-family ligand (e.g., determining or
estimating an increase or decrease in B and/or T cell proliferation
or tritiated thymidine labeling). By the invention, a cell
expressing the TR21 polypeptide can be contacted with either an
endogenous or exogenously administered TNF-family ligand.
[0686] TR21-like effects of potential agonists and antagonists may
by measured, for instance, by determining activity of a second
messenger system following interaction of the candidate molecule
with a cell or appropriate cell preparation, and comparing the
effect with that of TR21 or molecules that elicit the same effects
as TR21. Second messenger systems that may be useful in this regard
include but are not limited to AMP guanylate cyclase, ion channel
or phosphoinositide hydrolysis second messenger systems.
[0687] Another example of an assay for TR21 antagonists is a
competitive assay that combines TR21 and a potential antagonist
with membrane-bound ligand molecules or recombinant TR21 ligand
molecules under appropriate conditions for a competitive inhibition
assay. TR21 can be labeled, such as by radioactivity, such that the
number of TR21 molecules bound to a ligand molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0688] Agonists according to the present invention include
naturally occurring and synthetic compounds such as, for example,
the CD40 ligand, neutral amino acids, zinc, estrogen, androgens,
viral genes (such as Adenovirus E1B, Baculovirus p.sup.35 and IAP,
Cowpox virus crmA, Epstein-Barr virus BHRF1, LMP-1, African swine
fever virus LMW5-HL, and Herpesvirus y1 34.5), calpain inhibitors,
cysteine protease inhibitors, and tumor promoters (such as PMA,
Phenobarbital, and .alpha.-Hexachlorocyclohexane).
[0689] Antagonist according to the present invention include
naturally occurring and synthetic compounds such as, for example,
TNF family ligand peptide fragments, transforming growth factor,
neurotransmitters (such as glutamate, dopamine,
N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells
and antimetabolites. Preferred agonists include chemotherapeutic
drugs such as, for example, cisplatin, doxorubicin, bleomycin,
cytosine arabinoside, nitrogen mustard, methotrexate and
vincristine. Others include ethanol and -amyloid peptide. (Science
267:1457-1458 (1995)). Further preferred agonists include TR21
polypeptides of the invention, polyclonal and monoclonal antibodies
raised against the TR21 polypeptide, or a fragment thereof. Such
agonist antibodies raised against a TNF-family receptor are
disclosed in L. A. Tartaglia et al., Proc. Natl. Acad. Sci. USA
88:9292-9296 (1991); and L. A. Tartaglia and D. V. Goeddel, J.
Biol. Chem. 267:4304-4307(1992). See, also, PCT Application WO
94/09137.
[0690] Potential antagonists include small organic molecules,
peptides, polypeptides (e.g., IL-13), and antibodies that bind to a
polypeptide of the invention and thereby inhibit or extinguish its
activity. Potential antagonists also may be small organic
molecules, a peptide, a polypeptide such as a closely related
protein or antibody that binds the same sites on a binding
molecule, such as a ligand molecule, without inducing TR21 induced
activities, thereby preventing the action of TR21 by excluding TR21
from binding.
[0691] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, Fla. (1988). Antisense
technology can be used to control gene expression through antisense
DNA or RNA, or through triple-helix formation. Antisense techniques
are discussed for example, in Okano, J. Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance Lee et al., Nucleic Acids Research 6:
3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et
al., Science 251: 1360 (1991). The methods are based on binding of
a polynucleotide to a complementary DNA or RNA. For example, the 5'
coding portion of a polynucleotide that encodes the extracellular
domain of the polypeptide of the present invention may be used to
design an antisense RNA oligonucleotide of from about 10 to 40 base
pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of TR21. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into TR21 polypeptide. The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of TR21.
[0692] In one embodiment, the TR21 antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid (RNA) of the
invention. Such a vector would contain a sequence encoding the TR21
antisense nucleic acid. Such a vector can remain episomal or become
chromosomally integrated, as long as it can be transcribed to
produce the desired antisense RNA. Such vectors can be constructed
by recombinant DNA technology methods standard in the art. Vectors
can be plasmid, viral, or others know in the art, used for
replication and expression in vertebrate cells. Expression of the
sequence encoding TR21, or fragments thereof, can be by any
promoter known in the art to act in vertebrate, preferably human
cells. Such promoters can be inducible or constitutive. Such
promoters include, but are not limited to, the SV40 early promoter
region (Bemoist and Chambon, Nature 29:304-310 (1981)), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpes
thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
78:1441-1445 (1981)), the regulatory sequences of the
metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)),
etc.
[0693] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a TR21 gene. However, absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA," referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex; in the case of double stranded TR21
antisense nucleic acids, a single strand of the duplex DNA may thus
be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the
length of the antisense nucleic acid. Generally, the larger the
hybridizing nucleic acid, the more base mismatches with a TR21 RNA
it may contain and still form a stable duplex (or triplex as the
case may be). One skilled in the art can ascertain a tolerable
degree of mismatch by use of standard procedures to determine the
melting point of the hybridized complex.
[0694] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
1994, Nature 372:333-335. Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of TR21
shown in FIG. 1, respectively, could be used in an antisense
approach to inhibit translation of endogenous TR21 mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of TR21 mRNA, antisense nucleic acids
should be at least six nucleotides in length, and are preferably
oligonucleotides ranging from 6 to about 50 nucleotides in length.
In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[0695] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 (1987); PCT
Publication No. WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0696] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0697] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0698] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0699] In yet another embodiment, the antisense oligonucleotide is
an alpha-anomeric oligonucleotide. An alpha-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual beta-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids
Res. 15:6625-6641 (1987)). The oligonucleotide is a
2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res.
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,
FEBS Lett. 215:327-330 (1997)).
[0700] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(Nucl. Acids Res. 16:3209 (1988)), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451 (1988)), etc.
[0701] While antisense nucleotides complementary to the TR21 coding
region sequence could be used, those complementary to the
transcribed untranslated region are most preferred.
[0702] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al.,
Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site-specific recognition sequence can be used to destroy TR21
mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead
ribozymes cleave mRNAs at locations dictated by flanking regions
that form complementary base pairs with the target mRNA. The sole
requirement is that the target mRNA have the following sequence of
two bases: 5'-UG-3'. The construction and production of hammerhead
ribozymes is well known in the art and is described more fully in
Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous
potential hammerhead ribozyme cleavage sites within the nucleotide
sequence TR21 (FIG. 1). Preferably, the ribozyme is engineered so
that the cleavage recognition site is located near the 5' end of
the TR21 mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts.
[0703] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express TR21 in vivo. DNA constructs encoding the ribozyme may be
introduced into the cell in the same manner as described above for
the introduction of antisense encoding DNA. A preferred method of
delivery involves using a DNA construct "encoding" the ribozyme
under the control of a strong constitutive promoter, such as, for
example, pol III or pol II promoter, so that transfected cells will
produce sufficient quantities of the ribozyme to destroy endogenous
TR21 messages and inhibit translation. Since ribozymes unlike
antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
[0704] Endogenous gene expression can also be reduced by
inactivating or "knocking out" the TR21 gene and/or its promoter
using targeted homologous recombination. (E.g., see Smithies et
al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell
51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of
which is incorporated by reference herein in its entirety). For
example, a mutant, non-functional polynucleotide of the invention
(or a completely unrelated DNA sequence) flanked by DNA homologous
to the endogenous polynucleotide sequence (either the coding
regions or regulatory regions of the gene) can be used, with or
without a selectable marker and/or a negative selectable marker, to
transfect cells that express polypeptides of the invention in vivo.
In another embodiment, techniques known in the art are used to
generate knockouts in cells that contain, but do not express the
gene of interest. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the targeted
gene. Such approaches are particularly suited in research and
agricultural fields where modifications to embryonic stem cells can
be used to generate animal offspring with an inactive targeted gene
(e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra).
However this approach can be routinely adapted for use in humans
provided the recombinant DNA constructs are directly administered
or targeted to the required site in vivo using appropriate viral
vectors that will be apparent to those of skill in the art. The
contents of each of the documents recited in this paragraph is
herein incorporated by reference in its entirety.
[0705] In other embodiments, antagonists according to the present
invention include soluble forms of TR21 (e.g., fragments of TR21
shown in FIG. 1 (SEQ ID NO:2) that include one or more copies of
the cysteine rich motif from the extracellular domain of TR21).
Such soluble forms of the TR21, which may be naturally occurring or
synthetic, antagonize TR21 mediated signaling by competing with
native TR21 for binding to TR21 ligands (e.g., Neutrokine-alpha
(See, U.S. application Ser. No. 60/188,208)), and/or by forming a
multimer that may or may not be capable of binding the receptor,
but which is incapable of inducing signal transduction. Preferably,
these antagonists inhibit TR21 mediated stimulation of lymphocyte
(e.g., B-cell) proliferation, differentiation, and/or activation.
Antagonists of the present invention also include antibodies
specific for TNFR-family receptors and TR21-Fc fusion proteins.
[0706] By a "TNF-family ligand" is intended naturally occurring,
recombinant, and synthetic ligands that are capable of binding to a
member of the TNF receptor family and inducing and/or blocking the
ligand/receptor signaling pathway. Members of the TNF ligand family
include, but are not limited to, TNF-alpha, lymphotoxin-alpha
(LT-alpha, also known as TNF-beta), LT-beta (found in complex
heterotrimer LT-alpha2-beta), FasL, CD40L, (TNF-gamma
(International Publication No. WO 96/14328), AIM-I (International
Publication No. WO 97/33899), AIM-II (International Publication No.
WO 97/34911), APRIL (International Publication Number WO 97/33902;
J. Exp. Med. 188(6):1185-1190) (1998)), endokine-alpha
(International Publication No. WO 98/07880), Neutrokine-alpha
(International Publication No. WO 98/18921), CD27L, CD30L, 4-1BBL,
OX40L, CD27, CD30, 4-1BB, OX40, and nerve growth factor (NGF). In
specific embodiments, the TNF-family ligand is Neutrokine-alpha, or
fragments or variants thereof. In other specific embodiments, the
TNF-family ligand is APRIL or fragments or variants thereof.
[0707] Antagonists of the present invention also include antibodies
specific for TNF-family ligands or the TR21 polypeptides of the
invention. Antibodies according to the present invention may be
prepared by any of a variety of standard methods using TR21
immunogens of the present invention. As indicated, such TR21
immunogens include the complete TR21 polypeptides depicted in FIG.
1 (SEQ ID NO:2) and TR21 polypeptide fragments comprising, for
example, the cysteine rich domain, extracellular domain,
transmembrane domain, and/or intracellular domain, or any
combination thereof.
[0708] Polyclonal and monoclonal antibody agonists or antagonists
according to the present invention can be raised according to the
methods disclosed herein and/or known in the art, such as, for
example, those methods described in Tartaglia and Goeddel, J. Biol
Chem. 267(7):4304-4307(1992)); Tartaglia et al., Cell 73:213-216
(1993)), and PCT Application WO 94/09137 and are preferably
specific to (i.e., bind uniquely to polypeptides of the invention
having the amino acid sequence of SEQ ID NO:2.
[0709] In a preferred method, antibodies according to the present
invention are InAbs. Such InAbs can be prepared using hybridoma
technology (Kohler and Milstein, Nature 256:495-497 (1975) and U.S.
Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1988; Monoclonal Antibodies and Hybridomas: A New Dimension in
Biological Analyses, Plenum Press, New York, N.Y., 1980; Campbell,
"Monoclonal Antibody Technology," In: Laboratory Techniques in
Biochemistry and Molecular Biology, Volume 13 (Burdon et al.,
eds.), Elsevier, Amsterdam (1984)).
[0710] Antagonists according to the present invention include
soluble forms of TR21, i.e., TR21 fragments that include one or
more of the cysteine rich motif from the extracellular region of
the full-length receptor (or variants thereof). Such soluble forms
of the receptor, which may be naturally occurring or synthetic,
antagonize TR21 mediated signaling by competing with the cell
surface TR21 for binding to TNF-family ligands (e.g.,
Neutrokine-alpha). Thus, soluble forms of the receptor that include
one or more copies of the cysteine-rich motif of TR21 are novel
cytokines capable of inhibiting TR21 mediated signaling induced by
TNF-family ligands. These soluble forms are preferably expressed as
dimers or trimers, since these have been shown to be superior to
monomeric forms of soluble receptor as antagonists, e.g., IgGFc-TNF
receptor family fusions. Other such cytokines are known in the art
and include Fas B (a soluble form of the mouse Fas receptor) that
acts physiologically to limit apoptosis induced by Fas ligand (D.
P. Hughes and I. N. Crispe, J. Exp. Med. 182:1395-1401 (1995)).
[0711] The techniques of gene shuffling, motif shuffling, exon
shuffling, and/or codon-shuffling (collectively referred to as "DNA
shuffling") may be employed to modulate the activities of TR21
thereby effectively generating agonists and antagonists of TR21.
See generally, International Publication No. WO 99/29902, U.S. Pat.
Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and
Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama,
Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques
24(2):308-13 (1998) (each of these patents and publications are
hereby incorporated by reference). In one embodiment, alteration of
TR21 polynucleotides and corresponding polypeptides may be achieved
by DNA shuffling. DNA shuffling involves the assembly of two or
more DNA segments into a desired TR21 molecule by homologous, or
site-specific, recombination. In another embodiment, TR21
polynucleotides and corresponding polypeptides may be altered by
being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In
another embodiment, one or more components, motifs, sections,
parts, domains, fragments, etc., of TR21 may be recombined with one
or more components, motifs, sections, parts, domains, fragments,
etc. of one or more heterologous molecules. In preferred
embodiments, the heterologous molecules are include, but are not
limited to, TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as
TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta),
OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma
(International Publication No. WO 96/14328), TRAIL, AIM-II
(International Publication No. WO 97/34911), APRIL (J. Exp. Med.
188(6):1185-1190 (1998)), endokine-alpha (International Publication
No. WO 98/07880), Neutrokine alpha (International Publication No.
WO 98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble
forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International
Publication No. WO 96/34095), DR3 (International Publication No. WO
97/33904), DR4 (International Publication No. WO 98/32856), TR5
(International Publication No. WO 98/30693), TR6 (International
Publication No. WO 98/30694), TR7 (International Publication No. WO
98/41629), TRANK, TR9 (International Publication No. WO 98/56892),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153. In further preferred
embodiments, the heterologous molecules are any member of the TNF
family.
[0712] Proteins and other compounds which bind the TR21 domains are
also candidate agonists and antagonists according to the present
invention. Such binding compounds can be "captured" using the yeast
two-hybrid system (Fields and Song, Nature 340:245-246 (1989)). A
modified version of the yeast two-hybrid system has been described
by Roger Brent and his colleagues (Gyuris, Cell 75:791-803 (1993);
Zervos et al., Cell 72:223-232 (1993)). Preferably, the yeast
two-hybrid system is used according to the present invention to
capture compounds which bind to the extracellular domain,
intracellular, transmembrane, and the cysteine rich domain of TR21.
Such compounds are good candidate agonists and antagonists of the
present invention.
[0713] For example, using the two-hybrid assay described above, the
extracellular or intracellular domain of the TR21, or a portion
thereof (e.g., the cysteine rich domain), may be used to identify
cellular proteins which interact with TR21 the receptor in vivo.
Such an assay may also be used to identify ligands with potential
agonistic or antagonistic activity of TR21 receptor function. This
screening assay has previously been used to identify protein which
interact with the cytoplasmic domain of the murine TNF-RII and led
to the identification of two receptor associated proteins. Rothe et
al., Cell 78:681 (1994). Such proteins and amino acid sequences
which bind to the cytoplasmic domain of the TR21 are good candidate
agonist and antagonist of the present invention.
[0714] Other screening techniques include the use of cells which
express the polypeptide of the present invention (for example,
transfected CHO cells) in a system which measures extracellular pH
changes caused by receptor activation, for example, as described in
Science, 246:181-296 (1989). In another example, potential agonists
or antagonists may be contacted with a cell which expresses the
polypeptide of the present invention and a second messenger
response, e.g., signal transduction may be measured to determine
whether the potential antagonist or agonist is effective.
[0715] Agonists according to the present invention include
naturally occurring and synthetic compounds such as, for example,
TNF family ligand peptide fragments, transforming growth factor,
neurotransmitters (such as glutamate, dopamine,
N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells
and antimetabolites. Preferred agonists include chemotherapeutic
drugs such as, for example, cisplatin, doxorubicin, bleomycin,
cytosine arabinoside, nitrogen mustard, methotrexate and
vincristine. Others include ethanol and -amyloid peptide. (Science
267:1457-1458 (1995)).
[0716] Preferred agonists are fragments of TR21 polypeptides of the
invention which stimulate lymphocyte (e.g., B cell) proliferation,
differentiation and/or activation. Further preferred agonists
include polyclonal and monoclonal antibodies raised against the
TR21 polypeptides of the invention, or a fragment thereof. Such
agonist antibodies raised against a TNF-family receptor are
disclosed in Tartaglia et al., Proc. Natl. Acad. Sci. USA
88:9292-9296 (1991); and Tartaglia et al., J. Biol. Chem.
267:4304-4307(1992). See, also, PCT Application WO 94/09137.
[0717] In an additional embodiment, immunoregulatory molecules such
as, for example, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13,
IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha, may be used as
agonists of TR21 polypeptides of the invention which stimulate
lymphocyte (e.g., B cell) proliferation, differentiation and/or
activation. In a specific embodiment, IL4 and/or IL10 are used to
enhance the TR21-mediated proliferation of B cells.
[0718] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the polypeptides of the invention. The
engineered cells which express and preferably secrete the
polypeptides of the invention can be introduced into the patient
systemically, e.g., in the circulation, or intraperitoneally.
[0719] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[0720] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well-known
techniques to prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form that while allowing for an
exchange of components with the immediate extracellular environment
does not allow the introduced cells to be recognized by the host
immune system.
[0721] In yet another embodiment of the invention, the activity of
TR21 polypeptide can be reduced using a "dominant negative." To
this end, constructs which encode, for example, defective TR21
polypeptide, such as, for example, mutants lacking all or a portion
of the TNF-conserved domain, can be used in gene therapy approaches
to diminish the activity of TR21 on appropriate target cells. For
example, nucleotide sequences that direct host cell expression of
TR21 polypeptide in which all or a portion of the TNFR-conserved
domain is altered or missing can be introduced into monocytic cells
or other cells or tissues (either by in vivo or ex vivo gene
therapy methods described herein or otherwise known in the art).
Alternatively, targeted homologous recombination can be utilized to
introduce such deletions or mutations into the subject's endogenous
TR21 gene in monocytes. The engineered cells will express
non-functional TR21 polypeptides (i.e., a receptor (e.g., multimer)
that may be capable of binding, but which is incapable of inducing
signal transduction).
Diagnostic Assays
[0722] The compounds of the present invention are useful for
diagnosis or treatment of various immune system-related disorders
in mammals, preferably humans. Such disorders include but are not
limited to tumors (e.g., B cell and monocytic cell leukemias and
lymphomas) and tumor metastasis, infections by bacteria, viruses
and other parasites, immunodeficiencies, inflammatory diseases,
lymphadenopathy, autoimmune diseases, and graft versus host
disease.
[0723] TR21 is expressed in B cells and spleen. For a number of
immune system-related disorders, substantially altered (increased
or decreased) levels of TR21 gene expression can be detected in
immune system tissue or other cells or bodily fluids (e.g., sera,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to a "standard" TR21
gene expression level, that is, the TR21 expression level in immune
system tissues or bodily fluids from an individual not having the
immune system disorder. Thus, the invention provides a diagnostic
method useful during diagnosis of an immune system disorder, which
involves measuring the expression level of the gene encoding the
TR21 polypeptide in immune system tissue or other cells or body
fluid from an individual and comparing the measured gene expression
level with a standard TR21 gene expression level, whereby an
increase or decrease in the gene expression level(s) compared to
the standard is indicative of an immune system disorder or normal
activation, proliferation, differentiation, and/or death.
[0724] In particular, it is believed that certain tissues in
mammals with cancer of cells or tissue of the immune system express
significantly enhanced or reduced levels of normal or altered TR21
polypeptide and mRNA encoding the TR21 polypeptide when compared to
a corresponding "standard" level. Further, it is believed that
enhanced or depressed levels of the TR21 polypeptide can be
detected in certain body fluids (e.g., sera, plasma, urine, and
spinal fluid) or cells or tissue from mammals with such a cancer
when compared to sera from mammals of the same species not having
the cancer.
[0725] For example, as disclosed herein, TR21 are expressed in B
cells. Accordingly, polynucleotides of the invention (e.g.,
polynucleotide sequences complementary to all or a portion of TR21
mRNA) and antibodies (and antibody fragments) directed against the
polypeptides of the invention may be used to quantitate or
qualitate concentrations of cells of B cell lineage (e.g., B cell
leukemia cells) expressing TR21 on their cell surfaces. These
antibodies additionally have diagnostic applications in detecting
abnormalities in the level of TR21 gene expression, or
abnormalities in the structure and/or temporal, tissue, cellular,
or subcellular location of TR21. These diagnostic assays may be
performed in vivo or in vitro, such as, for example, on blood
samples, biopsy tissue or autopsy tissue.
[0726] Thus, the invention provides a diagnostic method useful
during diagnosis of a immune system disorder, including cancers of
this system, which involves measuring the expression level of the
gene encoding the TR21 polypeptide in immune system tissue or other
cells or body fluid from an individual and comparing the measured
gene expression level with a standard TR21 gene expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of an immune system
disorder.
[0727] Where a diagnosis of a disorder in the immune system,
including diagnosis of a tumor, has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting enhanced or
depressed TR21 gene expression will experience a worse clinical
outcome relative to patients expressing the gene at a level nearer
the standard level.
[0728] By "assaying the expression level of the gene encoding the
TR21 polypeptide" is intended qualitatively or quantitatively
measuring or estimating the level of the TR21 polypeptide or the
level of the mRNA encoding the TR21 polypeptide in a first
biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the TR21 polypeptide level or mRNA level in
a second biological sample). Preferably, the TR21 polypeptide level
or mRNA level in the first biological sample is measured or
estimated and compared to a standard TR21 polypeptide level or mRNA
level, the standard being taken from a second biological sample
obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having a disorder of the immune system. As will be appreciated in
the art, once a standard TR21 polypeptide level or mRNA level is
known, it can be used repeatedly as a standard for comparison.
[0729] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source containing TR21 receptor protein (including portions
thereof) or mRNA. As indicated, biological samples include body
fluids (such as sera, plasma, urine, synovial fluid and spinal
fluid) which contain free extracellular domains of the TR21
polypeptide, immune system tissue, and other tissue sources found
to express complete or free extracellular domain of the TR21
receptor. Methods for obtaining tissue biopsies and body fluids
from mammals are well known in the art. Where the biological sample
is to include mRNA, a tissue biopsy is the preferred source.
[0730] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels
of mRNA encoding the TR21 polypeptide are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0731] The present invention also relates to diagnostic assays such
as quantitative and diagnostic assays for detecting levels of TR21
receptor protein, or the soluble form thereof, in a biological
sample (e.g., cells and tissues), including determination of normal
and abnormal levels of polypeptides. Thus, for instance, a
diagnostic assay in accordance with the invention for detecting
over-expression of TR21, or soluble form thereof, compared to
normal control tissue samples may be used to detect the presence of
tumors, for example. Assay techniques that can be used to determine
levels of a protein, such as a TR21 protein of the present
invention, or a soluble form thereof, in a sample derived from a
host are well-known to those of skill in the art. Such assay
methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays. Assaying TR21 protein
levels in a biological sample can occur using any art-known
method.
[0732] Assaying TR21 polypeptide levels in a biological sample can
occur using antibody-based techniques. For example, TR21
polypeptide expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al., J. Cell. Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting TR21 polypeptide gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0733] The tissue or cell type to be analyzed will generally
include those which are known, or suspected, to express the TR21
gene (such as, for example, cells of B cell lineage and the spleen)
or cells or tissue which are known, or suspected, to express the
TR21 ligand gene (such as, for example, cells of monocytic
lineage). The protein isolation methods employed herein may, for
example, be such as those described in Harlow and Lane (Harlow, E.
and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is
incorporated herein by reference in its entirety. The isolated
cells can be derived from cell culture or from a patient. The
analysis of cells taken from culture may be a necessary step in the
assessment of cells that could be used as part of a cell-based gene
therapy technique or, alternatively, to test the effect of
compounds on the expression of the TR21 gene or TR21 ligand
gene.
[0734] For example, antibodies, or fragments of antibodies, such as
those described herein, may be used to quantitatively or
qualitatively detect the presence of TR21 gene products or
conserved variants or peptide fragments thereof. This can be
accomplished, for example, by immunofluorescence techniques
employing a fluorescently labeled antibody coupled with light
microscopic, flow cytometric, or fluorimetric detection.
[0735] The antibodies (or fragments thereof), TR21 polypeptides,
and/or TR21 ligands (e.g., Neutrokine-alpha) of the present
invention may, additionally, be employed histologically, as in
immunofluorescence, immunoelectron microscopy or non-immunological
assays, for in situ detection of TR21 gene products or conserved
variants or peptide fragments thereof, or for TR21 binding to TR21
ligand. In situ detection may be accomplished by removing a
histological specimen from a patient, and applying thereto a
labeled antibody or TR21 polypeptide of the present invention. The
antibody (or fragment) or TR21 polypeptide is preferably applied by
overlaying the labeled antibody (or fragment) onto a biological
sample. Through the use of such a procedure, it is possible to
determine not only the presence of the TR21 gene product, or
conserved variants or peptide fragments, or TR21 polypeptide
binding, but also its distribution in the examined tissue. Using
the present invention, those of ordinary skill will readily
perceive that any of a wide variety of histological methods (such
as staining procedures) can be modified in order to achieve such in
situ detection.
[0736] Immunoassays and non-immunoassays for TR21 gene products or
conserved variants or peptide fragments thereof will typically
comprise incubating a sample, such as a biological fluid, a tissue
extract, freshly harvested cells, or lysates of cells which have
been incubated in cell culture, in the presence of a detectably
labeled antibody capable of binding TR21 gene products or conserved
variants or peptide fragments thereof, and detecting the bound
antibody by any of a number of techniques well-known in the
art.
[0737] Immunoassays and non-immunoassays for TR21 ligand gene
products or conserved variants or peptide fragments thereof will
typically comprise incubating a sample, such as a biological fluid,
a tissue extract, freshly harvested cells, or lysates of cells
which have been incubated in cell culture, in the presence of a
detectable or labeled TR21 polypeptide capable of identifying TR21
ligand gene products or conserved variants or peptide fragments
thereof, and detecting the bound TR21 polypeptide by any of a
number of techniques well-known in the art.
[0738] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled anti-TR21 antibody or detectable TR21
polypeptide. The solid phase support may then be washed with the
buffer a second time to remove unbound antibody or polypeptide.
Optionally the antibody is subsequently labeled. The amount of
bound label on solid support may then be detected by conventional
means.
[0739] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0740] The binding activity of a given lot of anti-TR21 antibody or
TR21 polypeptide may be determined according to well-known methods.
Those skilled in the art will be able to determine operative and
optimal assay conditions for each determination by employing
routine experimentation.
[0741] In addition to assaying TR21 polypeptide levels or
polynucleotide levels in a biological sample obtained from an
individual, TR21 polypeptide or polynucleotide can also be detected
in vivo by imaging. For example, in one embodiment of the
invention, TR21 polypeptide is used to image monocytic leukemias or
lymphomas. In another embodiment, TR21 polynucleotides of the
invention and/or anti-TR21 antibodies (e.g., polynucleotides
complementary to all or a portion of TR21 mRNA) are used to image B
cell leukemias or lymphomas.
[0742] Antibody labels or markers for in vivo imaging of TR21
polypeptide include those detectable by X-radiography, NMR, MRI,
CAT-scans or ESR. For X-radiography, suitable labels include
radioisotopes such as barium or cesium, which emit detectable
radiation but are not overtly harmful to the subject. Suitable
markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma. Where in vivo imaging is used to detect enhanced levels
of TR21 polypeptide for diagnosis in humans, it may be preferable
to use human antibodies or "humanized" chimeric monoclonal
antibodies. Such antibodies can be produced using techniques
described herein or otherwise known in the art. For example methods
for producing chimeric antibodies are known in the art. See, for
review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et
al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature
312:643 (1984); Neuberger et al., Nature 314:268 (1985).
[0743] Additionally, any TR21 polypeptide whose presence can be
detected, can be administered. For example, TR21 polypeptides
labeled with a radio-opaque or other appropriate compound can be
administered and visualized in vivo, as discussed, above for
labeled antibodies. Further such TR21 polypeptides can be utilized
for in vitro diagnostic procedures.
[0744] A TR21 polypeptide-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging
moiety, such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for immune system disorder. It will be understood in
the art that the size of the subject and the imaging system used
will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain TR21 protein. In vivo tumor
imaging is described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
[0745] With respect to antibodies, one of the ways in which the
anti-TR21 antibody can be detectably labeled is by linking the same
to an enzyme and using the linked product in an enzyme immunoassay
(EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)",
1978, Diagnostic Horizons 2:1-7, Microbiological Associates
Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin.
Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523
(1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca
Raton, Fla.; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay,
Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will
react with an appropriate substrate, preferably a chromogenic
substrate, in such a manner as to produce a chemical moiety which
can be detected, for example, by spectrophotometric, fluorimetric
or by visual means. Enzymes which can be used to detectably label
the antibody include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. Additionally, the detection can be
accomplished by calorimetric methods which employ a chromogenic
substrate for the enzyme. Detection may also be accomplished by
visual comparison of the extent of enzymatic reaction of a
substrate in comparison with similarly prepared standards.
[0746] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect TR21
through the use of a radioimmunoassay (RIA) (see, for example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by means including, but not
limited to, a gamma counter, a scintillation counter, or
autoradiography.
[0747] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and
fluorescamine.
[0748] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0749] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0750] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in, which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
Chromosome Assays
[0751] The nucleic acid molecules of the present invention are also
valuable for chromosome identification.
[0752] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a TR21 receptor
gene. This can be accomplished using a variety of well-known
techniques and libraries, which generally are available
commercially. The genomic DNA is then used for in situ chromosome
mapping using well-known techniques for this purpose.
[0753] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes.
[0754] Fluorescence in situ hybridization ("FISH") of a CDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 50 or 60 bp. For a review of this technique, see
Verma et al., Human Chromosomes: a Manual of Basic Techniques,
Pergamon Press, New York (1988).
[0755] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available on
line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0756] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0757] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
Illustrative Examples
Example 1
Protein Fusions of TR21
[0758] TR21 polypeptides of the invention are optionally fused to
other proteins. These fusion proteins can be used for a variety of
applications. For example, fusion of TR21 polypeptides to His-tag,
HA-tag, protein A, IgG domains, and maltose binding protein
facilitates purification. (See EP A 394,827; Traunecker, et al.,
Nature 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and
albumin increases the half-life time in vivo. Nuclear localization
signals fused to TR21 polypeptides can target the protein to a
specific subcellular localization, while covalent heterodimer or
homodimers can increase or decrease the activity of a fusion
protein. Fusion proteins can also create chimeric molecules having
more than one function. Finally, fusion proteins can increase
solubility and/or stability of the fused protein compared to the
non-fused protein. All of the types of fusion proteins described
above can be made using techniques known in the art or by using or
routinely modifying the following protocol, which outlines the
fusion of a polypeptide to an IgG molecule.
[0759] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below (SEQ ID NO:3). These primers also
preferably contain convenient restriction enzyme sites that will
facilitate cloning into an expression vector, preferably a
mammalian expression vector.
[0760] For example, if the pC4 (Accession No. 209646) expression
vector is used, the human Fc portion can be ligated into the BamHI
cloning site. Note that the 3' BamHI site should be destroyed.
Next, the vector containing the human Fc portion is re-restricted
with BamHI, linearizing the vector, and TR21 polynucleotide is
ligated into this BamHI site. Note that the polynucleotide is
cloned without a stop codon otherwise a fusion protein will not be
produced.
[0761] If the naturally occurring signal sequence is used to
produce the secreted protein, pC4 does not need a second signal
peptide. Alternatively, if the naturally occurring signal sequence
is not used, the vector can be modified to include a heterologous
signal sequence. (See e.g., WO 96/34891.)
Human IgG Fc Region:
TABLE-US-00004 [0762] (SEQ ID NO: 3)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 2
Isolation of Antibody Fragments Directed Against Polypeptides of
the Present Invention from a Library of scFvs
[0763] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against polypeptides of the present invention
to which the donor may or may not have been exposed (see e.g., U.S.
Pat. No. 5,885,793 incorporated herein in its entirety by
reference).
Rescue of the Library
[0764] A library of scFvs is constructed from the RNA of human PBLs
as described in WO92/01047. To rescue phages displaying antibody
fragments, approximately 10.sup.9 E. coli harboring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 ug/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D.
of 0.8 with shaking. Five ml of this culture is used to innoculate
50 ml of 2.times.TY-AMP-GLU, 2.times.108 TU of .DELTA. gene 3
helper phage (M13 .DELTA. gene III, see WO92/01047) are added and
the culture incubated at 37.degree. C. for 45 minutes without
shaking and then at 37.degree. C. for 45 minutes with shaking. The
culture is centrifuged at 4000 r.p.m. for 10 minutes and the pellet
resuspended in 2 liters of 2.times.TY containing 100 ug/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phages are
prepared as described in WO92/01047.
[0765] M13 .DELTA. gene III is prepared as follows: M13 .DELTA.
gene III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 .DELTA. gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are pelleted (IEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100 ug
ampicillin/ml and 25 ug kanamycin/ml (2.times.TY-AMP-KAN) and grown
overnight, shaking at 37.degree. C. Phage particles are purified
and concentrated from the culture medium by two PEG-precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through
a 0.45 um filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
Panning of the Library
[0766] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 mg/ml or 10 mg/ml of a polypeptide of the present
invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at
37.degree. C. and then washed 3 times in PBS. Approximately
10.sup.13 TU of phage are applied to the tube and incubated for 30
minutes at room temperature tumbling on an over and under turntable
and then left to stand for another 1.5 hours. Tubes are washed 10
times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are
eluted by adding 1 ml of 100 mM triethylamine and rotating 15
minutes on an under and over turntable after which the solution is
immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4.
Phages are then used to infect 10 ml of mid-log E. coli TG1 by
incubating eluted phages with bacteria for 30 minutes at 37.degree.
C. The E. coli are then plated on TYE plates containing 1% glucose
and 100 ug/ml ampicillin. The resulting bacterial library is then
rescued with .DELTA. gene III helper phage as described above to
prepare phage for a subsequent round of selection. This process is
then repeated for a total of 4 rounds of affinity purification with
tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20
times with PBS for rounds 3 and 4.
Characterization of Binders
[0767] Eluted phage from the 3rd and 4th rounds of selection are
used to infect E. coli HB 2151 and soluble scFv is produced (Marks,
et al., 1991) from single colonies for assay. ELISAs are performed
with microtiter plates coated with either 10 pg/ml of the
polypeptide of the present invention in 50 mM bicarbonate pH 9.6.
Clones positive in ELISA are further characterized by PCR
fingerprinting (see e.g., WO92/01047) and then by sequencing.
Example 3
Production of a TR21 Antibody
a) Hybridoma Technology
[0768] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) As one
example of such methods, cells expressing TR21 are administered to
an animal to induce the production of sera containing polyclonal
antibodies. In a preferred method, a preparation of TR21 protein is
prepared and purified to render it substantially free of natural
contaminants. Such a preparation is then introduced into an animal
in order to produce polyclonal antisera of greater specific
activity.
[0769] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or protein binding fragments
thereof). Such monoclonal antibodies can be prepared using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In
general, such procedures involve immunizing an animal (preferably a
mouse) with TR21 polypeptide or, more preferably, with a secreted
TR21 polypeptide-expressing cell. Such cells may be cultured in any
suitable tissue culture medium; however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented with
10% fetal bovine serum (inactivated at about 56.degree. C.), and
supplemented with about 10 g/l of nonessential amino acids, about
1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.
[0770] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP2O), available
from the ATCC.TM.. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981).) The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the TR21 polypeptide.
[0771] Alternatively, additional antibodies capable of binding to
TR21 polypeptide can be produced in a two-step procedure using
anti-idiotypic antibodies. Such a method makes use of the fact that
antibodies are themselves antigens, and therefore, it is possible
to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the TR21 protein-specific
antibody can be blocked by TR21. Such antibodies comprise
anti-idiotypic antibodies to the TR21 protein-specific antibody and
can be used to immunize an animal to induce formation of further
TR21 protein-specific antibodies.
[0772] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, secreted TR21 protein-binding fragments
can be produced through the application of recombinant DNA
technology or through synthetic chemistry.
[0773] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. (See, for review, Morrison, Science 229:1202 (1985); Oi et
al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985)).
b) Isolation of Antibody Fragments Directed Against TR21 from a
Library of scFvs
[0774] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against TR21 to which the donor may or may not
have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated
herein in its entirety by reference).
[0775] Rescue of the Library
[0776] A library of scFvs is constructed from the RNA of human PBLs
as described in WO92/01047. To rescue phages displaying antibody
fragments, approximately 10.sup.9 E. coli harboring the phagemid
are used to inoculate 50 ml of 2.times.TY containing 1% glucose and
100 ug/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D.
of 0.8 with shaking. Five ml of this culture is used to innoculate
50 ml of 2.times.TY-AMP-GLU, 2.times.10.sup.8 TU of delta gene 3
helper (M13 delta gene III, see WO92/01047) are added and the
culture incubated at 37.degree. C. for 45 minutes without shaking
and then at 37.degree. C. for 45 minutes with shaking. The culture
is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100 ug/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phages are
prepared as described in WO92/01047.
[0777] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100 ug
ampicillin/ml and 25 ug kanamycin/ml (2.times.TY-AMP-KAN) and grown
overnight, shaking at 37.degree. C. Phage particles are purified
and concentrated from the culture medium by two PEG-precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through
a 0.45 um filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
[0778] Panning of the Library
[0779] Immunotubes (Nunc) are coated overnight in PBS with 4 ml of
either 100 ug/ml or 10 ug/ml of a polypeptide of the present
invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at
37.degree. C. and then washed 3 times in PBS. Approximately
10.sup.13 TU of phage is applied to the tube and incubated for 30
minutes at room temperature tumbling on an over and under turntable
and then left to stand for another 1.5 hours. Tubes are washed 10
times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are
eluted by adding 1 ml of 100 mM triethylamine and rotating 15
minutes on an under and over turntable after which the solution is
immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4.
Phages are then used to infect 10 ml of mid-log E. coli TG1 by
incubating eluted phage with bacteria for 30 minutes at 37.degree.
C. The E. coli are then plated on TYE plates containing 1% glucose
and 100 .mu.g/ml ampicillin. The resulting bacterial library is
then rescued with delta gene 3 helper phage as described above to
prepare phage for a subsequent round of selection. This process is
then repeated for a total of 4 rounds of affinity purification with
tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20
times with PBS for rounds 3 and 4.
[0780] Characterization of Binders
[0781] Eluted phages from the 3rd and 4th rounds of selection are
used to infect E. coli HB 2151 and soluble scFv is produced (Marks,
et al., 1991) from single colonies for assay. ELISAs are performed
with microtiter plates coated with either 10 pg/ml of the
polypeptide of the present invention in 50 mM bicarbonate pH 9.6.
Clones positive in ELISA are further characterized by PCR
fingerprinting (see e.g., WO92/01047) and then by sequencing.
Example 4
Method of Detecting Abnormal Levels of TR21 in a Biological
Sample
[0782] TR21 polypeptides can be detected in a biological sample,
and if an increased or decreased level of TR21 is detected, this
polypeptide is a marker for a particular phenotype. Methods of
detection are numerous, and thus, it is understood that one skilled
in the art can modify the following assay to fit their particular
needs.
[0783] For example, antibody-sandwich ELISA assays are used to
detect TR21 in a sample, preferably a biological sample. Wells of a
microtiter plate are coated with specific antibodies to TR21, at a
final concentration of 0.2 to 10 ug/ml. The antibodies are either
monoclonal or polyclonal and are produced using technique known in
the art. The wells are blocked so that non-specific binding of TR21
to the well is reduced.
[0784] The coated wells are then incubated for >2 hours at RT
with a sample containing TR21. Preferably, serial dilutions of the
sample should be used to validate results. The plates are then
washed three times with deionized or distilled water to remove
unbound TR21.
[0785] Next, 50 ul of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[0786] 75 ul of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution is then added to
each well and incubated 1 hour at room temperature to allow
cleavage of the substrate and fluorescence. The fluorescence is
measured using a microtiter plate reader. A standard curve is
prepared using the experimental results from serial dilutions of a
control sample with the sample concentration plotted on the X-axis
(log scale) and fluorescence or absorbance on the Y-axis (linear
scale). The TR21 polypeptide concentration in a sample is then
interpolated using the standard curve based on the measured
fluorescence of that sample.
Example 5
Method of Treating Decreased Levels of TR21
[0787] The present invention relates to a method for treating an
individual in need of a decreased level of TR21 biological activity
in the body comprising, administering to such an individual a
composition comprising a therapeutically effective amount of TR21
antagonist. Preferred antagonists for use in the present invention
are TR21-specific antibodies.
[0788] Moreover, it will be appreciated that conditions caused by a
decrease in the standard or normal expression level of TR21 in an
individual can be treated by administering TR21, preferably in a
soluble and/or secreted form. Thus, the invention also provides a
method of treatment of an individual in need of an increased level
of TR21 polypeptide comprising administering to such an individual
a pharmaceutical composition comprising an amount of TR21 to
increase the biological activity level of TR21 in such an
individual.
[0789] For example, a patient with decreased levels of TR21
polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide
for six consecutive days. Preferably, the polypeptide is in a
soluble and/or secreted form.
Example 6
Method of Treating Increased Levels of TR21
[0790] The present invention also relates to a method for treating
an individual in need of an increased level of TR21 biological
activity in the body comprising administering to such an individual
a composition comprising a therapeutically effective amount of TR21
or an agonist thereof.
[0791] Antisense technology is used to inhibit production of TR21.
This technology is one example of a method of decreasing levels of
TR21 polypeptide, preferably a soluble and/or secreted form, due to
a variety of etiologies, such as cancer.
[0792] For example, a patient diagnosed with abnormally increased
levels of TR21 is administered intravenously antisense
polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21
days. This treatment is repeated after a 7-day rest period if the
is determined to be well tolerated.
Example 7
Method of Treatment Using Gene Therapy--Ex Vivo
[0793] One method of gene therapy transplants fibroblasts, which
are capable of expressing soluble and/or mature TR21 polypeptides,
onto a patient. Generally, fibroblasts are obtained from a subject
by skin biopsy. The resulting tissue is placed in tissue-culture
medium and separated into small pieces. Small chunks of the tissue
are placed on a wet surface of a tissue culture flask,
approximately ten pieces are placed in each flask. The flask is
turned upside down, closed tight and left at room temperature over
night. After 24 hours at room temperature, the flask is inverted
and the chunks of tissue remain fixed to the bottom of the flask
and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin
and streptomycin) is added. The flasks are then incubated at
37.degree. C. for approximately one week.
[0794] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[0795] pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0796] The CDNA encoding TR21 can be amplified using PCR primers
which correspond to the 5' and 3' end encoding sequences
respectively. Preferably, the 5' primer contains an EcoRI site and
the 3' primer includes a HindIII site. Equal quantities of the
Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is then used to transform E. coli HB101, which are then plated onto
agar containing kanamycin for the purpose of confirming that the
vector contains properly inserted TR21.
[0797] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the TR21 gene is then added
to the media and the packaging cells transduced with the vector.
The packaging cells now produce infectious viral particles
containing the TR21 gene (the packaging cells are now referred to
as producer cells).
[0798] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether TR21 protein is produced.
[0799] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 8
Method of Treatment Using Gene Therapy--In Vivo
[0800] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) TR21 sequences
into an animal to increase or decrease the expression of the TR21
polypeptide. The TR21 polynucleotide may be operatively linked to a
promoter or any other genetic elements necessary for the expression
of the TR21 polypeptide by the target tissue. Such gene therapy and
delivery techniques and methods are known in the art, see, for
example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622,
5,705,151, 5,580,859; Tabata H. et al., Cardiovasc. Res. 35:470-479
(1997); Chao J. et al., Pharmacol. Res. 35:517-522 (1997); Wolff J.
A. Neuromuscut. Disord. 7:314-318 (1997); Schwartz B. et al., Gene
Ther. 3:405-411 (1996); Tsurumi Y. et al., Circulation 94:3281-3290
(1996) (incorporated herein by reference).
[0801] The TR21 polynucleotide constructs may be delivered by any
method that delivers injectable materials to the cells of an
animal, such as, injection into the interstitial space of tissues
(heart, muscle, skin, lung, liver, intestine and the like). The
TR21 polynucleotide constructs can be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0802] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, LIPOFECTIN.TM.
or precipitating agents and the like. However, the TR21
polynucleotides may also be delivered in liposome formulations
(such as those taught in Felgner P. L., et al Ann. NY Acad. Sci.
772:126-139 (1995), and Abdallah B., et al. Biol. Cell 85(1):1-7
(1995)) which can be prepared by methods well known to those
skilled in the art.
[0803] The TR21 polynucleotide vector constructs used in the gene
therapy method are preferably constructs that will not integrate
into the host genome nor will they contain sequences that allow for
replication. Any strong promoter known to those skilled in the art
can be used for driving the expression of DNA. Unlike other gene
therapy techniques, one major advantage of introducing naked
nucleic acid sequences into target cells is the transitory nature
of the polynucleotide synthesis in the cells. Studies have shown
that non-replicating DNA sequences can be introduced into cells to
provide production of the desired polypeptide for periods of up to
six months.
[0804] The TR21 polynucleotide construct can be delivered to the
interstitial space of tissues within the animal, including of
muscle, skin, brain, lung, liver, spleen, bone marrow, thymus,
heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous
system, eye, gland, and connective tissue. Interstitial space of
the tissues comprises the intercellular fluid, mucopolysaccharide
matrix among the reticular fibers of organ tissues, elastic fibers
in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that same matrix within connective tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space
occupied by the plasma of the circulation and the lymph fluid of
the lymphatic channels. Delivery to the interstitial space of
muscle tissue is preferred for the reasons discussed below. They
may be conveniently delivered by injection into the tissues
comprising these cells. They are preferably delivered to and
expressed in persistent, non-dividing cells which are
differentiated, although delivery and expression may be achieved in
non-differentiated or less completely differentiated cells, such
as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells are particularly competent in their ability to take up
and express polynucleotides.
[0805] For the naked TR21 polynucleotide injection, an effective
dosage amount of DNA or RNA will be in the range of from about 0.05
g/kg body weight to about 50 mg/kg body weight. Preferably the
dosage will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration. The
preferred route of administration is by the parenteral route of
injection into the interstitial space of tissues. However, other
parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
TR21 polynucleotide constructs can be delivered to arteries during
angioplasty by the catheter used in the procedure.
[0806] The dose response effects of injected TR21 polynucleotide in
muscle in vivo is determined as follows. Suitable TR21 template DNA
for production of mRNA coding for TR21 polypeptide is prepared in
accordance with a standard recombinant DNA methodology. The
template DNA, which may be either circular or linear, is either
used as naked DNA or complexed with liposomes. The quadriceps
muscles of mice are then injected with various amounts of the
template DNA.
[0807] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The TR21 template DNA is
injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge
needle over one minute, approximately 0.5 cm from the distal
insertion site of the muscle into the knee and about 0.2 cm deep. A
suture is placed over the injection site for future localization,
and the skin is closed with stainless steel clips.
[0808] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for TR21 protein expression. A time course
for TR21 protein expression may be done in a similar fashion except
that quadriceps from different mice are harvested at different
times. Persistence of TR21 DNA in muscle following injection may be
determined by Southern blot analysis after preparing total cellular
DNA and HIRT supernatants from injected and control mice. The
results of the above experimentation in mice can be use to
extrapolate proper dosages and other treatment parameters in humans
and other animals using TR21 naked DNA.
Example 9
Gene Therapy Using Endogenous TR21 Gene
[0809] Another method of gene therapy according to the present
invention involves operably associating the endogenous TR21
sequence with a promoter via homologous recombination as described,
for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;
International Publication Number WO 96/29411; International
Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad.
Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989). This method involves the activation of a gene
which is present in the target cells, but which is not expressed in
the cells, or is expressed at a lower level than desired.
Polynucleotide constructs are made which contain a promoter and
targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous TR21, flanking the promoter. The targeting
sequence will be sufficiently near the 5' end of TR21 so the
promoter will be operably linked to the endogenous sequence upon
homologous recombination. The promoter and the targeting sequences
can be amplified using PCR. Preferably, the amplified promoter
contains distinct restriction enzyme sites on the 5' and 3' ends.
Preferably, the 3' end of the first targeting sequence contains the
same restriction enzyme site as the 5' end of the amplified
promoter and the 5' end of the second targeting sequence contains
the same restriction site as the 3' end of the amplified
promoter.
[0810] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[0811] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[0812] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous TR21 sequence. This results in the expression of
TR21 in the cell. Expression may be detected by immunological
staining, or any other method known in the art.
[0813] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2 HPO.sub.4, 6 mM dextrose). The cells are
recentrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[0814] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the TR21
locus, plasmid pUC 18 (MBI Fermentas, Amherst, N.Y.) is digested
with HindIII. The CMV promoter is amplified by PCR with an XbaI
site on the 5' end and a BamHI site on the 3'end. Two TR21
non-coding sequences are amplified via PCR: one TR21 non-coding
sequence (TR21 fragment 1) is amplified with a HindIII site at the
5' end and an Xba site at the 3'end; the other TR21 non-coding
sequence (TR21 fragment 2) is amplified with a BamHI site at the
5'end and a HindIII site at the 3'end. The CMV promoter and TR21
fragments are digested with the appropriate enzymes (CMV
promoter--XbaI and BamHI; TR21 fragment 1--XbaI; TR21 fragment
2--BamHI) and ligated together. The resulting ligation product is
digested with HindIII, and ligated with the HindIII-digested pUC18
plasmid.
[0815] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5..times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[0816] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37.degree. C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[0817] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 10
Bioassay for the Effect of TR21 Polypeptides, Agonists, or
Antagonists on Hematopoietic Progenitor Cells and/or
Differentiation
[0818] Mouse bone marrow cells may be used as target cells to
examine the effect of TR21 polypeptides of the invention on
hematopoietic progenitor cells and/or differentiation. Briefly,
unfractionated bone marrow cells are first washed 2.times. with a
serum-free IMDM that is supplemented with 10% (V/V) BIT (Bovine
serum albumin, Insulin and Transferrin supplement from Stem Cell
Technologies, Vancouver, Canada). The washed cells are then
resuspended in the same growth medium and plated in the 96-well
tissue culture plate (5.times.10.sup.4 cells/well) in 0.2 ml of the
above medium in the presence or absence of cytokines and TR21. Stem
cell factor (SCF) and IL-3 are included as positive mediators of
cell proliferation. Cells are allowed to grow in a low oxygen
environment (5% CO.sub.2, 7% O.sup.2, and 88% N.sub.2) tissue
culture incubator for 6 days. On the sixth day, 0.5 .mu.Ci of
Tritiated thymidine is added to each well and incubation is
continued for an additional 16-18 hours, at which point the cells
are harvested. The level of radioactivity incorporated into
cellular DNA is determined by scintillation spectrometry and
reflects the amount of cell proliferation.
[0819] The studies described in this example test the activity of
TR21 polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
TR21 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of TR21. Potential agonists would be expected to
inhibit hematopoietic cell proliferation in the presence of SCF
and/or IL3 and/or to increase the inhibition of cell proliferation
in the presence of cytokines and TR21 in this assay. Potential
antagonists would be expected to reduce the inhibition of cell
proliferation in the presence of cytokines and TR21 in this
assay.
Example 11
Bioassay for the Effect of TR21 Polypeptides, Agonists or
Antagonists on IL-3 and SCF Stimulated Proliferation and
Differentiation of Hematopoietic Progenitor Cells
[0820] To determine if TR21 polypeptides of the invention inhibit
specific hematopoietic lineages, mouse bone marrow cells are first
washed 2.times. with a serum-free IMDM that is supplemented with
10% (V/V) BIT (Bovine serum albumin, Insulin and Transferrin
supplement from Stem Cell Technologies, Vancouver, Canada). The
washed cells are then resuspended in the same growth medium and
plated in the 96-well tissue culture plate (5.times.10.sup.4
cells/well) in 0.2 ml of the above medium in the presence of IL-3
(1 ng/ml) plus SCF (5 ng/ml) with or without TR21. Cells are
allowed to grow in a low oxygen environment (5% CO.sub.2, 7%
O.sup.2, and 88% N.sub.2) tissue culture incubator, and after 7
days, analyzed for expression of differentiation antigens by
staining with various monoclonal antibodies and FACScan.
[0821] The studies described in this example test the activity of
TR21 polypeptides of the invention. However, one skilled in the art
could easily modify the exemplified studies to test the activity of
TR21 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of TR21. Potential agonists tested in this assay would
be expected to inhibit cell proliferation in the presence of
cytokines and/or to increase the inhibition of cell proliferation
in the presence of cytokines and TR21. Potential antagonists tested
in this assay would be expected to reduce the inhibition of cell
proliferation in the presence of cytokines and TR21.
Example 12
Effect of TR21 on IL-3 and SCF Stimulated Proliferation and
Differentiation of Lin-Population of Bone Marrow Cells
[0822] A population of mouse bone marrow cells enriched in
primitive hematopoietic progenitors can be obtained using a
negative selection procedure, where the committed cells of most of
the lineages are removed using a panel of monoclonal antibodies
(anti cd11b, CD4, CD8, CD45R and Gr-1 antigens) and magnetic beads.
The resulting population of cells (lineage depleted cells) are
plated (5.times.10.sup.4 cells/ml) in the presence or absence of
TR21 polypeptide of the invention (in a range of concentrations) in
a growth medium supplemented with IL-3 (5 ng/ml) plus SCF (100
ng/ml). After seven days of incubation at 37.degree. C. in a
humidified incubator (5% CO.sub.2, 7% O.sup.2, and 88% N.sub.2
environment), cells are harvested and assayed for the HPP-CFC, and
immature progenitors. In addition, cells are analyzed for the
expression of certain differentiation antigens by FACScan. Colony
data is expressed as mean number of colonies.+-.SD) and are
obtained from assays performed in six dishes for each population of
cells.
Example 13
Assays to Detect Stimulation or Inhibition of B Cell Proliferation
and Differentiation
[0823] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL5, IL6, IL-7, IL10, IL-13, IL14 and IL15.
Interestingly, these signals are by themselves weak effectors but
can, in combination with various co-stimulatory proteins, induce
activation, proliferation, differentiation, homing, tolerance and
death among B cell populations. One of the most well studied
classes of B-cell co-stimulatory proteins is the TNF-superfamily.
Within this family CD40, CD27, and CD30 along with their respective
ligands CD154, CD70, and CD153 have been found to regulate a
variety of immune responses. Assays which allow for the detection
and/or observation of the proliferation and differentiation of
these B-cell populations and their precursors are valuable tools in
determining the effects various proteins may have on these B-cell
populations in terms of proliferation and differentiation. Listed
below are two assays designed to allow for the detection of the
differentiation, proliferation, or inhibition of B-cell populations
and their precursors.
In vitro Assay
[0824] Purified TR21 polypeptides of the invention (e.g., soluble
TR21) or agonists or antagonists thereof, are assessed for their
ability to induce activation, proliferation, differentiation or
inhibition and/or death in B-cell populations and their precursors.
The activity of TR21 polypeptides, or agonists or antagonists
thereof on purified human tonsillar B cells, measured qualitatively
over the dose range from 0.1 to 10,000 ng/ml, is assessed in a
standard B-lymphocyte co-stimulation assay in which purified
tonsillar B cells are cultured in the presence of either
formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized
anti-human IgM antibody as the priming agent. Second signals such
as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit
B cell proliferation as measured by tritiated-thymidine
incorporation. Novel synergizing agents can be readily identified
using this assay. The assay involves isolating human tonsillar B
cells by magnetic bead (MACS) depletion of CD3-positive cells. The
resulting cell population is greater than 95% B cells as assessed
by expression of CD45R(B220). Various dilutions of each sample are
placed into individual wells of a 96-well plate to which are added
10.sup.5 B-cells suspended in culture medium (RPMI 1640 containing
10% FBS, 5.times.10.sup.-5M .beta.ME, 100 U/ml penicillin, 10 ug/ml
streptomycin, and 10.sup.-5 dilution of SAC) in a total volume of
150 ul. Proliferation or inhibition is quantitated by a 20 h pulse
(1 uCi/well) with .sup.3H-thymidine (6.7 Ci/mM) beginning 72 h post
factor addition. The positive and negative controls are IL2 and
medium respectively.
In vivo Assay
[0825] BALB/c mice are injected (i.p.) twice per day with buffer
only, or 2 mg/Kg of TR21 polypeptide (e.g., soluble TR21) or
agonists or antagonists thereof. Mice receive this treatment for 4
consecutive days, at which time they are sacrificed and various
tissues and serum collected for analyses. Comparison of H&E
sections from normal and TR21 polypeptide-treated spleens identify
the results of the activity of TR21 polypeptide on spleen cells,
such as the diffusion of peri-arterial lymphatic sheaths, and/or
significant increases in the nucleated cellularity of the red pulp
regions, which may indicate the activation of the differentiation
and proliferation of B-cell populations. Immunohistochemical
studies using a B cell marker, anti-CD45R(B220), are used to
determine whether any physiological changes to splenic cells, such
as splenic disorganization, are due to increased B-cell
representation within loosely defined B-cell zones that infiltrate
established T-cell regions.
[0826] Flow cytometric analyses of the spleens from TR21
polypeptide-treated mice is used to indicate whether TR21
polypeptide specifically increases the proportion of ThB+,
CD45R(B220) dull B cells over that which is observed in control
mice.
[0827] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and TR21 polypeptide-treated mice.
[0828] The studies described in this example test the activity in
TR21 polypeptide. However, one skilled in the art could easily
modify the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), and agonists, and/or
antagonists of TR21.
Example 14
Assay for TR21 Polypeptide Inhibition of B Cell Proliferation in an
in vitro Co-Stimulatory Assay
[0829] This example provides a co-stimulatory assay using
Staphylococcus Aureus Cowan 1 (SAC) as priming agent and
Neutrokine-alpha (International Application Publication No. WO
98/18921) or IL-2 as a second signal to assay for TR21 polypeptide
antagonists of Neutrokine-alpha (or IL-2) mediated B cell
proliferation.
[0830] A soluble TR21 polypeptide is prepared (e.g., a soluble form
of TR21 corresponding to a portion of the TR21 extracellular domain
linked to the Fc portion of a human IgG1 immunoglobulin molecule).
The ability of this protein to alter the proliferative response of
human B cells is assessed in a standard co-stimulatory assay.
Briefly, human tonsillar B cells are purified by magnetic bead
(MACS) depletion of CD3-positive cells. The resulting cell
population is routinely greater than 95% B cells as assessed by
expression of CD19 and CD20 staining. Various dilutions of
rHuNeutrokine-alpha (International Application Publication No. WO
98/18921) or rHuIL2 are placed into individual wells of a 96-well
plate to which is added 10.sup.5 B cells suspended in culture
medium (RPMI 1640 containing 10% FBS, 5.times.10.sup.-5M 2ME, 100
U/ml penicillin, 10 ug/ml streptomycin, and 10.sup.-5 dilution of
formalin-fixed Staphylococcus aureus Cowan I (SAC) also known as
Pansorbin (Pan)) in a total volume of 150 ul. The TR21 polypeptide
is then added at various concentrations and the plates are placed
in the incubator (37.degree. C. 5% CO.sub.2, 95% humidity) for
three days. Proliferation is quantitated by a 20 h pulse (1
.mu.Ci/well) of .sup.3H-thymidine (6.7 Ci/mM) beginning 72 h post
factor addition. The positive and negative controls are SAC exposed
B cells with rHuNeutrokine-alpha (or rHuIL2) and medium (in the
absence of the TR21 polypeptide), respectively.
[0831] Antagonists of rHuNeutrokine-alpha (or rHuIL2) mediated B
cell proliferation demonstrate a reduced level of B cell
proliferation in the samples containing the TR21 polypeptides when
compared to the positive control.
Example 15
T Cell Proliferation Assay
[0832] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
.mu.l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control mAb (B33.1) overnight at 4.degree. C. (1 .mu.g/ml in 0.05M
bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC
are isolated by F/H gradient centrifugation from human peripheral
blood and added to quadruplicate wells (5.times.10.sup.4/well) of
mAb coated plates in RPMI containing 10% FCS and P/S in the
presence of varying concentrations of TR21 protein (total volume
200 .mu.l). Relevant protein buffer and medium alone are controls.
After 48 hr. culture at 37.degree. C., plates are spun for 2 min.
at 1000 rpm and 100 .mu.l of supernatant is removed and stored
-20.degree. C. for measurement of IL-2 (or other cytokines) if
effect on proliferation is observed. Wells are supplemented with
100 .mu.l of medium containing 0.5 .mu.Ci of .sup.3H-thymidine and
cultured at 37.degree. C. for 18-24 hr. Wells are harvested and
incorporation of .sup.3H-thymidine used as a measure of
proliferation. Anti-CD3 alone is the positive control for
proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce
proliferation of T cells is used as the negative controls for the
effects of TR21 proteins.
[0833] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 16
Effect of TR21 on the Expression of MHC Class II, Costimulatory and
Adhesion Molecules and Cell Differentiation of Monocytes and
Monocyte-Derived Human Dendritic Cells
[0834] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[0835] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of TR21
or LPS (positive control), washed with PBS containing 1% BSA and
0.02 mM sodium azide, and then incubated with 1:20 dilution of
appropriate FITC- or PE-labeled monoclonal antibodies for 30
minutes at 4.degree. C. After an additional wash, the labeled cells
are analyzed by flow cytometry on a FACScan (Becton Dickinson).
[0836] Effect on the production of cytokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Th1 helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (10.sup.6/ml)
are treated with increasing concentrations of TR21 for 24 hours.
LPS (100 ng/ml) is added to the cell culture as positive control.
Supernatants from the cell cultures are then collected and analyzed
for IL-12 content using commercial ELISA kit (e.g., R & D
Systems (Minneapolis, Minn.)). The standard protocols provided with
the kits are used.
[0837] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increase expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[0838] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of TR21 or LPS (positive control), washed with PBS
containing 1% BSA and 0.02 mM sodium azide, and then incubated with
1:20 dilution of appropriate FITC- or PE-labeled monoclonal
antibodies for 30 minutes at 4.degree. C. After an additional wash,
the labeled cells are analyzed by flow cytometry on a FACScan
(Becton Dickinson).
[0839] Monocyte activation and/or increased survival. Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. TR21, agonists, or
antagonists of TR21 can be screened using the three assays
described below. For each of these assays, Peripheral blood
mononuclear cells (PBMC) are purified from single donor leukopacks
(American Red Cross, Baltimore, Md.) by centrifugation through a
HISTOPAQUE.TM. gradient (Sigma). Monocytes are isolated from PBMC
by counterflow centrifugal elutriation.
[0840] Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from internally regulated
process (apoptosis). Addition to the culture of activating factors,
such as TNF-alpha dramatically improves cell survival and prevents
DNA fragmentation. Propidium iodide (PI) staining is used to
measure apoptosis as follows. Monocytes are cultured for 48 hours
in polypropylene tubes in serum-free medium (positive control), in
the presence of 100 ng/ml TNF-alpha (negative control), and in the
presence of varying concentrations of the compound to be tested.
Cells are suspended at a concentration of 2.times.10.sup.6/ml in
PBS containing PI at a final concentration of 5 .mu.g/ml, and then
incubated at room temperature for 5 minutes before FAC Scan
analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
[0841] Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.10.sup.5 cells/ml with increasing concentrations of TR21
and under the same conditions, but in the absence of TR21. For
IL-12 production, the cells are primed overnight with IFN-.gamma.
(100 U/ml) in presence of TR21. LPS (10 ng/ml) is then added.
Conditioned media are collected after 24 h and kept frozen until
use. Measurement of TNF-.alpha., IL-10, MCP-1 and IL-8 is then
performed using a commercially available ELISA kit (e.g., R & D
Systems (Minneapolis, Minn.)) applying the standard protocols
provided with the kit.
[0842] Oxidative burst. Purified monocytes are plated in 96-well
plate at 2-1.times.10.sup.5 cell/well. Increasing concentrations of
TR21 are added to the wells in a total volume of 0.2 ml culture
medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days
incubation, the plates are centrifuged and the medium is removed
from the wells. To the macrophage monolayers, 0.2 ml per well of
phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer
pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is
added, together with the stimulant (200 nM PMA). The plates are
incubated at 37.degree. C. for 2 hours and the reaction is stopped
by adding 20 .mu.l 1N NaOH per well. The absorbance is read at 610
nm. To calculate the amount of H.sub.2O.sub.2 produced by the
macrophages, a standard curve of a H.sub.2O.sub.2 solution of known
molarity is performed for each experiment.
[0843] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 17
The Effect of TR21 on the Growth of Vascular Endothelial Cells
[0844] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, 8 units/ml heparin. TR21 protein of SEQ ID NO.
2, and positive controls, such as VEGF and basic FGF (bFGF) are
added, at varying concentrations. On days 4 and 6, the medium is
replaced. On day 8, cell number is determined with a Coulter
Counter. An increase in the number of HUVEC cells indicates that
TR21 may proliferate vascular endothelial cells.
[0845] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 18
Stimulatory Effect of TR21 on the Proliferation of Vascular
Endothelial Cells
[0846] For evaluation of mitogenic activity of growth factors, the
calorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
).sub.2H-tetrazolium) assay with the electron coupling reagent PMS
(phenazine methosulfate) was performed (CellTiter 96 AQ,
PROMEGA.TM.). Cells are seeded in a 96-well plate (5,000
cells/well) in 0.1 ml serum-supplemented medium and are allowed to
attach overnight. After serum-starvation for 12 hours in 0.5% FBS,
conditions (bFGF, VEGF.sub.165 or TR21 in 0.5% FBS) with or without
Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS
mixture (1:0.05) are added per well and allowed to incubate for 1
hour at 37.degree. C. before measuring the absorbance at 490 nm in
an ELISA plate reader. Background absorbance from control wells
(some media, no cells) is subtracted, and seven wells are performed
in parallel for each condition. See, Leak et al. In Vitro Cell.
Dev. Biol 30A:512-518 (1994).
[0847] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 19
Inhibition of PDGF-Induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
[0848] HAoSMC proliferation can be measured, for example, by BrdU
incorporation. Briefly, subconfluent, quiescent cells grown on the
4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP.
Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdU.
After 24 h, immunocytochemistry is performed using the BrdU
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdU antibody at
4.degree. C. for 2 h after exposing to denaturing solution and then
with the streptavidin-peroxidase and diaminobenzidine. After
counterstaining with hematoxylin, the cells are mounted for
microscopic examination, and the Positive-positive cells are
counted. The Positive index is calculated as a percent of the
Positive-positive cells to the total cell number. In addition, the
simultaneous detection of the Positive staining (nucleus) and the
FITC uptake (cytoplasm) is performed for individual cells by the
concomitant use of bright field illumination and dark field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem.
6;27](36):21985-21992 (1996).
[0849] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 20
Stimulation of Endothelial Migration
[0850] This example will be used to explore the possibility that
TR21 may stimulate lymphatic endothelial cell migration.
[0851] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, MD;
Falk, W., Goodwin, R. H. J., and Leonard, E. J. "A 48 well micro
chemotaxis assembly for rapid and accurate measurement of leukocyte
migration." J. Immunological Methods 1980;33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 um (Nucleopore Corp. Cambridge, MA) are coated with 0.1% gelatin
for at least 6 hours at room temperature and dried under sterile
air. Test substances are diluted to appropriate concentrations in
M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul
of the final dilution is placed in the lower chamber of the
modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC
or BMEC cultures are washed and trypsinized for the minimum time
required to achieve cell detachment. After placing the filter
between lower and upper chamber, 2.5.times.10.sup.5 cells suspended
in 50 ul M199 containing 1% FBS are seeded in the upper
compartment. The apparatus is then incubated for 5 hours at
37.degree. C. in a humidified chamber with 5% CO2 to allow cell
migration. After the incubation period, the filter is removed and
the upper side of the filter with the non-migrated cells is scraped
with a rubber policeman. The filters are fixed with methanol and
stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park,
Ill.). Migration is quantified by counting cells of three random
high-power fields (40.times.) in each well, and all groups are
performed in quadruplicate.
[0852] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 21
Stimulation of Nitric Oxide Production by Endothelial Cells
[0853] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation. Thus,
TR21 activity can be assayed by determining nitric oxide production
by endothelial cells in response to TR21.
[0854] Nitric oxide is measured in 96-well plates of confluent
microvascular endothelial cells after 24 hours starvation and a
subsequent 4 hr exposure to various levels of a positive control
(such as VEGF-1) and TR21. Nitric oxide in the medium is determined
by use of the Griess reagent to measure total nitrite after
reduction of nitric oxide-derived nitrate, by nitrate reductase.
The effect of TR21 on nitric oxide release is examined on
HUVEC.
[0855] Briefly, NO release from cultured HUVEC monolayer is
measured with a NO-specific polarographic electrode connected to a
NO meter (Iso-NO, World Precision Instruments Inc.). Calibration of
the NO element is performed according to the following
equation:
2 KNO.sub.2+2 KI+2 H.sub.2SO.sub.4 6 2 NO+I.sub.2+2 H.sub.2O+2
K.sub.2SO.sub.4
[0856] The standard calibration curve is obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
nmol/L) into the calibration solution containing KI and
H.sub.2SO.sub.4. The specificity of the Iso-NO electrode to NO is
previously determined by measurement of NO from authentic NO gas.
The culture medium is removed and HUVECs are washed twice with
Dulbecco's phosphate buffered saline. The cells are then bathed in
5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the
cell plates are kept on a slide warmer (Lab Line Instruments Inc.)
to maintain the temperature at 37.degree. C. The NO sensor probe is
inserted vertically into the wells, keeping the tip of the
electrode 2 mm under the surface of the solution, before addition
of the different conditions. S-nitroso acetyl penicillamin (SNAP)
is used as a positive control. The amount of released NO is
expressed as picomoles per 1.times.10.sup.6 endothelial cells. All
values reported are means of four to six measurements in each group
(number of cell culture wells). See, Leak et al. Biochem. and
Biophys. Res. Comm. 217:96-105 (1995).
[0857] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 22
Effect of TR21 on Cord Formation in Angiogenesis
[0858] Another step in angiogenesis is cord formation, marked by
differentiation of endothelial cells. This bioassay measures the
ability of microvascular endothelial cells to form capillary-like
structures (hollow structures) when cultured in vitro.
[0859] CADMEC (microvascular endothelial cells) are purchased from
Cell Applications, Inc. as proliferating (passage 2) cells and are
cultured in Cell Applications' CADMEC Growth Medium and used at
passage 5. For the in vitro angiogenesis assay, the wells of a
48-well cell culture plate are coated with Cell Applications'
Attachment Factor Medium (200 .mu.l/well) for 30 min. at 37.degree.
C. CADMEC are seeded onto the coated wells at 7,500 cells/well and
cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 .mu.g Cell Applications' Chord Formation Medium
containing control buffer or TR21 (0.1 to 100 ng/ml) and the cells
are cultured for an additional 48 hr. The numbers and lengths of
the capillary-like chords are quantitated through use of the
Boeckeler VIA-170 video image analyzer. All assays are done in
triplicate.
[0860] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. .beta.-esteradiol (1 ng/ml) is used as a negative control.
The appropriate buffer (without protein) is also utilized as a
control.
[0861] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 23
Angiogenic Effect on Chick Chorioallantoic Membrane
[0862] Chick chorioallantoic membrane (CAM) is a well-established
system to examine angiogenesis. Blood vessel formation on CAM is
easily visible and quantifiable. The ability of TR21 to stimulate
angiogenesis in CAM can be examined.
[0863] Fertilized eggs of the White Leghorn chick (Gallus gallus)
and the Japanese quail (Coturnix coturnix) are incubated at
37.8.degree. C. and 80% humidity. Differentiated CAM of 16-day-old
chick and 13-day-old quail embryos is studied with the following
methods.
[0864] On Day 4 of development, a window is made into the eggshell
of chick eggs. The embryos are checked for normal development and
the eggs sealed with cellotape. They are further incubated until
Day 13. THERMANOX.TM. coverslips (Nunc, Naperville, Ill.) are cut
into disks of about 5 mm in diameter. Sterile and salt-free growth
factors, and the protein to be tested, are dissolved in distilled
water and about 3.3 mg/5 ml are pipetted on the disks. After
air-drying, the inverted disks are applied on CAM. After 3 days,
the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde
and rinsed in 0.12 M sodium cacodylate buffer. They are
photographed with a stereo microscope [Wild M8] and embedded for
semi- and ultrathin sectioning as described above. Controls are
performed with carrier disks alone.
[0865] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 24
Angiogenesis Assay Using a MATRIGEL.TM. Implant in Mouse
[0866] In order to establish an in vivo model for angiogenesis to
test TR21 protein activities, mice and rats are implanted
subcutaneously with methylcellulose disks containing either 20 mg
of BSA (negative control), 1 mg of TR21, or 0.5 mg of VEGF-1
(positive control). The negative control disks should contain
little vascularization, while the positive control disks should
show signs of vessel formation.
[0867] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 25
Rescue of Ischemia in Rabbit Lower Limb Model
[0868] To study the in vivo effects of TR21 on ischemia, a rabbit
hindlimb ischemia model is created by surgical removal of one
femoral arteries as described previously (Takeshita, S. et al., Am.
J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery
results in retrograde propagation of thrombus and occlusion of the
external iliac artery. Consequently, blood flow to the ischemic
limb is dependent upon collateral vessels originating from the
internal iliac artery (Takeshita, S. et al., Am. J. Pathol
147:1649-1660 (1995)). An interval of 10 days is allowed for
post-operative recovery of rabbits and development of endogenous
collateral vessels. At 10 day post-operatively (day 0), after
performing a baseline angiogram, the internal iliac artery of the
ischemic limb is transfected with 500 mg naked TR21 expression
plasmid by arterial gene transfer technology using a
hydrogel-coated balloon catheter as described (Riessen, R. et al.,
Hum Gene Ther. 4:749-758 (1993); Leclerc, G. et al., J. Clin.
Invest. 90: 936-944 (1992)). When TR21 is used in the treatment, a
single bolus of 500 mg TR21 protein or control is delivered into
the internal iliac artery of the ischemic limb over a period of 1
min. through an infusion catheter. On day 30, various parameters
are measured in these rabbits: (a) BP ratio--The blood pressure
ratio of systolic pressure of the ischemic limb to that of normal
limb; (b) Blood Flow and Flow Reserve--Resting FL: the blood flow
during undilated condition and Max FL: the blood flow during fully
dilated condition (also an indirect measure of the blood vessel
amount) and Flow Reserve is reflected by the ratio of max FL:
resting FL; (c) Angiographic Score--This is measured by the
angiogram of collateral vessels. A score is determined by the
percentage of circles in an overlaying grid that with crossing
opacified arteries divided by the total number m the rabbit thigh;
(d) Capillary density--The number of collateral capillaries
determined in light microscopic sections taken from hindlimbs.
[0869] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 26
Rat Ischemic Skin Flap Model
[0870] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. TR21 expression, during the skin
ischemia, is studied using in situ hybridization.
[0871] The study in this model is divided into three parts as
follows: [0872] Ischemic skin [0873] Ischemic skin wounds [0874]
Normal wounds
[0875] The experimental protocol includes: [0876] Raising a
3.times.4 cm, single pedicle full-thickness random skin flap
(myocutaneous flap over the lower back of the animal). [0877] An
excisional wounding (4-6 mm in diameter) in the ischemic skin
(skin-flap). [0878] Topical treatment with TR21 of the excisional
wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various
dosage ranges: 1 mg to 100 mg. [0879] Harvesting the wound tissues
at day 3, 5, 7, 10, 14 and 21 post-wounding for histological,
immunohistochemical, and in situ studies.
[0880] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 27
Peripheral Arterial Disease Model
[0881] Angiogenic therapy using TR21 is a novel therapeutic
strategy to obtain restoration of blood flow around the ischemia in
case of peripheral arterial diseases. The experimental protocol
includes:
[0882] a) One side of the femoral artery is ligated to create
ischemic muscle of the hindlimb, the other side of hindlimb serves
as a control.
[0883] b) TR21 protein, in a dosage range of 20 mg -500 mg, is
delivered intravenously and/or intramuscularly 3 times (perhaps
more) per week for 2-3 weeks.
[0884] c) The ischemic muscle tissue is collected after ligation of
the femoral artery at 1, 2, and 3 weeks for the analysis of TR21
expression and histology. Biopsy is also performed on the other
side of normal muscle of the contralateral hindlimb.
[0885] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 28
Ischemic Myocardial Disease Model
[0886] TR21 is evaluated as a potent mitogen capable of stimulating
the development of collateral vessels, and restructuring new
vessels after coronary artery occlusion. Alteration of TR21
expression is investigated in situ. The experimental protocol
includes:
[0887] a) The heart is exposed through a left-side thoracotomy in
the rat. Immediately, the left coronary artery is occluded with a
thin suture (6-0) and the thorax is closed.
[0888] b) TR21 protein, in a dosage range of 20 mg -500 mg, is
delivered intravenously and/or intramuscularly 3 times (perhaps
more) per week for 2-4 weeks.
[0889] c) Thirty days after the surgery, the heart is removed and
cross-sectioned for morphometric and in situ analyzes.
[0890] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 29
Rat Corneal Wound Healing Model
[0891] This animal model shows the effect of TR21 on
neovascularization. The experimental protocol includes:
[0892] a) Making a 1-1. 5 mm long incision from the center of
cornea into the stromal layer.
[0893] b) Inserting a spatula below the lip of the incision facing
the outer corner of the eye.
[0894] c) Making a pocket (its base is 1-1.5 mm form the edge of
the eye).
[0895] d) Positioning a pellet, containing 50 ng-5 ug of TR21,
within the pocket.
[0896] e) TR21 treatment can also be applied topically to the
corneal wounds in a dosage range of 20 mg -500 mg (daily treatment
for five days).
[0897] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 30
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[0898] A. Diabetic db+/db+ Mouse Model.
[0899] To demonstrate that TR21 accelerates the healing process,
the genetically diabetic mouse model of wound healing is used. The
full thickness wound healing model in the db+/db+ mouse is a well
characterized, clinically relevant and reproducible model of
impaired wound healing. Healing of the diabetic wound is dependent
on formation of granulation tissue and re-epithelialization rather
than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389
(1992); Greenhaigh, D. G. et al., Am. J. Pathol. 136:1235
(1990)).
[0900] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol.
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol. 120:1375-1377 (1978)).
[0901] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
[0902] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates are used in
this study (Jackson Laboratories). The animals are purchased at 6
weeks of age and were 8 weeks old at the beginning of the study.
Animals are individually housed and received food and water ad
libitum. All manipulations are performed using aseptic techniques.
The experiments are conducted according to the rules and guidelines
of Human Genome Sciences, Inc. Institutional Animal Care and Use
Committee and the Guidelines for the Care and Use of Laboratory
Animals.
[0903] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[0904] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[0905] TR21 is administered using at a range different doses of
TR21, from 4 mg to 500 mg per wound per day for 8 days in vehicle.
Vehicle control groups received 50 mL of vehicle solution.
[0906] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
[0907] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) TR21.
[0908] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 was 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations were made
using the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[0909] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface
(5mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with TR21.
This assessment included verification of the presence of cell
accumulation, inflammatory cells, capillaries, fibroblasts,
re-epithelialization and epidermal maturity (Greenhalgh, D. G. et
al., Am. J. Pathol 136:1235 (1990)). A calibrated lens micrometer
is used by a blinded observer.
[0910] Tissue sections are also stained immunohistochemically with
a polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
[0911] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA antibody (1:50) with
an ABC Elite detection system. Human colon cancer served as a
positive tissue control and human brain tissue is used as a
negative tissue control. Each specimen included a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[0912] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
B. Steroid Impaired Rat Model
[0913] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl, S. M.
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahl, S. M. et
al., J. Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med.
147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability (Ebert,
R. H., et al., An. Intern. Med. 37:701-705 (1952)), fibroblast
proliferation, and collagen synthesis (Beck, L. S. et al., Growth
Factors. 5: 295-304 (1991); Haynes, B. F. et al., J. Clin. Invest.
61: 703-797 (1978)) and producing a transient reduction of
circulating monocytes (Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Anti-inflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989)). The systemic
administration of steroids to impaired wound healing is a well
establish phenomenon in rats (Beck, L. S. et al., Growth Factors.
5: 295-304 (1991); Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Anti-inflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989); Pierce, G. F. et al.,
Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
[0914] To demonstrate that TR21 can accelerate the healing process,
the effects of multiple topical applications of TR21 on full
thickness excisional skin wounds in rats in which healing has been
impaired by the systemic administration of methylprednisolone is
assessed.
[0915] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and were 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
[0916] The wounding protocol is followed according to section A,
above. On the day of wounding, animals are anesthetized with an
intramuscular injection of ketamine (50 mg/kg) and xylazine (5
mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[0917] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day
8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue was no longer visible and the wound is covered
by a continuous epithelium.
[0918] TR21 is administered using at a range different doses of
TR21, from 4 mg to 500 mg per wound per day for 8 days in vehicle.
Vehicle control groups received 50 mL of vehicle solution.
[0919] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
[0920] Four groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) were evaluated: 1) Untreated group 2)
Vehicle placebo control 3) TR21 treated groups.
[0921] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 was 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations were made using the
following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[0922] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining was performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin was improved by treatment with TR21. A calibrated
lens micrometer is used by a blinded observer to determine the
distance of the wound gap.
[0923] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[0924] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
Example 31
Lymphedema Animal Model
[0925] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of TR21 in lymphangiogenesis and
re-establishment of the lymphatic circulatory system in the rat
hind limb. Effectiveness is measured by swelling volume of the
affected limb, quantification of the amount of lymphatic
vasculature, total blood plasma protein, and histopathology. Acute
lymphedema is observed for 7-10 days. Perhaps more importantly, the
chronic progress of the edema is followed for up to 3-4 weeks.
[0926] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs are shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements are made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[0927] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated or suture ligated.
[0928] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located.
[0929] The 2 proximal and 2 distal lymphatic vessels and distal
blood supply of the popliteal node are then and ligated by
suturing. The popliteal lymph node, and any accompanying adipose
tissue, is then removed by cutting connective tissues.
[0930] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of 0.5 cm around the leg. Skin also may be anchored by suturing
to underlying muscle when necessary.
[0931] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[0932] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people then those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[0933] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), both legs are shaved and
equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software (ChenVictor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[0934] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[0935] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs were amputated using a
guillotine, then both experimental and control legs were cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint was disarticulated and the foot was
weighed.
[0936] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle was observed under fluorescent microscopy
for lymphatics. Other immuno/histological methods are currently
being evaluated.
[0937] The studies described in this example test the activity in
TR21 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of TR21
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of TR21.
[0938] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0939] The entire disclosure of each document cited (including
patents, patent applications, journal articles, abstracts,
laboratory manuals, books, or other disclosures) in the Background
of the Invention, Detailed Description, and Examples is hereby
incorporated herein by reference.
[0940] Further, the Sequence Listing submitted herewith, in both
computer and paper forms, is hereby incorporated by reference in
its entirety.
[0941] Moreover, the disclosure of U.S. Provisional Patent
Application Ser. No. 60/315,357 is hereby incorporated by reference
in its entirety.
Sequence CWU 1
1
51899DNAhuman 1gcaccatgag gcgagggccc cggagcctgc ggggcaggga
cgcgccagcc cccacgccct 60gcgtcccggc cgagtgcttc gacctgctgg tccgccactg
cgtggcctgc gggctcctgc 120gcacgccgcg gccgaaaccg gccggggcca
gcagccctgc gcccaggacg gcgctgcagc 180cgcaggagtc ggtgggcgcg
ggggccggcg aggcggcgct gcccctgccc gggctgctct 240ttggcgcccc
cgcgctgctg ggcctggcac tggtcctggc gctggtcctg gtgggtctgg
300tgagctggag gcggcgacag cggcggcttc gcggcgcgtc ctccgcagag
gcccccgacg 360gagacaagga cgccccagag cccctggaca aggtcatcat
tctgtctccg ggaatctctg 420atgccacagc tcctgcctgg cctcctcctg
gggaagaccc aggaaccacc ccacctggcc 480acagtgtccc tgtgccagcc
acagagctgg gctccactga actggtgacc accaagacgg 540ccggccctga
gcaacaatag cagggagccg gcaggaggtg gcccctgccc tccctctgga
600cccccagcca ggggcttgga aatcaaattc agctcttcac tccagcatgc
acatgccctc 660tttctgggac caggctaacc ctgcagaagc acagacacta
cagaccacag cattcagccc 720ccatggagtt tggtgtgctt gcctttggct
tcagacctca ccatctttga cagcccttga 780aggtggtagc ccagctcctg
ttcctgtgcc ttcaaaaggc tggggcacta tgagtaaaag 840accgctttta
aaatggggaa ggcaccatta agccaaaatg aatctgaaaa aagacaaaa
8992184PRThuman 2Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp
Ala Pro Ala Pro1 5 10 15Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu
Leu Val Arg His Cys 20 25 30Val Ala Cys Gly Leu Leu Arg Thr Pro Arg
Pro Lys Pro Ala Gly Ala 35 40 45Ser Ser Pro Ala Pro Arg Thr Ala Leu
Gln Pro Gln Glu Ser Val Gly 50 55 60Ala Gly Ala Gly Glu Ala Ala Leu
Pro Leu Pro Gly Leu Leu Phe Gly65 70 75 80Ala Pro Ala Leu Leu Gly
Leu Ala Leu Val Leu Ala Leu Val Leu Val 85 90 95Gly Leu Val Ser Trp
Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser 100 105 110Ser Ala Glu
Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp 115 120 125Lys
Val Ile Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala 130 135
140Trp Pro Pro Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His
Ser145 150 155 160Val Pro Val Pro Ala Thr Glu Leu Gly Ser Thr Glu
Leu Val Thr Thr 165 170 175Lys Thr Ala Gly Pro Glu Gln Gln
1803733DNAhuman 3gggatccgga gcccaaatct tctgacaaaa ctcacacatg
cccaccgtgc ccagcacctg 60aattcgaggg tgcaccgtca gtcttcctct tccccccaaa
acccaaggac accctcatga 120tctcccggac tcctgaggtc acatgcgtgg
tggtggacgt aagccacgaa gaccctgagg 180tcaagttcaa ctggtacgtg
gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240aggagcagta
caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact
300ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca
acccccatcg 360agaaaaccat ctccaaagcc aaagggcagc cccgagaacc
acaggtgtac accctgcccc 420catcccggga tgagctgacc aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct 480atccaagcga catcgccgtg
gagtgggaga gcaatgggca gccggagaac aactacaaga 540ccacgcctcc
cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg
600acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat
gaggctctgc 660acaaccacta cacgcagaag agcctctccc tgtctccggg
taaatgagtg cgacggccgc 720gactctagag gat 733417PRThuman 4Met Leu Gln
Asn Ser Ala Val Leu Leu Leu Leu Val Ile Ser Ala Ser1 5 10
15Ala522PRTartificial sequenceconsensus signal sequence 5Met Pro
Thr Trp Ala Trp Trp Leu Phe Leu Val Leu Leu Leu Ala Leu1 5 10 15Trp
Ala Pro Ala Arg Gly 20
* * * * *