U.S. patent application number 11/773491 was filed with the patent office on 2008-01-17 for galectin 11.
This patent application is currently assigned to Human Genome Science, Inc.. Invention is credited to ReinerL Gentz, Fu-Tong Lui, Jian Ni, Craig A. Rosen.
Application Number | 20080015149 11/773491 |
Document ID | / |
Family ID | 27671284 |
Filed Date | 2008-01-17 |
United States Patent
Application |
20080015149 |
Kind Code |
A1 |
Ni; Jian ; et al. |
January 17, 2008 |
Galectin 11
Abstract
The present invention relates to novel galectin 11 proteins
which are members of the galectin superfamily. In particular,
isolated nucleic acid molecules are provided encoding the human
galectin 11 proteins. Galectin 11 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 galectin 11 activity. Also
provided are diagnostic and therapeutic methods.
Inventors: |
Ni; Jian; (Germantown,
MD) ; Gentz; ReinerL; (Gauting, DE) ; Rosen;
Craig A.; (Laytosville, MD) ; Lui; Fu-Tong;
(San Diego, CA) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Science, Inc.
Rockville
MD
La Jolla Institute for Allergy and Immunology
San Diego
CA
|
Family ID: |
27671284 |
Appl. No.: |
11/773491 |
Filed: |
July 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11246980 |
Oct 11, 2005 |
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11773491 |
Jul 5, 2007 |
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10455366 |
Jun 6, 2003 |
7041803 |
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11246980 |
Oct 11, 2005 |
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09557170 |
Apr 21, 2000 |
6605699 |
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10455366 |
Jun 6, 2003 |
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09109864 |
Jul 6, 1998 |
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09557170 |
Apr 21, 2000 |
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09010146 |
Jan 21, 1998 |
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09109864 |
Jul 6, 1998 |
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60034205 |
Jan 21, 1997 |
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60034204 |
Jan 21, 1997 |
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60169932 |
Dec 10, 1999 |
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60130390 |
Apr 21, 1999 |
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Current U.S.
Class: |
424/133.1 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/7.1; 435/70.1;
514/1.7; 514/19.3; 530/350; 530/387.9; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
A61P 37/00 20180101; C07K 14/70535 20130101; C12N 9/6475 20130101;
A61P 11/06 20180101; A61K 2039/505 20130101; A61K 38/00 20130101;
A61P 35/00 20180101; C12N 2799/026 20130101; C07K 16/18 20130101;
C07K 14/4726 20130101; C07K 16/2851 20130101; C12N 9/6491 20130101;
A61P 29/00 20180101; C12N 9/6489 20130101; A61P 37/08 20180101;
C07K 14/47 20130101 |
Class at
Publication: |
514/012 ;
435/252.3; 435/254.2; 435/320.1; 435/325; 435/007.1; 435/070.1;
530/350; 530/387.9; 536/023.5 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 11/06 20060101 A61P011/06; A61P 29/00 20060101
A61P029/00; A61P 35/00 20060101 A61P035/00; A61P 37/00 20060101
A61P037/00; A61P 37/08 20060101 A61P037/08; C07H 21/04 20060101
C07H021/04; C07K 14/435 20060101 C07K014/435; C07K 16/18 20060101
C07K016/18; C12N 15/63 20060101 C12N015/63; C12N 5/10 20060101
C12N005/10; C12P 21/00 20060101 C12P021/00; G01N 33/53 20060101
G01N033/53 |
Claims
1. An isolated polynucleotide comprising a nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 1 to 133 of SEQ ID NO:2; (b) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 2 to 133 of SEQ ID NO:2; (c) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 1 to 275 of SEQ ID NO:25; (d) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 2 to 275 of SEQ ID NO:25; (e) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 1 to 296 of SEQ ID NO:27; (f) a nucleotide sequence
which is at least 95% identical to a nucleotide sequence encoding
amino acids 2 to 296 of SEQ ID NO:27; and (g) the complement of
(a), (b), (c), (d), (e), or (f).
2. The isolated polynucleotide of claim 1 wherein said nucleotide
sequence is (a) or a complementary sequence thereto.
3. The isolated polynucleotide of claim 1 wherein said nucleotide
sequence is (c) or a complementary sequence thereto.
4. The isolated polynucleotide of claim 1 wherein the nucleotide is
sequence (e) or a complementary sequence thereto.
5. A method of making a recombinant vector comprising inserting the
isolated polynucleotide of claim 1 into a vector.
6. A recombinant vector comprising the polynucleotide of claim
1.
7. A genetically engineered host cell comprising the polynucleotide
of claim 1.
8. A method for producing a galectin 11 polypeptide, comprising
culturing the genetically engineered host cell of claim 7 under
conditions suitable to produce the polypeptide, and recovering said
polypeptide.
9. An isolated galectin 11 polypeptide comprising an amino acid
sequence selected from the group consisting of: (a) an amino acid
sequence which is at least 95% identical to amino acids 1 to 133 of
SEQ ID NO:2; (b) an amino acid sequence which is at least 95%
identical to amino acids 1 to 275 of SEQ ID NO:25; (c) an amino
acid sequence which is at least 95% identical to amino acids 1 to
296 of SEQ ID NO:27 (d) amino acids 1 to 121 of SEQ ID NO:25; (e)
amino acids 1 to 142 of SEQ ID NO:27; and (f) amino acids 151 to
275 of SEQ ID NO:25.
10. The isolated polypeptide of claim 9 wherein said amino acid
sequence is (a).
11. The isolated polypeptide of claim 9 wherein said amino acid
sequence is (b).
12. A pharmaceutical composition comprising the polypeptide of
claim 9, and a pharmaceutically acceptable carrier.
13. An isolated antibody that binds specifically to the polypeptide
of claim 9.
14. An isolated antibody that binds specifically to the polypeptide
of claim 10.
15. A method of detecting a galectin 11 polypeptide in a sample,
comprising: a) contacting said sample with an antibody according to
claim 13, and b) detecting the presence of said antibody bound to
said polypeptide.
16. A method of detecting a galectin 11 polypeptide in a sample,
comprising: a) contacting said sample with an antibody according to
claim 14, and b) detecting the presence of said antibody bound to
said polypeptide.
17. A method of treatment of a cell growth disorder in a mammal,
comprising administering a therapeutically effective amount of the
polypeptide of claim 9 to said mammal.
18. The method of claim 17, wherein said disorder is selected from
the group consisting of cancer, autoimmune diseases, inflammatory
diseases, asthma, and allergic diseases.
19. A method of regulating cell growth or differentiation in a
mammal, comprising administering a galectin 11 polypeptide of claim
9 to the mammal in an amount sufficient to suppress cell growth or
differentiation.
20. A method of regulating cell growth or differentiation in a
mammal, comprising administering a galectin 11 polynucleotide of
claim 1 to the mammal in an amount sufficient to suppress cell
growth or differentiation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/246,980, filed Oct. 11, 2005, which is a divisional of U.S.
application Ser. No. 10/455,366, filed Jun. 6, 2003, which is a
divisional of U.S. application Ser. No. 09/557,170, filed Apr. 21,
2000, which is a continuation-in-part of U.S. application Ser. No.
09/109,864 (abandoned), filed Jul. 6, 1998, which is a
continuation-in-part of U.S. application Ser. No. 09/010,146
(abandoned), filed Jan. 21, 1998, which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/034,205, filed Jan. 21, 1997 and of U.S. Provisional Application
No. 60/034,204, filed Jan. 21, 1997; and said U.S. application Ser.
No. 09/557,170 also claims benefit under 35 U.S.C. .sctn. 119(e) of
U.S. Provisional Application No. 60/169,932, filed Dec. 10, 1999
and of U.S. Provisional Application No. 60/130,390, filed Apr. 21,
1999. Each of the aforementioned non-provisional and provisional
applications is hereby incorporated by reference in its
entirety.
STATEMENT UNDER 37 C.F.R. .sctn. 1.77(b)(4)
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text document. The document is
entitled "PF354P2D2C1_SeqListing.txt" (26,553 bytes, created Jul.
2, 2007), and is hereby incorporated by reference in its entirety
herein.
FIELD OF THE INVENTION
[0003] The present invention relates to a novel galectin. More
specifically, isolated nucleic acid molecules are provided encoding
human galectin 11. Galectin 11 polypeptides are also provided, as
are vectors, host cells, recombinant methods for producing the
same, and antibodies to galectin 11 polypeptides. The invention
further relates to screening methods for identifying agonists and
antagonists of galectin 11 activity. Also provided are diagnostic
methods for detecting cell growth disorders and therapeutic methods
for cell growth disorders, including autoimmune diseases, cancer,
and inflammatory diseases.
BACKGROUND OF THE INVENTION
[0004] Lectins are proteins that bind to specific carbohydrate
structures and can thus recognize particular glycoconjugates.
Barondes et al., J. Biol. Chem. 269(33):20807-20810 (1994).
Galectins are members of a family of .beta.-galactoside-binding
lectins with related amino acid sequences (For review see, Barondes
et al., Cell 76:597-598 (1994); Barondes et al., J. Biol. Chem.
269(33):20807-20810 (1994)). Although a large number of
glycoproteins containing .beta.-galactoside sugars are produced by
the cell, only a few will bind to known galectins in vitro. Such
apparent binding specificity suggests a highly specific functional
role for the galectins.
[0005] Galectin 1 (conventionally termed LGALS1 for lectin,
galactoside-binding, soluble-1, but which is also known as: L-14-1,
L-14, RL-14.5, galaptin, MGBP, GBP, BBL, CHA, HBP, HPL, HLBP 14,
rIML-1) is a homodimer with a subunit molecular mass of 14,500
Daltons. Galectin 1 is expressed abundantly in smooth and skeletal
muscle, and to a lesser extent in many other cell types (Couraud et
al., J. Biol. Chem. 264:1310-1316 (1989). Galectin 1 is thought to
specifically bind laminin, a highly polylactosaminated cellular
glycoprotein, as well as the highly polylactosaminated
lysosome-associated membrane proteins (LAMPs). Galectin 1 has also
been shown to bind specifically to a lactosamine-containing
glycolipid found on olfactory neurons and to integrin
a.sub.7b.sub.1 on skeletal muscle cells.
[0006] Other members of the Galectin family have also been
reported. Galectin 2 was originally found in hepatoma and is a
homodimer with a subunit molecular mass of 14,650 Daltons (Gitt et
al., J. Biol. Chem. 267:10601-10606 (1992)). Galectin 3 (a.k.a.,
Mac-2, EPB, CBP-35, CBP-30, and L-29) is abundant in activated
macrophages and epithelial cells and is a monomer with an apparent
molecular mass between 26,320 and 30,300 Daltons (Cherayil et al.,
Proc. Natl. Acad. Sci. USA 87: 7324-7326 (1990)). Galectin 3 has
been observed to bind specifically to laminin, immunoglobulin E and
its receptor, and bacterial lipopolysaccharides. Galectin 4 has a
molecular mass of 36,300 Daltons and contains two
carbohydrate-binding domains within a single polypeptide chain (Oda
et al., J. Biol. Chem. 268:5929-5939 (1993)). Galectins 5 and 6 are
discussed in Barondes et al., Cell 76:597-598 (1994). Human
Galectin 7 has a molecular mass of 15,073 Daltons and is found
mainly in stratified squamous epithelium (Madsen et al., J. Biol.
Chem. 270(11):5823-5829 (1995)).
[0007] Animal lectins, in general, often function in modulating
cell-cell and cell-matrix interactions. Galectin 1 has been shown
to either promote or inhibit cell adhesion depending upon the cell
type in which it is present. Galectin 1 inhibits cell-matrix
interactions in skeletal muscle presumably, by galectin 1-mediated
disruption of laminin-integrin a.sub.7b.sub.1 interactions (Cooper
et al., J. Cell Biol. 115:1437-1448 (1991)). In several
non-skeletal muscle cell types, Galectin 1 promotes cell-matrix
adhesion possibly by cross-linking cell surface and substrate
glycoconjugates (Zhou et al., Arch. Bioch. Biophys. 300:6-17
(1993); Skrincosky et al., Cancer Res. 53:2667-2675 (1993)).
[0008] Galectin 1 also participates in regulating cell
proliferation (Wells et al., Cell 64:91-97 (1991)) and some immune
functions (Offner et al., J. Neuroimmunol. 28:177-184 (1990)).
Galectin 1 induces the release of tumor necrosis factor from
macrophages (Kajikawa et al., Life Sci. 39:1177-1181 (1986).
Galectin 1 has also been demonstrated to have therapeutic activity
against autoimmune diseases in animal models for experimental
myasthenia gravis, and experimental autoimmune encephalomyelitis
(Levi et al., Eur. J. Immunol. 13:500-507 (1983); and Offner et
al., J. Neuroimmunol. 28:177-184 (1990), respectively).
Additionally, galectin 1 has been shown to regulate immune response
by mediating apoptosis of T cells (Perillo et al., Nature
378:736-739 (1995)).
[0009] Galectin 3 promotes the growth of cells cultured under
restrictive culture conditions (Yang et al., Proc. Natl. Acad. Sci.
USA 93:6737-6742 (June 1996)). Galectin 3 expression in cells
confers resistance to apoptosis which indicates that galectin 3
could be a cell death suppresser which interferes in a common
pathway of apoptosis. Id. Galectin 3 has also been observed to
function in modulating cell-adhesion, as well as in the activation
of certain immune cells by cross-linking IgE and IgE receptors.
[0010] Recently, a galectin-like antigen designated HOM-HD-21 was
found to be highly expressed in a Hodgkin's Disease cDNA library
and another galectin, termed PCTA-1, was identified as a specific
cell surface marker on human prostate cancer cell lines and
patient-derived carcinomas.
[0011] Thus, galectins have been observed to be involved in the
regulation of immune cell activity, as well as in such diverse
processes as cell adhesion, proliferation, inflammation,
autoimmunity, and metastasis of tumor cells. Accordingly, there is
a need in the art for the identification of novel galectins which
can serve as useful tools in the development of therapeutics and
diagnostics for regulating immune response, inflammatory disease
and cancer.
SUMMARY OF THE INVENTION
[0012] The present invention provides isolated nucleic acid
molecules comprising, or alternatively consisting of, a
polynucleotide encoding the galectin 11 polypeptide having the
amino acid sequence shown in FIG. 1 (SEQ ID NO:2), the amino acid
sequence encoded by the cDNA clone deposited in a bacterial host as
ATCC Deposit Number 209053, on May 16, 1997, and fragments,
variants, derivatives, and analogs thereof.
[0013] The present invention also provides isolated nucleic acid
molecules comprising a polynucleotide encoding the galectin 11
polypeptide having the amino acid sequence shown in FIGS. 6A-B (SEQ
ID NO:14), referred to herein sometimes as "Galectin-11.alpha." and
fragments, variants, derivatives, and analogs thereof.
[0014] The present invention also provides isolated nucleic acid
molecules comprising a polynucleotide encoding the galectin 11
polypeptide having the amino acid sequence shown in FIGS. 6A-B and
8 (SEQ ID NO:16), referred to herein sometimes as
"Galectin-11.beta.", and fragments, variants, derivatives, and
analogs thereof.
[0015] The galectin 11 of FIG. 1 (SEQ ID NOS:1 and 2), the galectin
11.alpha. of FIGS. 6A-B (SEQ ID NOS:24 and 25), and the galectin
11.beta. of FIGS. 7-8 (SEQ ID NOS:26 and 27) are often referred to
herein collectively as, e.g., "galectin 11."
[0016] The galectin 11 polynucleotide of FIG. 1 (SEQ ID NO:1), the
galectin 11.alpha. polynucleotide of FIGS. 6A-B (SEQ ID NO:24), and
the galectin 11.beta. polynucleotide of FIG. 7 (SEQ ID NO:26) are
often referred to herein collectively as, e.g., "galectin 11
polynucleotides."
[0017] The present invention also relates to recombinant vectors
which include the isolated nucleic acid molecules of the invention,
and to host cells containing the recombinant vectors, as well as to
methods of making such vectors and host cells and for using them
for production of galectin 11 polypeptides by recombinant
techniques.
[0018] The invention further provides isolated galectin 11
polypeptides, including galectin 11 of SEQ ID NO:2 and galectin
11.alpha. and .beta., having an amino acid sequence encoded by a
polynucleotide described herein and antibodies which bind these
polypeptides. The galectin 11 polypeptide of FIG. 1 (SEQ ID NO:2),
the galectin 11.alpha. polypeptide of FIGS. 6A-B (SEQ ID NO:25),
and the galectin 11.beta. polypeptide of FIG. 7 (SEQ ID NO:27) are
often referred to herein collectively as, e.g., "galectin 11
polypeptides."
[0019] The present invention also provides screening methods for
identifying compounds capable of enhancing or inhibiting a cellular
response, such as, for example, apoptosis, induced by galectin 11.
Generally, these methods involve contacting galectin 11, the
candidate compound, and a cell which expresses a galectin 11
ligand, assaying a cellular response resulting from the binding of
galectin 11 with the ligand, and comparing the cellular response to
a standard, the standard being assayed when contact of galectin 11
and the galectin 11 ligand is made in the absence of the candidate
compound; whereby, an increased cellular response over the standard
indicates that the compound is an agonist and a decreased cellular
response over the standard indicates that the compound is an
antagonist.
[0020] In another aspect, a screening assay for agonists and
antagonists is provided which involves determining the effect a
candidate compound has on galectin 11 binding to a
.beta.-galactoside sugar. In particular, the method involves
contacting a .beta.-galactoside sugar with a galectin 11
polypeptide and a candidate compound and determining whether
galectin 11 binding to the .beta.-galactoside sugar is increased or
decreased due to the presence of the candidate compound.
[0021] The invention also provides diagnostic methods useful during
diagnosis of disorders associated with elevated, decreased, or
otherwise aberrant expression of galectin 11.
[0022] The invention further provides for methods for treating an
individual in need of an increased level of galectin 11 activity in
the body comprising, administering to such an individual a
composition comprising a therapeutically effective amount of an
isolated galectin 11 polypeptide, fragment, variant, derivative, or
analog of the invention, or an agonist thereof.
[0023] In another embodiment, the invention provides for methods
for treating an individual in need of a decreased level of galectin
11 activity in the body comprising, administering to such an
individual a composition comprising a therapeutically effective
amount of a galectin 11 fragment, variant, derivative, analog or
antibody of the invention or galectin 11 antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and
deduced amino acid sequence (SEQ ID NO:2) of galectin 11. The
protein has a deduced molecular mass of about 14.8 kDa. The
complementary strand of the nucleotide sequence of SEQ ID NO:1 is
shown in SEQ ID NO:12.
[0025] FIG. 2 shows the regions of similarity between the amino
acid sequences of the galectin 11 protein (HJACE54) (SEQ ID NO:2),
rat galectin 5 (SEQ ID NO:3), and human galectin 8 (SEQ ID NO:4).
Identical amino acids shared between the galectins are shaded,
while conservative amino acid changes are boxed. By examining the
regions of amino acids shaded and/or boxed, the skilled artisan can
readily identify conserved domains between the two polypeptides.
These conserved domains are preferred embodiments of the present
invention.
[0026] FIG. 3 shows structural and functional features of galectin
11 (SEQ ID NO:2) predicted using the default parameters of the
indicated computer programs. Alpha, beta, turn and coil regions;
hydrophilicity and hydrophobicity; amphipathic regions; flexible
regions; antigenic index and surface probability are shown. In the
Antigenic Index--Jameson-Wolf graph, the positive peaks indicate
locations of the highly antigenic regions of the galectin 11
protein, i.e., regions from which epitope-bearing peptides of the
invention can be obtained. The domains defined by these graphs are
contemplated by the present invention, including for example, amino
acid residues 65-70 and 118-124 in FIG. 1 (SEQ ID NO:2), which
correspond to the shown highly antigenic regions of the galectin 11
polypeptide.
[0027] The data presented in FIG. 3 are also represented in tabular
form in Table I. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIII. The column headings refer to
the following features of the amino acid sequence presented in FIG.
3, 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: Alpha, Amphipathic
Regions--Eisenberg; X: Beta, Amphipathic Regions--Eisenberg; XI:
Flexible Regions--Karplus-Schulz; XII: Antigenic
Index--Jameson-Wolf; and XIII: Surface Probability Plot--Emini.
[0028] FIG. 4. Structure of human galectin 11 gene. The human
galectin 11 gene is located on chromosome 11. This figure shows the
structure of the region of chromosome 11 containing the galectin 11
gene and discloses the number of nucleotides corresponding to the
transcribed (shaded) and untranscribed (open) portions of this
region of the chromosome. The human galectin 11 gene contains 5
exons. The translation initiation site is located on the second
exon. The nucleotide numbering identified in exons designated by
roman numerals correspond to that presented in FIG. 1 (SEQ ID
NO:1).
[0029] FIG. 5A is a bar graph showing that transfection of Jurkat
cells with a galectin 11 expression construct (pEF-Leg11) induces
apoptosis of transfected cells. Shaded bars represent % apoptosis
of Jurkat cells that have been transfected with the galectin 11
expression construct, whereas open bars represent % apoptosis of
Jurkat cells that have been transfected with the pEF control
vector. Apoptosis was measured by two-color cytometry using
mitoTracker Red.
[0030] FIG. 5B is a bar graph showing the survival of GFP positive
cells that have been successfully transfected, 4 days after
transfection. The survival of the transfected cells was examined
after co-transfection with either the control vector (pEF1), or the
galectin 11 expression vector (pEF-Leg11). There were about 4 times
more surviving GFP positive cells after transfection with pEF1 than
with pEF-Leg11.
[0031] FIGS. 6A-B shows the nucleotide sequence (SEQ ID NO:24) and
deduced amino acid sequence (SEQ ID NO:25) of the complete galectin
11.alpha. cDNA and protein, respectively.
[0032] FIG. 7 is a schematic showing the relative positions of the
8 exons which comprise the galectin-11 gene. Also shown is the
difference created by alternative splicing between
galectin-11.alpha. and galectin-11.beta. (galectin-11.alpha. being
7 nucleotides longer at the 5' terminus of exon 2) resulting in
divergent N-termini between the variants. Nucleotide residues
136-147 of SEQ ID NO:24 (galectin-11.alpha.) and nucleotide
residues 136-140 of SEQ ID NO:26 (galectin-11.beta.) are shown.
[0033] FIG. 8 shows the difference between the polypeptide
sequences of galectin-11.alpha. and galectin-11.beta.. The complete
nucleotide and amino acid sequences of galectin-11.beta. are shown
in the sequence listing as SEQ ID NOS: 26 and 27, respectively.
Amino acid residues 1-29 of SEQ ID NO:25 (galectin-11.alpha.) and
amino acid residues 1-50 of SEQ ID NO:27 (galectin-11.beta.) are
shown.
DETAILED DESCRIPTION
[0034] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a galectin 11
polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID
NO:2), FIGS. 6A-B (SEQ ID NO:25), or FIGS. 6A-B and 8 (SEQ ID
NO:27) which were determined by sequencing cloned cDNAs. The
nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) was obtained by
sequencing the HJACE54 plasmid which was deposited on May 16, 1997
at the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va., and given accession number 209053. The
galectin 11 polypeptides of the present invention share sequence
homology with rat galectin 5, chicken galectin 3, and human
galectin 8 gene products (see, e.g., FIG. 2; SEQ ID NOS: 3-4).
[0035] The invention further provides for fragments, variants,
derivatives and analogs of galectin 11 polynucleotides and
polypeptides encoded thereby, and antibodies which bind these
polypeptides.
DEFINITIONS
[0036] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0037] "Functional activity" or "biological activity" refers to
galectin 11 polypeptides, fragments, derivatives, variants, and
analogs, exhibiting activity similar, but not necessarily identical
to, an activity of a galectin 11 polypeptide, including mature
forms, as measured in a particular biological assay, with or
without dose dependency. In the case where dose dependency does
exist, it need not be identical to that of the galectin 11
polypeptide, but rather substantially similar to the
dose-dependence in a given activity as compared to the galectin 11
polypeptide (i.e., the candidate polypeptide will exhibit greater
activity or not more than about 25-fold less and, preferably, not
more than about tenfold less activity, and most preferably, not
more than about three-fold less activity relative to the galectin
11 polypeptide.) Such functional activities include, but are not
limited to, biological activity (such as, for example, the ability
to bind a .beta.-galactoside sugar, the ability to agglutinate
trypsin-treated rabbit erythrocytes and/or to induce apoptosis),
antigenicity (ability to bind or compete with a galectin 11
polypeptide for binding to an anti-galectin 11 antibody),
immunogenicity (ability to generate antibody which binds to a
galectin 11 polypeptide), the ability to form dimers with galectin
11 polypeptides of the invention, and the ability to bind to other
galectins and/or a receptor or ligand for galectin 11.
Polynucleotides encoding polypeptides having galectin 11 functional
or biological activity, and the complementary strand of these
polynucleotides are also encompassed by the invention.
[0038] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications have been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0039] "Polypeptide" refers to any peptide or protein comprising
two or more amino acids joined to each other by peptide bonds or
modified peptide bonds, i.e., peptide isosteres. "Polypeptide"
refers to both short chains, commonly referred to as peptides,
oligopeptides or oligomers, and to longer chains, generally
referred to as proteins. Polypeptides may contain amino acids other
than the 20 gene-encoded amino acids. "Polypeptides" include amino
acid sequences modified either by natural processes, such as
posttranslational processing, or by chemical modification
techniques which are well known in the art. Such modifications are
well described in basic texts and in more detailed monographs, as
well as in a voluminous research literature. Modifications can
occur anywhere in a polypeptide, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini. 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
galectin 11 polypeptide. Also, a given galectin 11 polypeptide may
contain many types of modifications. Galectin 11 polypeptides may
be branched as a result of ubiquitination, and they may be cyclic,
with or without branching. Cyclic, branched and branched cyclic
polypeptides may result from posttranslation natural 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 phosphotidylinositol, 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, 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 and Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, 1983; Seifter et al., "Analysis for
protein modifications and nonprotein cofactors", Meth Enzymol
182:626-646 (1990) and Rattan et al., "Protein Synthesis:
Posttranslational Modifications and Aging", Ann NY Acad Sci
663:48-62 (1992).)
[0040] "Variant" as the term is used herein, is a polynucleotide or
polypeptide that differs from a reference polynucleotide or
polypeptide respectively, but retains functional or biological
activity of galectin 11. A typical variant of a polynucleotide
differs in nucleotide sequence from another, reference
polynucleotide. Changes in the nucleotide sequence of the variant
may or may not alter the amino acid sequence of a polypeptide
encoded by the reference polynucleotide. Nucleotide changes may
result in amino acid substitutions, additions, deletions, fusions
and truncations in the polypeptide encoded by the reference
sequence, as discussed below. A typical variant of a polypeptide
differs in amino acid sequence from another, reference polypeptide.
Generally, differences are limited so that the sequences of the
reference polypeptide and the variant are closely similar overall
and, in many regions, identical. A variant and reference
polypeptide may differ in amino acid sequence by one or more
substitutions, additions, deletions in any combination. A
substituted or inserted amino acid residue may or may not be one
encoded by the genetic code. A variant of a polynucleotide or
polypeptide may be a naturally occurring such as an allelic
variant, or it may be a variant that is not known to occur
naturally. Non-naturally occurring variants of polynucleotides and
polypeptides may be made by mutagenesis techniques or by direct
synthesis.
[0041] "Antibodies" as used herein includes polyclonal and
monoclonal antibodies, chimeric, single chain, and humanized
antibodies, as well as Fab fragments, including the products of an
Fab or other immunoglobulin expression library.
Nucleic Acid Molecules
[0042] The galectin 11 nucleotide sequence identified as SEQ ID
NO:1 was assembled from partially homologous ("overlapping")
sequences obtained from the deposited clone. The overlapping
sequences were assembled into a single contiguous sequence of high
redundancy resulting in a final sequence identified as SEQ ID
NO:1.
[0043] Therefore, SEQ ID NO:1 and the translated SEQ ID NO:2 are
sufficiently accurate and otherwise suitable for a variety of uses
well known in the art and described further below. For instance,
SEQ ID NO:1 is useful for designing nucleic acid hybridization
probes that will detect nucleic acid sequences contained in SEQ ID
NO:1 or the cDNA contained in the deposited clone. These probes
will also hybridize to nucleic acid molecules in biological
samples, thereby enabling a variety of forensic and diagnostic
methods of the invention. Similarly, polypeptides identified from
SEQ ID NO:2 may be used, for example, to generate antibodies which
bind specifically to proteins galectin 11.
[0044] Further, unless otherwise indicated, all nucleotide
sequences determined by sequencing a DNA molecule herein were
determined using an automated DNA sequencer (such as the Model 373
from Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0045] Accordingly, for those applications requiring precision in
the nucleotide sequence or the amino acid sequence, the present
invention provides not only the generated nucleotide sequence
identified as SEQ ID NO:1 and the predicted translated amino acid
sequence identified as SEQ ID NO:2, but also a sample of plasmid
DNA containing a human cDNA of galectin 11 deposited with the ATCC.
The nucleotide sequence of the deposited galectin 11 clone can
readily be determined by sequencing the deposited clone in
accordance with known methods. The predicted galectin 11 amino acid
sequence can then be verified from such deposits. Moreover, the
amino acid sequence of the protein encoded by the deposited clone
can also be directly determined by peptide sequencing or by
expressing the protein in a suitable host cell containing the
deposited human galectin 11 cDNA, collecting the protein, and
determining its sequence.
[0046] Using the information provided herein, such as the
nucleotide sequence in FIG. 1, a nucleic acid molecule of the
present invention encoding a galectin 11 polypeptide may be
obtained using standard cloning and screening procedures, such as
those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecule described
in FIG. 1 (SEQ ID NO:1) was discovered in a cDNA library derived
from G1 phase Jurkat T-cells. This gene was also identified in cDNA
libraries generated from human neutrophil and human infant adrenal
gland. Polynucleotides of the invention can also be obtained from
natural sources such as mRNA or genomic DNA using techniques known
in the art, or can be chemically synthesized using techniques known
in the art.
[0047] The human galectin 11 gene is located on chromosome 11 and
contains 5 exons (see, e.g., FIG. 4). The nucleotide sequence of
the galectin 11 cDNA of FIG. 1 (SEQ ID NO:1) is 865 nucleotides in
length (830 nucleotides discounting the poly A tail of the cDNA)
which encodes a predicted open reading frame of 133 amino acid
residues. There is a predicted initiation codon at nucleotides
49-51 of the nucleotide sequence depicted in FIG. 1 (SEQ ID NO:1),
located on the second exon of the gene. The galectin 11 protein
shown in FIG. 1 (SEQ ID NO:2) shares homology with the translation
product of rat galectin 5, chicken galectin 3, and human galectin 8
(see, e.g., FIG. 2). Additionally, as further discussed below,
galectin 11 induces apoptosis of transfected T-cells (see Example 5
and FIGS. 5A and 5B). These findings indicate that galectin 11
functions in a manner similar to other previously characterized
galectins and therefore, that galectin 11 is important in the
regulation of cell growth disorders, autoimmune diseases, cancer,
and inflammatory diseases.
[0048] The nucleotide sequence of the galectin 11 cDNA of FIGS.
6A-B (SEQ ID NO:24) is 1337 nucleotides in length. This is one of
two alternatively spliced forms of galectin 11 and is referred to
as galectin 11.alpha.. The other form, galectin 11.beta., differs
only in the loss of 7 nucleotides (nucleotides 136-142 as shown in
FIGS. 6A-B (SEQ ID NO:24)). See FIG. 7. The sequence of galectin 11
is shown in the sequence listing as SEQ ID NO:26. The resulting
translation products of these splice variants are believed to
differ only at the N-terminus. The amino acid sequences of galectin
11.alpha. and .beta. are shown in the sequence listing as SEQ ID
NOS:25 and 27, respectively. The differences between the two
proteins are highlighted in FIG. 8.
[0049] The galectin 11 polypeptide is comprised of two carbohydrate
binding domains (CARD domains) separated by a linker sequence. The
first carbohydrate binding domain consists of the first 121 amino
acid residues of galectin-11.alpha. (SEQ ID NO:25) and the first
142 amino acids of galectin 11.beta. (SEQ ID NO:27). The 29 amino
acid residues following the first CARD domain is the linker
sequence. Finally, the last 125 amino acid residues in each protein
is the C-terminal CARD domain. Preferred polypeptides of the
invention comprise either an N-terminal or C-terminal CARD domain.
Polynucleotides encoding such polypeptides are also provided.
[0050] Also provided in the present invention are allelic variants,
orthologs, and/or species homologs. Procedures known in the art can
be used to obtain full-length genes, allelic variants, splice
variants, full-length coding portions, orthologs, and/or species
homologs of genes corresponding to SEQ ID NO: 1-2, 24-25, 26-27, or
the deposited clone, using information from the sequences disclosed
herein or the clones deposited with the ATCC. For example, allelic
variants and/or species homologs may be isolated and identified by
making suitable probes or primers from the sequences provided
herein and screening a suitable nucleic acid source for allelic
variants and/or the desired homologue.
[0051] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, as well as the
variability of processing sites for different known proteins, the
predicted galectin 11 polypeptide encoded by the deposited cDNA
comprises about 133 amino acid residues, but may be anywhere in the
range of 125-150 amino acids.
[0052] 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 or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the complementary or anti-sense
strand.
[0053] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment (e.g., the natural environment if it is naturally
occurring), and thus is altered "by the hand of man" from its
natural state. 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 synthetically. In a specific embodiment,
"isolated" nucleic acid molecules of the invention comprise all or
a portion of the coding region of galectin 11, as disclosed in FIG.
1 (SEQ ID NO:1) or galectin 11.alpha. as disclosed in FIGS. 6A-B
(SEQ ID NO:24), or galectin 11.beta. as disclosed in SEQ ID NO:26.
The term "isolated" does not refer to genomic or cDNA libraries,
whole cell total or mRNA preparations, genomic DNA preparations
(including those separated by electrophoresis and transferred onto
blots), sheared whole cell genomic DNA preparations or other
compositions where the art demonstrates no distinguishing features
of the polynucleotide/sequences of the present invention.
[0054] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) or a
portion of an ORF shown in FIG. 1 or 6A-B (SEQ ID NO:1, 24, or 26);
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 galectin 11 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.
[0055] In specific embodiments, the invention provides isolated
nucleic acid molecules encoding the full length galectin 11
polypeptide depicted in FIG. 1 (SEQ ID NO:2), and galectin 11
nucleic acid molecules encoding the galectin 11 polypeptide
sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC Deposit No. 209053, on May 16, 1997. In a further
embodiment, nucleic acid molecules are provided encoding the full
length galectin 11 polypeptide lacking the N-terminal methionine.
The invention further provides an isolated nucleic acid molecule
having the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or the
nucleotide sequence of the galectin 11 cDNA contained in the
above-described deposited clone, or a nucleic acid molecule having
a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, have uses which
include, but are not limited to, probes for gene mapping by in situ
hybridization with chromosomes, and for detecting expression of the
galectin 11 gene in human tissue, for instance, by Northern blot
analysis. The invention further provides a polynucleotide encoding
a polypeptide comprising the full-length amino acid sequence shown
as SEQ ID NO:25 or 27, with or without an N-terminal
methoinine.
[0056] In specific embodiments, the polynucleotides of the
invention are at least 15, at least 30, at least 50, at least 100,
at least 125, at least 500, or at least 1000 continuous nucleotides
but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb,
10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a
further embodiment, polynucleotides of the invention comprise a
portion of the coding sequences, as disclosed herein, but do not
comprise all or a portion of any intron. In another embodiment, the
polynucleotides comprising coding sequences do not contain coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the
galectin 11 gene of interest on chromosome II). 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).
[0057] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA, the nucleotide sequence shown in FIGS. 1 and
6A-B (SEQ ID NOS:1, 24, and 26), or the complementary strand
thereto, is intended fragments of at least about 15 nt, and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably, at least about 40 nt in
length. 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 deposited cDNA or the nucleotide
sequence as shown in FIG. 1 (SEQ ID NO:1) or the cDNA shown in FIG.
6 (SEQ ID NOS:24 and 26) or the complementary strand thereto. Also
encompassed by the invention are DNA fragments comprising 50, 100,
150, 200, 250, 300, 350, 365, 370, 375, 380, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850 contiguous nucleotides of the sequence
shown in FIG. 1 (SEQ ID NO:1), the strand complementary thereto, or
contained in the deposited clone. The present invention also
encompasses fragments corresponding to most, if not all, of the
nucleotide sequence of the deposited cDNA or as shown in FIG. 1
(SEQ ID NO:1) or the complimentary strand thereto. In further
embodiments, the polynucleotide fragments of the invention comprise
a sequence which encodes amino acids 1-14, 1-20, 1-40, 1-66, 2-67,
3-8, 3-67, 5-108, 5-128, 10-17, 10-20, 12-16, 13-20, 13-68, 14-67,
23-40, 20-50, 40-108, 41-60, 47-61, 47-108, 47-128, 50-100, 61-80,
65-108, 65-128, 66-108, 76-88, 81-100, 88-108, 88-128, 95-101,
101-133, 108-120, 114-128, and/or 114-128 of the amino acid
sequence depicted in FIG. 1 (SEQ ID NO:2). In preferred
embodiments, polynucleotide fragments of the invention encode a
polypeptide which demonstrates a galectin 11 functional activity.
Fragments of the invention have numerous uses which include, but
are not limited to, diagnostic probes and primers as discussed
herein.
[0058] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the galectin 11 protein. In particular, such nucleic acid fragments
of the present invention include nucleic acid molecules encoding: a
polypeptide comprising amino acid residues from about 65-70 and
118-124 in FIG. 1 (SEQ ID NO:2). The inventors have determined that
the above polypeptide fragments are antigenic regions of the
galectin 11 protein. Methods for determining other such
epitope-bearing portions of the galectin 11 protein are described
in detail below.
[0059] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide which hybridizes under stringent hybridization
conditions to all or a portion of a galectin 11 polynucleotides
(including fragments) described herein, the complementary strand
thereof, the cDNA clone contained in ATCC Deposit No. 209053, on
May 16, 1997, or fragments thereof. 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 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at 65.degree. C.
[0060] Also contemplated are nucleic acid molecules that hybridize
to the galectin 11 polynucleotides under lower stringency
hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, lower stringency
conditions include an overnight incubation at 37 degree C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl;
0.2MNaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30%
formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes
at 50 degree C. with 1.times.SSPE, 0.1% SDS. In addition, to
achieve even lower stringency, washes performed following stringent
hybridization can be done at higher salt concentrations (e.g.
5.times.SSC).
[0061] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0062] 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, still more preferably at least about
30, 50, 60, 75, 100, 150, 175, 200, 250, 300, 350 nt preferable
about 30-70 nt, or 80-150 nucleotides, or the entire length of the
reference polynucleotide. 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 deposited cDNA or the
nucleotide sequence as depicted in FIG. 1 (SEQ ID NO:1). In
specific embodiments, the polynucleotide hybridizes to nucleotides
0-20, 0-25, 0-30, 0-50, 51-100, 80-100, 101-200, 201-300, 301-400,
401-450, 451-500, 501-550, 551-600, 601-700, 701-750, 751-780,
and/or 780-820 of the nucleotide sequence disclosed in FIG. 1 (SEQ
ID NO:1). In other specific embodiments, the polynucleotide
hybridizes to a nucleotide sequence which encodes amino acid
residues 1-14, 10-20, 20-50, 50-100, 100-133 of the amino acid
sequence depicted in FIG. 1 (SEQ ID NO:2). In specific embodiments,
the polynucleotide hybridizes to nucleotides 1-20, 1-25, 1-30,
1-50, 51-100, 80-100, 101-200, 201-300, 301-400, 401-450, 451-500,
501-550, 551-600, 601-700, 701-750, 751-800, 801-850, 851-900,
901-950, 951-1,000, 1,001-1050, 1,051-1,100, 1,101-1,150,
1,151-1,200, 1,201-1,250, and/or 1,251-1,337 of the nucleotide
sequence disclosed in SEQ ID NO:24. In other specific embodiments,
the polynucleotide hybridizes to a nucleotide sequence which
encodes amino acid residues 1-14, 10-20, 20-50, 50-100, 100-130,
130-160, 160-210, 210-240 and/or 240-275 of the amino acid sequence
depicted in SEQ ID NO:25. In specific embodiments, the
polynucleotide hybridizes to nucleotides 1-20, 1-25, 1-30, 1-50,
51-100, 80-100, 101-200, 201-300, 301-400, 401-450, 451-500,
501-550, 551-600, 601-700, 701-750, 751-800, 801-850, 851-900,
901-950, 951-1,000, 1,001-1050, 1,051-1,100, 1,101-1,150,
1,151-1,200, 1,201-1,250, and/or 1,251-1,330 of the nucleotide
sequence disclosed in Figure SEQ ID NO:26. In other specific
embodiments, the polynucleotide hybridizes to a nucleotide sequence
which encodes amino acid residues 1-14, 10-20, 20-50, 50-100,
100-130, 130-160, 160-210, 210-240, 240-270 and/or 270-296 of the
amino acid sequence depicted in SEQ ID NO:27. These polynucleotides
have uses which include, but are not limited to, diagnostic probes
and primers, as discussed above and in more detail below.
[0063] Of course, a polynucleotide which hybridizes only to a poly
A sequence (such as the 3' terminal poly(A) tract of the galectin
11 cDNA shown in FIG. 1 (SEQ ID NO:1), FIGS. 6A-B (SEQ ID NO:24) or
SEQ ID NO:26 or to a complementary stretch of T (or U) residues,
would not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone generated using an
oligo-dT primer).
[0064] As indicated, nucleic acid molecules of the present
invention which encode a galectin 11 polypeptide may include, but
are not limited to, those encoding the amino acid sequence of the
polypeptide, by itself; the coding sequence for the polypeptide and
additional sequences, such as those encoding an amino acid leader
or secretory sequence, such as a pre-, or pro- or prepro-protein
sequence; the coding sequence of the 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; an additional coding sequence which codes for
additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid 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 galectin 11 fused to Fc at the N- or
C-terminus.
[0065] The present invention is also directed to polynucleotide
fragments of the polynucleotides of the invention. In the present
invention, a "polynucleotide fragment" refers to a short
polynucleotide having a nucleic acid sequence which: is a portion
of that contained in a deposited clone, or encoding the polypeptide
encoded by the cDNA in a deposited clone; is a portion of that
shown in SEQ ID NO:1, 24, or 26 or the complementary strand
thereto, or is a portion of a polynucleotide sequence encoding the
polypeptide of SEQ ID NO:2, 25, or 27. The nucleotide fragments of
the invention are preferably at least about 15 nt, and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably, at least about 40 nt, at
least about 50 nt, at least about 75 nt, or at least about 150 nt
in length. A fragment "at least 20 nt in length," for example, is
intended to include 20 or more contiguous bases from the cDNA
sequence contained in a deposited clone or the nucleotide sequence
shown in SEQ ID NO:1, 24, or 26. In this context "about" includes
the particularly recited value, a value larger or smaller by
several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both termini. These nucleotide fragments have uses that include,
but are not limited to, as diagnostic probes and primers as
discussed herein. Of course, larger fragments (e.g., 50, 150, 500,
600, 2000 nucleotides) are preferred.
[0066] Moreover, representative examples of polynucleotide
fragments of the invention, include, for example, fragments
comprising, or alternatively consisting of, a sequence from about
nucleotide number 1-48, 49-99, 100-150, 151-201, 202-252, 253-303,
304-354, 355-405, 406-450, 451-501, and 502 to the end of SEQ ID
NO:1, or the complementary strand thereto, or the cDNA contained in
the deposited clone. 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. Preferably, these fragments encode a polypeptide
which has biological activity. More preferably, these
polynucleotides can be used as probes or primers as discussed
herein. Polynucleotides which hybridize to these nucleic acid
molecules under stringent hybridization conditions or lower
stringency conditions are also encompassed by the invention, as are
polypeptides encoded by these polynucleotides.
[0067] The exact formulation, route of administration and dosage of
the compounds of the invention to be administrated can be chosen by
the individual physician in view of the patient's condition (see
e.g., Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics." Ch. 1 p. 1). Other methods will be known to the
skilled artisan and are within the scope of the invention.
[0068] However, many polynucleotide sequences, such as EST
sequences, are publicly available and accessible through sequence
databases. Some of these sequences are related to SEQ ID NO:1 and
may have been publicly available prior to conception of the present
invention. Preferably, such related polynucleotides are
specifically excluded from the scope of the present invention. To
list every related sequence would be cumbersome. Accordingly,
preferably excluded from the present invention are one or more
polynucleotides comprising a nucleotide sequence described by the
general formula of a-b, where a is any integer between 1 to 851 of
SEQ ID NO:1, b is an integer of 15 to 865, where both a and b
correspond to the positions of nucleotide residues shown in SEQ ID
NO:1, and where the b is greater than or equal to a+14.
[0069] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode a
portion (i.e., fragments), analogs or derivatives of the galectin
11 protein. 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.
[0070] Such variants include those produced by nucleotide
substitutions, deletions or additions which may involve one or more
nucleotides Particularly preferred are variants in which the
nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14 15, 20, 25, 30, 35, 40, 50, or, 20-15, 15-10, 10-5 1-5, 1-3,
or 1-2 amino acids of a polypeptide of the invention are
substituted, deleted, or added in any combination. The variants may
be altered in coding regions, 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 deletion, which do not alter the properties and
activities of the galectin 11 protein or portions thereof. Also
especially preferred in this regard are conservative
substitutions.
[0071] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 75%, 80%, 85%, or 90% identical, and
more preferably at least 95%, 96%, 97%, 98% or 99% or 98-99%
identical to (a) a nucleotide encoding amino acids 1 to 133 of SEQ
ID NO:2; (b) a nucleotide encoding amino acids 2 to 133 of SEQ ID
NO:2; (c) a nucleotide sequence of the galectin 11 polypeptide
encoded by the cDNA contained in ATCC Deposit No. 209053; (d) a
nucleotide encoding amino acids 1 to 275 of SEQ ID NO:25; (e) a
nucleotide encoding amino acids 1 to 296 of SEQ ID NO:27; (f) a
nucleotide encoding amino acid residues 1 to 121 of SEQ ID NO:25;
(g) a nucleotide encoding amino acid residues 1 to 142 of SEQ ID
NO:27; (h) a nucleotide encoding amino acids 2 to 275 of SEQ ID
NO:25; (i) a nucleotide encoding amino acid residues 2 to 296 of
SEQ ID NO:27; (j) a nucleotide encoding amino acids 151 to 275 of
SEQ ID NO:25; or (k) fragments and other polynucleotide sequences
of the invention as described herein. Polynucleotides which
hybridize to these nucleic acid molecules under stringent
hybridization conditions or lower stringency conditions are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
[0072] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a galectin 11 polypeptide of the present invention 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 nucleotide mismatches per each 100
nucleotides of the reference nucleotide sequence encoding the
galectin 11 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 mutations 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 query sequence
may be an entire sequence shown of SEQ ID NO:1, the ORF (open
reading frame), or any fragment specified as described herein.
[0073] As a practical matter, whether any particular nucleic acid
molecule is at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% identical to, for instance, the nucleotide sequence shown in
SEQ ID NOS: 1, 24, and 26 or to the nucleotides sequence of the
deposited cDNA clone can be determined conventionally using known
computer programs, such as, for example, 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.
[0074] A preferred method for determining the best overall match
between a 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)). In a sequence alignment the query and subject sequences
are both DNA sequences. An RNA sequence can be compared by
converting U's to T's. The result of said global sequence alignment
is in percent identity. Preferred parameters used in a FASTDB
alignment of DNA sequences to calculate percent identify 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.
[0075] 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.
[0076] 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.
[0077] The galectin 11 variants may contain alterations in the
coding regions, non-coding regions, or both. Especially preferred
are polynucleotide variants containing alterations which produce
silent substitutions, additions, or deletions, but do not alter the
properties or activities of the encoded polypeptide. Nucleotide
variants produced by silent substitutions due to the degeneracy of
the genetic code are preferred. Moreover, variants in which 5-10,
1-5, or 1-2 amino acids are substituted, deleted, or added in any
combination are also preferred. Galectin 11 polynucleotide variants
can be produced for a variety of reasons, e.g., to optimize codon
expression for a particular host (change codons in the human mRNA
to those preferred by a bacterial host such as E. coli).
[0078] Naturally occurring galectin 11 variants are called "allelic
variants," and refer to 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).) These
allelic variants can vary at either the polynucleotide and/or
polypeptide level and are included in the present invention.
Alternatively, non-naturally occurring variants may be produced by
mutagenesis techniques or by direct synthesis.
[0079] Using known methods of protein engineering and recombinant
DNA technology, variants may be generated to improve or alter the
characteristics of the galectin 11 polypeptides. For instance, one
or more amino acids can be deleted from the N-terminus or
C-terminus of the secreted protein without substantial loss of
biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988 (1993), reported variant KGF proteins having heparin
binding activity even after deleting 3, 8, or 27 amino-terminal
amino acid residues. Similarly, Interferon gamma exhibited up to
ten times higher activity after deleting 8-10 amino acid residues
from the carboxy terminus of this protein. (Dobeli et al., J.
Biotechnology 7:199-216 (1988).)
[0080] Moreover, ample evidence demonstrates that variants often
retain a biological activity similar to that of the naturally
occurring protein. For example, Gayle and coworkers (J. Biol. Chem.
268:22105-22111 (1993)) conducted extensive mutational analysis of
human cytokine IL-1a. They used random mutagenesis to generate over
3,500 individual IL-1a mutants that averaged 2.5 amino acid changes
per variant over the entire length of the molecule. Multiple
mutations were examined at every possible amino acid position. The
investigators found that "[m]ost of the molecule could be altered
with little effect on either [binding or biological activity]."
(See, Abstract.) In fact, only 23 unique amino acid sequences, out
of more than 3,500 nucleotide sequences examined, produced a
protein that significantly differed in activity from wild-type.
[0081] Furthermore, even if deleting one or more amino acids from
the N-terminus or C-terminus of a polypeptide results in
modification or loss of one or more biological functions, other
biological activities may still be retained. For example, the
ability of a deletion variant to induce and/or to bind antibodies
which recognize the secreted form will likely be retained when less
than the majority of the residues of the secreted form are removed
from the N-terminus or C-terminus. Whether a particular polypeptide
lacking N- or C-terminal residues of a protein retains such
immunogenic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0082] Thus, the invention further includes galectin 11 polypeptide
variants which show substantial biological activity. Such variants
include deletions, insertions, inversions, repeats, and
substitutions selected according to general rules known in the art
so as have little effect on activity.
[0083] The present application is directed to nucleic acid
molecules at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% identical to the nucleic acid sequences disclosed herein (e.g.,
nucleic acid sequence shown in FIG. 1 or 6A-B (SEQ ID NO:1, 24, or
26), nucleic acid sequence of the deposited cDNA clone, and nucleic
acid sequences encoding a polypeptide having the amino acid
sequence of an N and/or C terminal deletion disclosed below as m-n
of SEQ ID NO:2, 25, or 27), irrespective of whether they encode a
polypeptide having galectin 11 functional activity. This is because
even where a particular nucleic acid molecule does not encode a
polypeptide having galectin 11 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 galectin 11
functional activity include, inter alia, (1) isolating the galectin
11 gene or allelic or splice variants thereof in a cDNA library;
(2) in situ hybridization (e.g., "FISH") to metaphase chromosomal
spreads to provide precise chromosomal location of the galectin 11
gene, as described in Verma et al., Human Chromosomes: A Manual of
Basic Techniques, Pergamon Press, New York (1988); (3) use in
linkage analysis as a marker for chromosome 11; and (4) Northern
Blot analysis for detecting galectin 11 mRNA expression in specific
tissues.
[0084] Preferred, however, are nucleic acid molecules having
sequences at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or
99% identical to the nucleic acid sequence disclosed herein, shown
in FIG. 1 or 6A-B (SEQ ID NO:1, 24, or 26), nucleic acid sequence
of the deposited cDNA clone, the nucleic acid encoding the
polypeptide shown in FIG. 1 or 6A-B (SEQ ID NO:2, 25, or 27), and
fragments thereof, which do, in fact, encode a polypeptide having
galectin 11 functional activity. By "a polypeptide having galectin
11 functional activity" is intended polypeptides exhibiting
activity similar, but not necessarily identical, to a functional
activity of the galectin 11 protein of the invention (e.g.,
complete (full-length) galactin 11, and mature galactin 11), as
measured in a particular assay. For example, galectin 11 protein
activity can be measured using a .beta.-galactoside sugar (e.g.,
thiodigalactoside or lactose) binding assay, an assay for apoptosis
and/or an assay for agglutination of trypsin-treated rabbit
erythrocytes, as further described below.
[0085] 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
75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to
the nucleic acid sequence of the deposited cDNA, the nucleic acid
sequence shown in FIG. 1 or 6A-B (SEQ ID NO:1, 24, or 26), the
nucleic acid encoding the polypeptide shown in FIG. 1 or 6A-B (SEQ
ID NO:2, 25, or 27), or fragment thereof, will encode "a
polypeptide having galectin 11 functional activity". In fact, since
numerous degenerate variants of these nucleotide sequences encode
the same polypeptide, this will be clear to the skilled artisan
even without performing the above described comparison 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 galectin 11 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), as
further described below.
[0086] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in 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.
[0087] The first strategy exploits the tolerance of amino acid
substitutions by natural selection during the process of evolution.
By comparing amino acid sequences in different species, conserved
amino acids can be identified. These conserved amino acids are
likely important for protein function. In contrast, the amino acid
positions where substitutions have been tolerated by natural
selection indicates that these positions are not critical for
protein function. Thus, positions tolerating amino acid
substitution could be modified while still maintaining biological
activity of the protein.
[0088] The second strategy uses genetic engineering to introduce
amino acid changes at specific positions of a cloned gene to
identify regions critical for protein function. For example, site
directed mutagenesis or alanine-scanning mutagenesis (introduction
of single alanine mutations at every residue in the molecule) can
be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The
resulting mutant molecules can then be tested for biological
activity.
[0089] As the authors state, these two strategies have revealed
that proteins are surprisingly tolerant of amino acid
substitutions. The authors further indicate which amino acid
changes are likely to be permissive at certain amino acid positions
in the protein. For example, most buried (within the tertiary
structure of the protein) amino acid residues require nonpolar side
chains, whereas few features of surface side chains are generally
conserved. Moreover, tolerated conservative amino acid
substitutions involve replacement of the aliphatic or hydrophobic
amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and
Glu; replacement of the amide residues Asn and Gln, replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic
residues Phe, Tyr, and Trp, and replacement of the small-sized
amino acids Ala, Ser, Thr, Met, and Gly.
[0090] For example, site directed changes at the amino acid level
of galectin 11 of FIG. 1 (SEQ ID NO:2) can be made by replacing a
particular amino acid with a conservative amino acid. Preferred
conservative mutations include: M1 replaced with A, G, I, L, S, T,
or V; S2 replaced with A, G, I, L, T, M, or V; R4 replaced with H,
or K; L5 replaced with A, G, I, S, T, M, or V; E6 replaced with D;
V7 replaced with A, G, I, L, S, T, or M; S10 replaced with A, G, I,
L, T, M, or V; H11 replaced with K, or R; A12 replaced with G, I,
L, S, T, M, or V; L13 replaced with A, G, I, S, T, M, or V; Q15
replaced with N; G16 replaced with A, I, L, S, T, M, or V; L17
replaced with A, G, I, S, T M, or V; S18 replaced with A, G, I, L,
T, M, or V; G20 replaced with A, I, L, S, T, M, or V; Q21 replaced
with N; V22 replaced with A, G, I, L, S, T, or M; I23 replaced with
A, G, L, S, T, M, or V; I24 replaced with A, G, L, S, T, M, or V;
V25 replaced with A, G, I, L, S, T, or M; R26 replaced with H, or
K; G27 replaced with A, I, L, 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; L30
replaced with A, G, I, S, T, M, or V; Q31 replaced with N; E32
replaced with D; K34 replaced with H, or R; H35 replaced with K, or
R; F36 replaced with W, or Y; T37 replaced with A, G, I, L, S, M,
or V; V38 replaced with A, G, I, L, S, T, or M; S39 replaced with
A, G, I, L, T, M, or V; L40 replaced with A, G, I, S, T, M, or V;
R41 replaced with H, or K; D42 replaced with E; Q43 replaced with
N; A44 replaced with G, I, L, S, T, M, or V; A45 replaced with G,
I, L, S, T, M, or V; H46 replaced with K, or R; A47 replaced with
G, I, L, S, T, M, or V; V49 replaced with A, G, I, L, S, T, or M;
T50 replaced with A, G, I, L, S, M, or V; L51 replaced with A, G,
I, S, T, M, or V; R52 replaced with H, or K; A53 replaced with G,
I, L, S, T, M, or V; S54 replaced with A, G, I, L, T, M, or V; F55
replaced with W, or Y; A56 replaced with G, I, L, S, T, M, or V;
D57 replaced with E; R58 replaced with H, or K; T59 replaced with
A, G, I, L, S, M, or V; L60 replaced with A, G, I, S, T, M, or V;
A61 replaced with G, I, L, S, T, M, or V; W62 replaced with F, or
Y; I63 replaced with A, G, L, S, T, M, or V; S64 replaced with A,
G, I, L, T, M, or V; R65 replaced with H, or K; W66 replaced with
F, or Y; G67 replaced with A, I, L, S, T, M, or V; Q68 replaced
with N; K69 replaced with H, or R; K70 replaced with H, or R; L71
replaced with A, G, I, S, T, M, or V; I72 replaced with A, G, L, S,
T, M, or V; S73 replaced with A, G, I, L, T, M, or V; A74 replaced
with G, I, L, S, T, M, or V; F76 replaced with W, or Y; L77
replaced with A, G, I, S, T, M, or V; F78 replaced with W, or Y;
Y79 replaced with F, or W; Q81 replaced with N; R82 replaced with
H, or K; F83 replaced with W, or Y; F84 replaced with W, or Y; E85
replaced with D; V86 replaced with A, G, I, L, S, T, or M; L87
replaced with A, G, I, S, T, M, or V; L88 replaced with A, G, I, S,
T, M, or V; L89 replaced with A, G, I, S, T, M, or V; F90 replaced
with W, or Y; Q91 replaced with N; E92 replaced with D; G93
replaced with A, I, L, S, T, M, or V; G94 replaced with A, I, L, S,
T, M, or V; L95 replaced with A, G, I, S, T, M, or V; K96 replaced
with H, or R; L97 replaced with A, G, I, S, T, M, or V; A98
replaced with G, I, L, S, T, M, or V; L99 replaced with A, G, I, S,
T, M, or V; N100 replaced with Q; G101 replaced with A, I, L, S, T,
M, or V; Q102 replaced with N; G103 replaced with A, I, L, S, T, M,
or V; L104 replaced with A, G, I, S, T, M, or V; G105 replaced with
A, I, L, S, T, M, or V; A106 replaced with G, I, L, S, T, M, or V;
T107 replaced with A, G, I, L, S, M, or V; S108 replaced with A, G,
I, L, T, M, or V; M109 replaced with A, G, I, L, S, T, or V; N110
replaced with Q; Q111 replaced with N; Q112 replaced with N; A113
replaced with G, I, L, S, T, M, or V; L114 replaced with A, G, I,
S, T, M, or V; E115 replaced with D; Q116 replaced with N; L117
replaced with A, G, I, S, T, M, or V; R118 replaced with H, or K;
E119 replaced with D; L120 replaced with A, G, I, S, T, M, or V;
R121 replaced with H, or K; I122 replaced with A, G, L, S, T, M, or
V; S123 replaced with A, G, I, L, T, M, or V; G124 replaced with A,
I, L, S, T, M, or V; S125 replaced with A, G, I, L, T, M, or V;
V126 replaced with A, G, I, L, S, T, or M; Q127 replaced with N;
L128 replaced with A, G, I, S, T, M, or V; Y129 replaced with F, or
W; V131 replaced with A, G, I, L, S, T, or M; H132 replaced with K,
or R; and/or S133 replaced with A, G, I, L, T, M, or V.
[0091] Using these same principles, similar conservative
substitutions can be made in the polypeptide of SEQ ID NO:25 or
27.
[0092] The resulting constructs can be routinely screened for
activities or functions described throughout the specification and
known in the art. Preferably, the resulting constructs have an
increased and/or a decreased galectin 11 activity or function,
while the remaining galectin 11 activities or functions are
maintained. More preferably, the resulting constructs have more
than one increased and/or decreased galectin 11 activity or
function, while the remaining galectin 11 activities or functions
are maintained.
[0093] Besides conservative amino acid substitution, variants of
galectin 11 include (i) substitutions with one or more of the
non-conserved amino acid residues, where the substituted amino acid
residues may or may not be one encoded by the genetic code, or (ii)
substitution with one or more of amino acid residues having a
substituent group, or (iii) fusion of the mature polypeptide with
another compound, such as a compound to increase the stability
and/or solubility of the polypeptide (for example, polyethylene
glycol), or (iv) fusion of the polypeptide with additional amino
acids, such as, for example, an IgG Fc fusion region peptide, or
leader or secretory sequence, or a sequence facilitating
purification. Such variant polypeptides are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0094] For example, galectin 11 polypeptide variants containing
amino acid substitutions of charged amino acids with other charged
or neutral amino acids may produce proteins with improved
characteristics, such as less aggregation. Aggregation of
pharmaceutical formulations both reduces activity and increases
clearance due to the aggregate's immunogenic activity. (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).)
[0095] For example, preferred non-conservative substitutions of
galectin 11 of FIG. 1 (SEQ ID NO:2) include: M1 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S2 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P3 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; R4 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L5 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; V7 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; P8 replaced with D, E, H, K, R, A, CG, I, L, S, T,
M, V, N, Q, F, W, Y, or C; C9 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or P; S10 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; H11 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A12 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L13 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; Q15 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; G16 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L17 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S18 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P19 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; G20 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q21 replaced with D, E, H, K, K, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; V22 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I23 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I24 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V25
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R26 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G27
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; L30 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; Q31 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; E32 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P33 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K34 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H35
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
F36 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; T37 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V38
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S39 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L40 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R41 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; D42 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q43 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A44
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A45 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H46 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A47 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P48 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V49 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T50 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L51 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; R52 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; A53 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; S54 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; F55 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; A56 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; D57 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; R58 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; T59 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L60 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A61 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W62
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
I63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S64
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R65 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W66
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
G67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q68
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; K69 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; K70 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; L71 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I72 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S73
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A74 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; F76 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L77
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F78 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Y79
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
P80 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; Q81 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; R82 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; F83 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; F84 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; E85 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V86 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; L88 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; F90 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; Q91 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; E92 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G93 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; G94 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; K96 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; L97 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A98 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L99
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N100 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G101
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q102 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G103
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L104 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G105 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A106 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T107 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S108 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; M109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N110 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; Q111 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; Q112 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; A113 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L114 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; Q116 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; L117 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; R118 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E119 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L120 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; R121 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; I122 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; S123 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V126
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q127 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L128
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y129 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C130
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; V131 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H132 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; and S133 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
Using these same principles, similar non-conservative substitutions
can be made in the polypeptide of SEQ ID NO:25 or 27.
[0096] The resulting constructs can be routinely screened for
activities or functions described throughout the specification and
known in the art. Preferably, the resulting constructs have an
increased and/or decreased galectin 11 activity or function, while
the remaining galectin 11 activities or functions are maintained.
More preferably, the resulting constructs have more than one
increased and/or decreased galectin 11 activity or function, while
the remaining galectin 11 activities or functions are
maintained.
[0097] Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6,
7, 8, 9 and 10) can be replaced with the substituted amino acids as
described above (either conservative or nonconservative). The
substituted amino acids can occur in the full length, mature, or
proprotein form of galectin 11 protein, as well as the N- and
C-terminal deletion mutants, having the general formula m-n,
[m.sup.1-n.sup.1, m.sup.1-n.sup.2, m.sup.1-n.sup.3,
m.sup.2-n.sup.1, m.sup.2-n.sup.2, m.sup.2-n.sup.3, m.sup.3-n.sup.1,
m.sup.3-n.sup.2 and m.sup.3-n.sup.3].
[0098] A further embodiment of the invention relates to a
polypeptide which comprises the amino acid sequence of a galectin
11 polypeptide having an amino acid sequence which contains at
least one amino acid substitution, but not more than 50 amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and still even more preferably, not more than 20
amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a polypeptide to have an
amino acid sequence which comprises the amino acid sequence of a
galectin 11 polypeptide, which contains at least one, but not more
than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In
specific embodiments, the number of additions, substitutions,
and/or deletions in the amino acid sequence of SEQ ID NOS: 2, 25,
or 27 or fragments thereof (e.g., the mature form and/or other
fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or
50-150, conservative amino acid substitutions are preferable.
Vectors, Host Cells and Protein Production
[0099] 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 polynucleotides and/or
recombinant vectors of the invention, and the production of
galectin 11 polypeptides and fragments, variants, derivatives, and
analogs thereof, by recombinant techniques.
[0100] Galectin 11 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.
[0101] In one embodiment, the DNA of the invention is operatively
associated with an appropriate heterologous regulatory element
(e.g., promoter or enhancer), 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 and enhancers will be known to the skilled
artisan.
[0102] In embodiments in which vectors contain expression
constructs, these 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 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.
[0103] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or G418 neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin 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, Streptococcus staphylococci Bacillus subtilis,
Streptomyces and Salmonella typhimurium cells; fungal cells, such
as yeast cells; insect cells such as Drosophila S2 and Spodoptera
Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK,
HEK293, and Bowes melanoma cells; and plant cells. Appropriate
culture mediums and conditions for the above-described host cells
are known in the art.
[0104] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host, and expression in the
host are routine skills in the art. A great variety of expression
vectors can be used to express galectin 11 polypeptides and
fragments, variants, derivatives, and analogs of the invention.
Such vectors include chromosomal, episomal and virus-derived
vectors e.g., vectors derived from bacterial plasmids, from
bacteriophage, from yeast episomes, from yeast chromosomal
elements, from viruses such as baculoviruses, papova viruses, such
as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as those derived from plasmid and
bacteriophage genetic elements, such as cosmids and phagemids, all
may be used for expression in accordance with this aspect of the
present invention. Generally, any vector suitable to maintain,
propagate or express polynucleotides to express a polypeptide in a
host may be used. The appropriate nucleotide sequence may be
inserted into an expression vector system by any of a variety of
known technique, such as for example, those set forth in Ausubel et
al., eds., 1989, Current Protocols in Molecular Biology, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc., New
York.
[0105] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0106] The present invention also relates to host cells containing
the vector constructs discussed 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., promoters and/or enhancers)
using techniques known in the art. As discussed above, 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 be chosen which modulates the
expression of the inserted gene sequences, or modifies and
processes 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.,
glycosylation, phosphorylation, cleavage) of proteins. Appropriate
cell lines can be chosen to ensure the desired modifications and
processing of the foreign protein expressed.
[0107] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the desired polypeptide using techniques known in
the art. These signals may be endogenous to the polypeptide or they
may be heterologous signals.
[0108] 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). It is
specifically contemplated that galectin 11 polypeptides may in fact
be expressed by a host cell lacking a recombinant vector.
[0109] The 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. For instance, 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.
Alternatively, such a fusion protein can be made by protein
synthetic techniques, e.g., by use of a peptide synthesizer.
[0110] 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 Fc portion proves to be a
hindrance to use in therapy and diagnosis, for example when the
fusion protein is to be used as antigen for immunizations. In drug
discovery, for example, human proteins, such as, hIL5--has been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. See, Bennett et al., J.
Md. Recog. 8:52-58 (1995) and Johanson et al., J. Biol. Chem.
270(16):9459-9471 (1995).
[0111] The galectin 11 protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification.
[0112] 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, plant,
insect, teleost, avian, and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue or may be
missing an initial methionine residue, in some cases as a result of
host-mediated processes. Thus, it is well known in the art that the
N-terminal methionine encoded by the translation initiation codon
generally is removed with high efficiency from any protein after
translation in all eukaryotic cells. While the N-terminal
methionine on most proteins also is efficiently removed in most
prokaryotes, for some proteins, this prokaryotic removal process is
inefficient, depending on the nature of the amino acid to which the
N-terminal methionine is covalently linked.
[0113] 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., galectin 11
coding sequence), and/or to include genetic material (e.g.,
heterologous polynucleotide sequences) that is operably associated
with galectin 11 polynucleotides of the invention, and which
activates, alters, and/or amplifies endogenous galectin 11
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 galectin 11 polynucleotide
sequences via homologous recombination, resulting in the formation
of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670,
issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31,
1998; International Publication No. WO 96/29411, published Sep. 26,
1996; International Publication No. WO 94/12650, published Aug. 4,
1994; 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).
[0114] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller et al., Nature, 310:105-111
(1984)). For example, a polypeptide corresponding to a fragment of
a galectin 11 polypeptide 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 galectin 11 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-methylamino acids, Ca-methylamino acids, Na-methylamino acids,
and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0115] The invention encompasses galectin 11 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.
[0116] 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.
[0117] Also provided by the invention are chemically modified
derivatives of the polypeptides of the invention 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
derivitization 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 proteins 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.
[0122] 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
(polypeptide) 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.
[0123] 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.
[0124] 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
monmethoxy 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.
[0125] 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.
[0126] 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).
[0127] The galectin 11 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 galectin 11 polypeptides of the invention,
their preparation, and compositions (preferably, Therapeutics)
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.
[0128] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only polypeptides corresponding to the amino acid
sequence of SEQ ID NO:2, or alternatively SEQ ID NO:25 or 27, or
encoded by the cDNA contained in the deposited clone (including
fragments, variants, splice variants, and fusion proteins,
corresponding to these as described herein). These homomers may
contain galectin 11 polypeptides having identical or different
amino acid sequences. In a specific embodiment, a homomer of the
invention is a multimer containing only galectin 11 polypeptides
having an identical amino acid sequence. In another specific
embodiment, a homomer of the invention is a multimer containing
galectin 11 polypeptides having different amino acid sequences. In
specific embodiments, the multimer of the invention is a homodimer
(e.g., containing galectin 11 polypeptides having identical or
different amino acid sequences) or a homotrimer (e.g., containing
galectin 11 polypeptides having identical and/or different amino
acid sequences). In additional embodiments, the homomeric multimer
of the invention is at least a homodimer, at least a homotrimer, or
at least a homotetramer.
[0129] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the galectin 11
polypeptides 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.
[0130] 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 polypeptides 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 polypeptides 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 galectin 11
polypeptides of the invention. Such covalent associations may
involve one or more amino acid residues contained in the
polypeptide sequence (e.g., that recited in SEQ ID NO:2, 25, or 27,
or contained in the polypeptide encoded by the clone HJACE54). In
one instance, the covalent associations are cross-linking between
cysteine residues located within the polypeptide sequences 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 galectin 11 fusion
protein. In one example, covalent associations are between 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 between the heterologous
sequence contained in a galectin 11-Fc fusion protein of the
invention (as described herein). In another specific example,
covalent associations of fusion proteins of the invention are
between heterologous polypeptide sequence from another protein that
is capable of forming covalently associated multimers, such as for
example, oseteoprotegerin (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
polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No.
5,073,627 (hereby incorporated by reference). Proteins comprising
multiple polypeptides of the invention separated by peptide linkers
may be produced using conventional recombinant DNA technology.
[0131] Another method for preparing multimer polypeptides of the
invention involves use of polypeptides of the invention fused to a
leucine zipper or isoleucine zipper polypeptide sequence. Leucine
zipper 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 proteins of the invention
are those described in PCT application WO 94/10308, hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the invention fused to a polypeptide sequence that
dimerizes or trimerizes in solution are expressed in suitable host
cells, and the resulting soluble multimeric fusion protein is
recovered from the culture supernatant using techniques known in
the art.
[0132] Trimeric polypeptides of the invention may offer the
advantage of enhanced biological activity. Preferred leucine zipper
moieties and isoleucine 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 polypeptides of the invention.
[0133] In further preferred embodiments, Galectin 11
polynucleotides of the invention are fused to a polynucleotide
encoding a "FLAG" polypeptide. Thus, a galectin 11-FLAG fusion
protein is encompassed by the present invention. The FLAG antigenic
polypeptide may be fused to an galectin 11 polypeptide of the
invention at either or both the amino or the carboxy terminus. In
preferred embodiments, a galectin 11-FLAG fusion protein is
expressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector
(available from Sigma, St. Louis, Mo., USA). See, Andersson, S., et
al., J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al.,
Proc. Natl. Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature
308:241 (1984) (each of which is hereby incorporated by reference).
In further preferred embodiments, a galectin 11-FLAG fusion protein
is detectable by anti-FLAG monoclonal antibodies (also available
from Sigma).
[0134] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
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 sequence of the polypeptides 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, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (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 polypeptide 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).
[0135] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides 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 (or hyrophobic or signal peptide) 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).
Galectin 11 Polypeptides and Fragments
[0136] The invention further provides an isolated galectin 11
polypeptide having the amino acid sequence encoded by the deposited
cDNA, the amino acid sequence depicted in FIG. 1 (amino acid
residues 1-133 of SEQ ID NO:2), the amino acid sequence depicted in
FIG. 1 less the amino terminal methionine (amino acid residues
2-133 of SEQ ID NO:2), polypeptides which are encoded by a
polynucleotide that hybridizes under stringent hybridization
conditions to a polynucleotide sequence encoding a polypeptide
having the amino acid sequence depicted in FIG. 1 (SEQ ID NO:2)
and/or contained in the deposited clone, and fragments, variants,
derivatives and analogs of these polypeptides.
[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
galectin 11 polypeptide can be substantially purified by the
one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
[0138] It will be recognized in the art that some amino acid
sequences of the galectin 11 polypeptide can be varied without
significant effect of 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.
[0139] Thus, the invention further includes variations of the
galectin 11 polypeptide which show substantial galectin 11
polypeptide functional activity or which include regions of
galectin 11 protein such as the protein portions discussed below.
Such mutants include deletions, insertions, inversions, repeats,
and type substitutions.
[0140] As indicated above, guidance concerning which amino acid
changes are likely to be phenotypically silent can be found in
Bowie et al., Deciphering the Message in Protein Sequences:
Tolerance to Amino Acid Substitutions, Science 247:1306-1310
(1990). Thus, a fragment, variant, derivative, or analog of the
polypeptide of FIG. 1 or 6A-B (SEQ ID NO:2, 25, or 27), or that
encoded by the deposited cDNA, include (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, variants, derivatives and
analogs are deemed to be within the scope of those skilled in the
art from the teachings herein.
[0141] Of particular interest are substitutions of one or more
charged amino acids with other 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
galectin 11 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)).
[0142] 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 2).
TABLE-US-00001 TABLE 2 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
[0143] In the specific embodiments, the number of additions,
substitutions and/or deletions in the amino acid sequence of FIG. 1
or 6A-B (SEQ ID NO:2, 25, or 27) and/or any of the polypeptide
fragments described herein is 50, 40, 35, 30, 25, 20, 15, 14, 13,
12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, or 15-20, 15-10, 5-10,
1-5, 1-3, or 1-2.
[0144] Amino acid residues of the galectin 11 polypeptide,
fragment, variant, derivative, or analog 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 or functional activity, such as,
receptor binding, .beta.-galactoside (e.g., thiodigalactoside or
lactose) binding, the ability to agglutinate trypsin-treated rabbit
erythrocytes, or the ability in vitro or in vivo to induce
apoptosis. 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)).
[0145] The present invention also encompasses polypeptides which
are at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%,
or, 97-99% identical to the polypeptides described above. By a
polypeptide having an amino acid sequence at least, for example,
95% "identical" to a reference amino acid sequence of a galectin 11
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
galectin 11 polypeptide. 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.
[0146] As a practical matter, whether any particular polypeptide is
at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%
identical to, for instance, the amino acid sequence shown in FIG. 1
or 6A-B (SEQ ID NO:2, 25, or 27), the amino acid sequence encoded
by deposited cDNA clone, or fragments thereof, 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.
[0147] In another 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.
[0148] The polypeptides of the present invention have uses which
include, but are not limited to, molecular weight marker on
SDS-PAGE gels or on molecular sieve gel filtration columns using
methods well known to those of skill in the art.
[0149] The present invention also encompasses fragments of the
above-described polypeptides of the invention. In specific
embodiments, these fragments are at least 20, 25, 30, 40, 50, 75,
90, 100, 110, 120, 125, or 130 amino acid residues in length.
[0150] In the present invention, a "polypeptide fragment" refers to
an amino acid sequence which is a portion of that contained in SEQ
ID NO:2, 25, or 27 or encoded by the cDNA contained in the
deposited clone. Protein (polypeptide) 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 comprising, or
alternatively consisting of, from about amino acid number 1-20,
21-40, 41-60, 61-80, 81-100, 101-120, or 121 to the end of the
coding region. Moreover, polypeptide fragments can be about 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids in
length. In this context "about" includes the particularly recited
ranges or values, and ranges or values larger or smaller by several
(5, 4, 3, 2, or 1) amino acids, at either extreme or at both
extremes. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0151] 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 galectin 11 ligand) may still be retained. For
example, the ability of shortened galectin 11 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. Whether a particular polypeptide
lacking N-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 an galectin 11 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
galectin 11 amino acid residues may often evoke an immune
response.
[0152] Accordingly, polypeptide fragments include the secreted
galectin 11 protein as well as the mature form. Further preferred
polypeptide fragments include the secreted galectin 11 protein or
the mature form having a continuous series of deleted residues from
the amino or the carboxy terminus, or both. For example, any number
of amino acids, ranging from 1-60, can be deleted from the amino
terminus of either the secreted galectin 11 polypeptide or the
mature form. Similarly, any number of amino acids, ranging from
1-30, can be deleted from the carboxy terminus of the secreted
galectin 11 protein or mature form. Furthermore, any combination of
the above amino and carboxy terminus deletions are preferred.
Similarly, polynucleotides encoding these polypeptide fragments are
also preferred.
[0153] In one embodiment, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the galectin 11 polypeptide
depicted in FIG. 1 or 6A-B (SEQ ID NO:2, 25, or 27) or encoded by
the cDNA of the deposited clone. Particularly, in one embodiment,
N-terminal deletions of the galectin 11 polypeptide can be
described by the general formula m to 133, where m is a integer
from 1 to 128 corresponding to the position of amino acid residue
identified in SEQ ID NO:2 and preferably, corresponds to one of the
N-terminal amino acid residues identified in the N-terminal
deletions specified herein. In specific embodiments, N-terminal
deletions of the galectin 11 polypeptide of the invention comprise,
or alternatively consist of, amino acid residues: S-2 to S-133; P-3
to S-133; R-4 to S-133; L-5 to S-133; E-6 to S-133; V-7 to S-133;
P-8 to S-133; C-9 to S-133; S-10 to S-133; H-11 to S-133; A-12 to
S-133; L-13 to S-133; P-14 to S-133; Q-15 to S-133; G-16 to S-133;
L-17 to S-133; S-18 to S-133; P-19 to S-133; G-20 to S-133; Q-21 to
S-133; V-22 to S-133; I-23 to S-133; 1-24 to S-133; V-25 to S-133;
R-26 to S-133; G-27 to S-133; L-28 to S-133; V-29 to S-133; L-30 to
S-133; Q-31 to S-133; E-32 to S-133; P-33 to S-133; K-34 to S-133;
H-35 to S-133; F-36 to S-133; T-37 to S-133; V-38 to S-133; S-39 to
S-133; L-40 to S-133; R-41 to S-133; D-42 to S-133; Q-43 to S-133;
A-44 to S-133; A-45 to S-133; H-46 to S-133; A-47 to S-133; P-48 to
S-133; V-49 to S-133; T-50 to S-133; L-51 to S-133; R-52 to S-133;
A-53 to S-133; S-54 to S-133; F-55 to S-133; A-56 to S-133; D-57 to
S-133; R-58 to S-133; T-59 to S-133; L-60 to S-133; A-61 to S-133;
W-62 to S-133; 1-63 to S-133; S-64 to S-133; R-65 to S-133; W-66 to
S-133; G-67 to S-133; Q-68 to S-133; K-69 to S-133; K-70 to S-133;
L-71 to S-133; 1-72 to S-133; S-73 to S-133; A-74 to S-133; P-75 to
S-133; F-76 to S-133; L-77 to S-133; F-78 to S-133; Y-79 to S-133;
P-80 to S-133; Q-81 to S-133; R-82 to S-133; F-83 to S-133; F-84 to
S-133; E-85 to S-133; V-86 to S-133; L-87 to S-133; L-88 to S-133;
L-89 to S-133; F-90 to S-133; Q-91 to S-133; E-92 to S-133; G-93 to
S-133; G-94 to S-133; L-95 to S-133; K-96 to S-133; L-97 to S-133;
A-98 to S-133; L-99 to S-133; N-100 to S-133; G-101 to S-133; Q-102
to S-133; G-103 to S-133; L-104 to S-133; G-105 to S-133; A-106 to
S-133; T-107 to S-133; S-108 to S-133; M-109 to S-133; N-110 to
S-133; Q-111 to S-133; Q-112 to S-133; A-113 to S-133; L-114 to
S-133; E-115 to S-133; Q-116 to S-133; L-117 to S-133; R-118 to
S-133; E-119 to S-133; L-120 to S-133; R-121 to S-133; 1-122 to
S-133; S-123 to S-133; G-124 to S-133; S-125 to S-133; V-126 to
S-133; Q-127 to S-133; and L-128 to S-133 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0154] 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 galectin 11 ligand) may
still be retained. For example the ability of the shortened
galectin 11 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 an galectin 11
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 galectin 11 amino acid
residues may often evoke an immune response.
[0155] Accordingly, further embodiments of the invention are
directed to C-terminal deletions of the galectin 11 polypeptide
described by the general formula I to n, where n is an integer from
6 to 132 corresponding to the position of amino acid residue
identified in SEQ ID NO:2 and preferably, corresponds to one of the
C-terminal amino acid residues identified in the C-terminal
deletions specified herein. In specific embodiments, C terminal
deletions of the galectin 11 polypeptide of the invention comprise,
or alternatively, consist of, amino acid residues: M-1 to H-132;
M-1 to V-131; M-1 to C-130; M-1 to Y-129; M-1 to L-128; M-1 to
Q-127; M-1 to V-126; M-1 to S-125; M-1 to G-124; M-1 to S-123; M-1
to 1-122; M-1 to R-121; M-1 to L-120; M-1 to E-119; M-1 to R-118;
M-1 to L-117; M-1 to Q-116; M-1 to E-115; M-1 to L-114; M-1 to
A-113; M-1 to Q-112; M-1 to Q-111; M-1 to N-110; M-1 to M-109; M-1
to S-108; M-1 to T-107; M-1 to A-106; M-1 to G-105; M-1 to L-104;
M-1 to G-103; M-1 to Q-102; M-1 to G-101; M-1 to N-100; M-1 to
L-99; M-1 to A-98; M-1 to L-97; M-1 to K-96; M-1 to L-95; M-1 to
G-94; M-1 to G-93; M-1 to E-92; M-1 to Q-91; M-1 to F-90; M-1 to
L-89; M-1 to L-88; M-1 to L-87; M-1 to V-86; M-1 to E-85; M-1 to
F-84; M-1 to F-83; M-1 to R-82; M-1 to Q-81; M-1 to P-80; M-1 to
Y-79; M-1 to F-78; M-1 to L-77; M-1 to F-76; M-1 to P-75; M-1 to
A-74; M-1 to S-73; M-1 to I-72; M-1 to L-71; M-1 to K-70; M-1 to
K-69; M-1 to Q-68; M-1 to G-67; M-1 to W-66; M-1 to R-65; M-1 to
S-64; M-1 to I-63; M-1 to W-62; M-1 to A-61; M-1 to L-60; M-1 to
T-59; M-1 to R-58; M-1 to D-57; M-1 to A-56; M-1 to F-55; M-1 to
S-54; M-1 to A-53; M-1 to R-52; M-1 to L-51; M-1 to T-50; M-1 to
V-49; M-1 to P-48; M-1 to A-47; M-1 to H-46; M-1 to A-45; M-1 to
A-44; M-1 to Q-43; M-1 to D-42; M-1 to R-41; M-1 to L-40; M-1 to
S-39; M-1 to V-38; M-1 to T-37; M-1 to F-36; M-1 to H-35; M-1 to
K-34; M-1 to P-33; M-1 to E-32; M-1 to Q-31; M-1 to L-30; M-1 to
V-29; M-1 to L-28; M-1 to G-27; M-1 to R-26; M-1 to V-25; M-1 to
1-24; M-1 to I-23; M-1 to V-22; M-1 to Q-21; M-1 to G-20; M-1 to
P-19; M-1 to S-18; M-1 to L-17; M-1 to G-16; M-1 to Q-15; M-1 to
P-14; M-1 to L-13; M-1 to A-12; M-1 to H-11; M-1 to S-10; M-1 to
C-9; M-1 to P-8; M-1 to V-7; and M-1 to E-6 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0156] In addition, any of the above listed N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
galectin 11 polypeptide. The invention also provides polypeptides
having one or more amino acids deleted from both the amino and the
carboxyl termini, which may be described generally as having
residues m-n of SEQ ID NO:2, where n and m are integers as
described above. Polynucleotides encoding these polypeptides are
also encompassed by the invention.
[0157] In preferred embodiments, the polypeptides of the invention
comprise, or alternatively, consist of, amino acid residues: M-1 to
L-40; M-1 to W-66; P-3 to L-40; L-5 to L-40; L-5 to S-108; L-5 to
L-128; P-3 to L-128; L-5 to L-128; L-5 to G-124; C-9 to C-130; L-13
to L-40; P-14 to L-40; L-40 to S-108; A-47 to S-108; A-47 to L-128;
R-65 to S-108; R-65 to L-128; L-88 to S-108; L-88 to L-128; S-108
to L-120; or L-114 to L-128 of SEQ ID NO:2. Polynucleotides
encoding these polypeptides are also encompassed by the
invention.
[0158] Also included are a nucleotide sequence encoding a
polypeptide consisting of a portion of the complete galectin 11
amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 209053, where this portion excludes any integer of
amino acid residues from 1 to about 123 amino acids from the amino
terminus of the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 209053, or any integer of amino
acid residues from 1 to about 123 amino acids from the carboxy
terminus, or any combination of the above amino terminal and
carboxy terminal deletions, of the complete amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 209053.
Polynucleotides encoding all of the above deletion mutant
polypeptide forms also are provided.
[0159] 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 galectin 11 polypeptide sequence set
forth herein m-n. In preferred embodiments, the application is
directed to proteins containing polypeptides at least 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98% or 99% identical to polypeptides
having the amino acid sequence of the specific galectin 11N- and
C-terminal deletions recited herein. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0160] Additional preferred polypeptide fragments comprise, or
alternatively consist of, the amino acid sequence of residues: M-1
to Q-15; S-2 to G-16; P-3 to L-17; R-4 to S-18; L-5 to P-19; E-6 to
G-20; V-7 to Q-21; P-8 to V-22; C-9 to I-23; S-10 to I-24; H-11 to
V-25; A-12 to R-26; L-13 to G-27; P-14 to L-28; Q-15 to V-29; G-16
to L-30; L-17 to Q-31; S-18 to E-32; P-19 to P-33; G-20 to K-34;
Q-21 to H-35; V-22 to F-36; I-23 to T-37; 1-24 to V-38; V-25 to
S-39; R-26 to L-40; G-27 to R-41; L-28 to D-42; V-29 to Q-43; L-30
to A-44; Q-31 to A-45; E-32 to H-46; P-33 to A-47; K-34 to P-48;
H-35 to V-49; F-36 to T-50; T-37 to L-51; V-38 to R-52; S-39 to
A-53; L-40 to S-54; R-41 to F-55; D-42 to A-56; Q-43 to D-57; A-44
to R-58; A-45 to T-59; H-46 to L-60; A-47 to A-61; P-48 to W-62;
V-49 to I-63; T-50 to S-64; L-51 to R-65; R-52 to W-66; A-53 to
G-67; S-54 to Q-68; F-55 to K-69; A-56 to K-70; D-57 to L-71; R-58
to I-72; T-59 to S-73; L-60 to A-74; A-61 to P-75; W-62 to F-76;
I-63 to L-77; S-64 to F-78; R-65 to Y-79; W-66 to P-80; G-67 to
Q-81; Q-68 to R-82; K-69 to F-83; K-70 to F-84; L-71 to E-85; 1-72
to V-86; S-73 to L-87; A-74 to L-88; P-75 to L-89; F-76 to F-90;
L-77 to Q-91; F-78 to E-92; Y-79 to G-93; P-80 to G-94; Q-81 to
L-95; R-82 to K-96; F-83 to L-97; F-84 to A-98; E-85 to L-99; V-86
to N-100; L-87 to G-101; L-88 to Q-102; L-89 to G-103; F-90 to
L-104; Q-91 to G-105; E-92 to A-106; G-93 to T-107; G-94 to S-108;
L-95 to M-109; K-96 to N-110; L-97 to Q-111; A-98 to Q-112; L-99 to
A-113; N-100 to L-114; G-101 to E-115; Q-102 to Q-116; G-103 to
L-117; L-104 to R-118; G-105 to E-119; A-106 to L-120; T-107 to
R-121; S-108 to 1-122; M-109 to S-123; N-110 to G-124; Q-111 to
S-125; Q-112 to V-126; A-113 to Q-127; L-114 to L-128; E-115 to
Y-129; Q-116 to C-130; L-117 to V-131; R-118 to H-132; and E-119 to
S-133 of SEQ ID NO:2. These polypeptide fragments may retain the
biological activity of galectin 11 polypeptides of the invention
and/or may be useful to generate or screen for antibodies, as
described further below. Polynucleotides encoding these polypeptide
fragments are also encompassed by the invention.
[0161] In another embodiment, the present invention further
provides polypeptides having one or more residues deleted from the
amino terminus of the amino acid sequence of the galectin 11
polypeptide depicted in SEQ ID NO:25 or encoded by the cDNA of the
deposited clone. Particularly, in one embodiment, N-terminal
deletions of the galectin 11 polypeptide can be described by the
general formula m to 275, where m is a integer from 2 to 270
corresponding to the position of amino acid residue identified in
SEQ ID NO:25 and preferably, corresponds to one of the N-terminal
amino acid residues identified in the N-terminal deletions
specified herein. In specific embodiments, N-terminal deletions of
the galectin 11 polypeptide of the invention comprise, or
alternatively consist of, amino acid residues: V-2 to S-275; M-3 to
S-275; L-4 to S-275; Q-5 to S-275; G-6 to S-275; V-7 to S-275; V-8
to S-275; P-9 to S-275; L-10 to S-275; D-11 to S-275; A-12 to
S-275; H-13 to S-275; R-14 to S-275; F-15 to S-275; Q-16 to S-275;
V-17 to S-275; D-18 to S-275; F-19 to S-275; Q-20 to S-275; C-21 to
S-275; G-22 to S-275; C-23 to S-275; S-24 to S-275; L-25 to S-275;
C-26 to S-275; P-27 to S-275; R-28 to S-275; P-29 to S-275; D-30 to
S-275; I-31 to S-275; A-32 to S-275; F-33 to S-275; H-34 to S-275;
F-35 to S-275; N-36 to S-275; P-37 to S-275; R-38 to S-275; F-39 to
S-275; H-40 to S-275; T-41 to S-275; T-42 to S-275; K-43 to S-275;
P-44 to S-275; H-45 to S-275; V-46 to S-275; 1-47 to S-275; C-48 to
S-275; N-49 to S-275; T-50 to S-275; L-51 to S-275; H-52 to S-275;
G-53 to S-275; G-54 to S-275; R-55 to S-275; W-56 to S-275; Q-57 to
S-275; R-58 to S-275; E-59 to S-275; A-60 to S-275; R-61 to S-275;
W-62 to S-275; P-63 to S-275; H-64 to S-275; L-65 to S-275; A-66 to
S-275; L-67 to S-275; R-68 to S-275; R-69 to S-275; G-70 to S-275;
S-71 to S-275; S-72 to S-275; F-73 to S-275; L-74 to S-275; 1-75 to
S-275; L-76 to S-275; F-77 to S-275; L-78 to S-275; F-79 to S-275;
G-80 to S-275; N-81 to S-275; E-82 to S-275; E-83 to S-275; V-84 to
S-275; K-85 to S-275; V-86 to S-275; S-87 to S-275; V-88 to S-275;
N-89 to S-275; G-90 to S-275; Q-91 to S-275; H-92 to S-275; F-93 to
S-275; L-94 to S-275; H-95 to S-275; F-96 to S-275; R-97 to S-275;
Y-98 to S-275; R-99 to S-275; L-100 to S-275; P-101 to S-275; L-102
to S-275; S-103 to S-275; H-104 to S-275; V-105 to S-275; D-106 to
S-275; T-107 to S-275; L-108 to S-275; G-109 to S-275; I110 to
S-275; F-111 to S-275; G-112 to S-275; D-113 to S-275; I-114 to
S-275; L-115 to S-275; V-116 to S-275; E-117 to S-275; A-118 to
S-275; V-119 to S-275; G-120 to S-275; F-121 to S-275; L-122 to
S-275; N-123 to S-275; I-124 to S-275; N-125 to S-275; P-126 to
S-275; F-127 to S-275; V-128 to S-275; E-129 to S-275; G-130 to
S-275; S-131 to S-275; R-132 to S-275; E-133 to S-275; Y-134 to
S-275; P-135 to S-275; A-136 to S-275; G-137 to S-275; H-138 to
S-275; P-139 to S-275; F-140 to S-275; L-141 to S-275; L-142 to
S-275; M-143 to S-275; S-144 to S-275; P-145 to S-275; R-146 to
S-275; L-147 to S-275; E-148 to S-275; V-149 to S-275; P-150 to
S-275; C-151 to S-275; S-152 to S-275; H-153 to S-275; A-154 to
S-275; L-155 to S-275; P-156 to S-275; Q-157 to S-275; G-158 to
S-275; L-159 to S-275; S-160 to S-275; P-161 to S-275; G-162 to
S-275; Q-163 to S-275; V-164 to S-275; 1-165 to S-275; 1-166 to
S-275; V-167 to S-275; R-168 to S-275; G-169 to S-275; L-170 to
S-275; V-171 to S-275; L-172 to S-275; Q-173 to S-275; E-174 to
S-275; P-175 to S-275; K-176 to S-275; H-177 to S-275; F-178 to
S-275; T-179 to S-275; V-180 to S-275; S-181 to S-275; L-182 to
S-275; R-183 to S-275; D-184 to S-275; Q-185 to S-275; A-186 to
S-275; A-187 to S-275; H-188 to S-275; A-189 to S-275; P-190 to
S-275; V-191 to S-275; T-192 to S-275; L-193 to S-275; R-194 to
S-275; A-195 to S-275; S-196 to S-275; F-197 to S-275; A-198 to
S-275; D-199 to S-275; R-200 to S-275; T-201 to S-275; L-202 to
S-275; A-203 to S-275; W-204 to S-275; 1-205 to S-275; S-206 to
S-275; R-207 to S-275; W-208 to S-275; G-209 to S-275; Q-210 to
S-275; K-211 to S-275; K-212 to S-275; L-213 to S-275; I214 to
S-275; S-215 to S-275; A-216 to S-275; P-217 to S-275; F-218 to
S-275; L-219 to S-275; F-220 to S-275; Y-221 to S-275; P-222 to
S-275; Q-223 to S-275; R-224 to S-275; F-225 to S-275; F-226 to
S-275; E-227 to S-275; V-228 to S-275; L-229 to S-275; L-230 to
S-275; L-231 to S-275; F-232 to S-275; Q-233 to S-275; E-234 to
S-275; G-235 to S-275; G-236 to S-275; L-237 to S-275; K-238 to
S-275; L-239 to S-275; A-240 to S-275; L-241 to S-275; N-242 to
S-275; G-243 to S-275; Q-244 to S-275; G-245 to S-275; L-246 to
S-275; G-247 to S-275; A-248 to S-275; T-249 to S-275; S-250 to
S-275; M-251 to S-275; N-252 to S-275; Q-253 to S-275; Q-254 to
S-275; A-255 to S-275; L-256 to S-275; E-257 to S-275; Q-258 to
S-275; L-259 to S-275; R-260 to S-275; E-261 to S-275; L-262 to
S-275; R-263 to S-275; 1-264 to S-275; S-265 to S-275; G-266 to
S-275; S-267 to S-275; V-268 to S-275; Q-269 to S-275; and L-270 to
S-275 of SEQ ID NO:25. Polynucleotides encoding such polypeptides
are also encompassed by the invention.
[0162] Further embodiments of the invention are directed to
C-terminal deletions of the galectin 11 polypeptide described by
the general formula I to n, where n is an integer from 6 to 275
corresponding to the position of amino acid residue identified in
SEQ ID NO:25 and preferably, corresponds to one of the C-terminal
amino acid residues identified in the C-terminal deletions
specified herein. In specific embodiments, C terminal deletions of
the galectin 11 polypeptide of the invention comprise, or
alternatively, consist of, amino acid residues: M-1 to H-274; M-1
to V-273; M-1 to C-272; M-1 to Y-271; M-1 to L-270; M-1 to Q-269;
M-1 to V-268; M-1 to S-267; M-1 to G-266; M-1 to S-265; M-1 to
I-264; M-1 to R-263; M-1 to L-262; M-1 to E-261; M-1 to R-260; M-1
to L-259; M-1 to Q-258; M-1 to E-257; M-1 to L-256; M-1 to A-255;
M-1 to Q-254; M-1 to Q-253; M-1 to N-252; M-1 to M-251; M-1 to
S-250; M-1 to T-249; M-1 to A-248; M-1 to G-247; M-1 to L-246; M-1
to G-245; M-1 to Q-244; M-1 to G-243; M-1 to N-242; M-1 to L-241;
M-1 to A-240; M-1 to L-239; M-1 to K-238; M-1 to L-237; M-1 to
G-236; M-1 to G-235; M-1 to E-234; M-1 to Q-233; M-1 to F-232; M-1
to L-231; M-1 to L-230; M-1 to L-229; M-1 to V-228; M-1 to E-227;
M-1 to F-226; M-1 to F-225; M-1 to R-224; M-1 to Q-223; M-1 to
P-222; M-1 to Y-221; M-1 to F-220; M-1 to L-219; M-1 to F-218; M-1
to P-217; M-1 to A-216; M-1 to S-215; M-1 to I-214; M-1 to L-213;
M-1 to K-212; M-1 to K-211; M-1 to Q-210; M-1 to G-209; M-1 to
W-208; M-1 to R-207; M-1 to S-206; M-1 to 1-205; M-1 to W-204; M-1
to A-203; M-1 to L-202; M-1 to T-201; M-1 to R-200; M-1 to D-199;
M-1 to A-198; M-1 to F-197; M-1 to S-196; M-1 to A-195; M-1 to
R-194; M-1 to L-193; M-1 to T-192; M-1 to V-191; M-1 to P-190; M-1
to A-189; M-1 to H-188; M-1 to A-187; M-1 to A-186; M-1 to Q-185;
M-1 to D-184; M-1 to R-183; M-1 to L-182; M-1 to S-181; M-1 to
V-180; M-1 to T-179; M-1 to F-178; M-1 to H-177; M-1 to K-176; M-1
to P-175; M-1 to E-174; M-1 to Q-173; M-1 to L-172; M-1 to V-171;
M-1 to L-170; M-1 to G-169; M-1 to R-168; M-1 to V-167; M-1 to
I-166; M-1 to I-165; M-1 to V-164; M-1 to Q-163; M-1 to G-162; M-1
to P-161; M-1 to S-160; M-1 to L-159; M-1 to G-158; M-1 to Q-157;
M-1 to P-156; M-1 to L-155; M-1 to A-154; M-1 to H-153; M-1 to
S-152; M-1 to C-151; M-1 to P-150; M-1 to V-149; M-1 to E-148; M-1
to L-147; M-1 to R-146; M-1 to P-145; M-1 to S-144; M-1 to M-143;
M-1 to L-142; M-1 to L-141; M-1 to F-140; M-1 to P-139; M-1 to
H-138; M-1 to G-137; M-1 to A-136; M-1 to P-135; M-1 to Y-134; M-1
to E-133; M-1 to R-132; M-1 to S-131; M-1 to G-130; M-1 to E-129;
M-1 to V-128; M-1 to F-127; M-1 to P-126; M-1 to N-125; M-1 to
I-124; M-1 to N-123; M-1 to L-122; M-1 to F-121; M-1 to G-120; M-1
to V-119; M-1 to A-118; M-1 to E-117; M-1 to V-116; M-1 to L-115;
M-1 to I-114; M-1 to D-113; M-1 to G-112; M-1 to F-111; M-1 to
I-110; M-1 to G-109; M-1 to L-108; M-1 to T-107; M-1 to D-106; M-1
to V-105; M-1 to H-104; M-1 to S-103; M-1 to L-102; M-1 to P-101;
M-1 to L-100; M-1 to R-99; M-1 to Y-98; M-1 to R-97; M-1 to F-96;
M-1 to H-95; M-1 to L-94; M-1 to F-93; M-1 to H-92; M-1 to Q-91;
M-1 to G-90; M-1 to N-89; M-1 to V-88; M-1 to S-87; M-1 to V-86;
M-1 to K-85; M-1 to V-84; M-1 to E-83; M-1 to E-82; M-1 to N-81;
M-1 to G-80; M-1 to F-79; M-1 to L-78; M-1 to F-77; M-1 to L-76;
M-1 to I-75; M-1 to L-74; M-1 to F-73; M-1 to S-72; M-1 to S-71;
M-1 to G-70; M-1 to R-69; M-1 to R-68; M-1 to L-67; M-1 to A-66;
M-1 to L-65; M-1 to H-64; M-1 to P-63; M-1 to W-62; M-1 to R-61;
M-1 to A-60; M-1 to E-59; M-1 to R-58; M-1 to Q-57; M-1 to W-56;
M-1 to R-55; M-1 to G-54; M-1 to G-53; M-1 to H-52; M-1 to L-51;
M-1 to T-50; M-1 to N-49; M-1 to C-48; M-1 to I-47; M-1 to V-46;
M-1 to H-45; M-1 to P-44; M-1 to K-43; M-1 to T-42; M-1 to T-41;
M-1 to H-40; M-1 to F-39; M-1 to R-38; M-1 to P-37; M-1 to N-36;
M-1 to F-35; M-1 to H-34; M-1 to F-33; M-1 to A-32; M-1 to 1-31;
M-1 to D-30; M-1 to P-29; M-1 to R-28; M-1 to P-27; M-1 to C-26;
M-1 to L-25; M-1 to S-24; M-1 to C-23; M-1 to G-22; M-1 to C-21;
M-1 to Q-20; M-1 to F-19; M-1 to D-18; M-1 to V-17; M-1 to Q-16;
M-1 to F-15; M-1 to R-14; M-1 to H-13; M-1 to A-12; M-1 to D-11;
M-1 to L-10; M-1 to P-9; M-1 to V-8; M-1 to V-7; and M-1 to G-6 all
of SEQ ID NO:25. Polynucleotides encoding such polypeptides are
also encompassed by the invention.
[0163] Further embodiments of the invention are directed to
polypeptide fragments comprising, or alternatively, consisting of,
amino acids described by the general formula m to n, where m and n
correspond to any one of the amino acid residues specified above
for these symbols, respectively. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0164] In yet another embodiment, the present invention further
provides polypeptides having one or more residues deleted from the
amino terminus of the amino acid sequence of the galectin 11
polypeptide depicted in SEQ ID NO:27. Particularly, in one
embodiment, N-terminal deletions of the galectin 11 polypeptide can
be described by the general formula m to 296, where m is an integer
from 2 to 291 corresponding to the position of amino acid residue
identified in SEQ ID NO:27 and preferably, corresponds to one of
the N-terminal amino acid residues identified in the N-terminal
deletions specified herein. In specific embodiments, N-terminal
deletions of the galectin 11 polypeptide of the invention comprise,
or alternatively consist of, amino acid residues: S-2 to S-296; F-3
to S-296; F-4 to S-296; S-5 to S-296; C-6 to S-296; S-7 to S-296;
G-8 to S-296; G-9 to S-296; S-10 to S-296; L-11 to S-296; C-12 to
S-296; H-13 to S-296; D-14 to S-296; D-15 to S-296; F-16 to S-296;
W-17 to S-296; R-18 to S-296; P-19 to S-296; A-20 to S-296; C-21 to
S-296; R-22 to S-296; Q-23 to S-296; D-24 to S-296; G-25 to S-296;
H-26 to S-296; A-27 to S-296; A-28 to S-296; R-29 to S-296; S-30 to
S-296; G-31 to S-296; P-32 to S-296; S-33 to S-296; R-34 to S-296;
C-35 to S-296; T-36 to S-296; Q-37 to S-296; V-38 to S-296; D-39 to
S-296; F-40 to S-296; Q-41 to S-296; C-42 to S-296; G-43 to S-296;
C-44 to S-296; S-45 to S-296; L-46 to S-296; C-47 to S-296; P-48 to
S-296; R-49 to S-296; P-50 to S-296; D-51 to S-296; 1-52 to S-296;
A-53 to S-296; F-54 to S-296; H-55 to S-296; F-56 to S-296; N-57 to
S-296; P-58 to S-296; R-59 to S-296; F-60 to S-296; H-61 to S-296;
T-62 to S-296; T-63 to S-296; K-64 to S-296; P-65 to S-296; H-66 to
S-296; V-67 to S-296; 1-68 to S-296; C-69 to S-296; N-70 to S-296;
T-71 to S-296; L-72 to S-296; H-73 to S-296; G-74 to S-296; G-75 to
S-296; R-76 to S-296; W-77 to S-296; Q-78 to S-296; R-79 to S-296;
E-80 to S-296; A-81 to S-296; R-82 to S-296; W-83 to S-296; P-84 to
S-296; H-85 to S-296; L-86 to S-296; A-87 to S-296; L-88 to S-296;
R-89 to S-296; R-90 to S-296; G-91 to S-296; S-92 to S-296; S-93 to
S-296; F-94 to S-296; L-95 to S-296; 1-96 to S-296; L-97 to S-296;
F-98 to S-296; L-99 to S-296; F-100 to S-296; G-101 to S-296; N-102
to S-296; E-103 to S-296; E-104 to S-296; V-105 to S-296; K-106 to
S-296; V-107 to S-296; S-108 to S-296; V-109 to S-296; N-110 to
S-296; G-111 to S-296; Q-112 to S-296; H-113 to S-296; F-114 to
S-296; L-115 to S-296; H-116 to S-296; F-117 to S-296; R-118 to
S-296; Y-119 to S-296; R-120 to S-296; L-121 to S-296; P-122 to
S-296; L-123 to S-296; S-124 to S-296; H-125 to S-296; V-126 to
S-296; D-127 to S-296; T-128 to S-296; L-129 to S-296; G-130 to
S-296; 1-131 to S-296; F-132 to S-296; G-133 to S-296; D-134 to
S-296; I-135 to S-296; L-136 to S-296; V-137 to S-296; E-138 to
S-296; A-139 to S-296; V-140 to S-296; G-141 to S-296; F-142 to
S-296; L-143 to S-296; N-144 to S-296; 1-145 to S-296; N-146 to
S-296; P-147 to S-296; F-148 to S-296; V-149 to S-296; E-150 to
S-296; G-151 to S-296; S-152 to S-296; R-153 to S-296; E-154 to
S-296; Y-155 to S-296; P-156 to S-296; A-157 to S-296; G-158 to
S-296; H-159 to S-296; P-160 to S-296; F-161 to S-296; L-162 to
S-296; L-163 to S-296; M-164 to S-296; S-165 to S-296; P-166 to
S-296; R-167 to S-296; L-168 to S-296; E-169 to S-296; V-170 to
S-296; P-171 to S-296; C-172 to S-296; S-173 to S-296; H-174 to
S-296; A-175 to S-296; L-176 to S-296; P-177 to S-296; Q-178 to
S-296; G-179 to S-296; L-180 to S-296; S-181 to S-296; P-182 to
S-296; G-183 to S-296; Q-184 to S-296; V-185 to S-296; I-186 to
S-296; I-187 to S-296; V-188 to S-296; R-189 to S-296; G-190 to
S-296; L-191 to S-296; V-192 to S-296; L-193 to S-296; Q-194 to
S-296; E-195 to S-296; P-196 to S-296; K-197 to S-296; H-198 to
S-296; F-199 to S-296; T-200 to S-296; V-201 to S-296; S-202 to
S-296; L-203 to S-296; R-204 to S-296; D-205 to S-296; Q-206 to
S-296; A-207 to S-296; A-208 to S-296; H-209 to S-296; A-210 to
S-296; P-211 to S-296; V-212 to S-296; T-213 to S-296; L-214 to
S-296; R-215 to S-296; A-216 to S-296; S-217 to S-296; F-218 to
S-296; A-219 to S-296; D-220 to S-296; R-221 to S-296; T-222 to
S-296; L-223 to S-296; A-224 to S-296; W-225 to S-296; 1-226 to
S-296; S-227 to S-296; R-228 to S-296; W-229 to S-296; G-230 to
S-296; Q-231 to S-296; K-232 to S-296; K-233 to S-296; L-234 to
S-296; 1-235 to S-296; S-236 to S-296; A-237 to S-296; P-238 to
S-296; F-239 to S-296; L-240 to S-296; F-241 to S-296; Y-242 to
S-296; P-243 to S-296; Q-244 to S-296; R-245 to S-296; F-246 to
S-296; F-247 to S-296; E-248 to S-296; V-249 to S-296; L-250 to
S-296; L-251 to S-296; L-252 to S-296; F-253 to S-296; Q-254 to
S-296; E-255 to S-296; G-256 to S-296; G-257 to S-296; L-258 to
S-296; K-259 to S-296; L-260 to S-296; A-261 to S-296; L-262 to
S-296; N-263 to S-296; G-264 to S-296; Q-265 to S-296; G-266 to
S-296; L-267 to S-296; G-268 to S-296; A-269 to S-296; T-270 to
S-296; S-271 to S-296; M-272 to S-296; N-273 to S-296; Q-274 to
S-296; Q-275 to S-296; A-276 to S-296; L-277 to S-296; E-278 to
S-296; Q-279 to S-296; L-280 to S-296; R-281 to S-296; E-282 to
S-296; L-283 to S-296; R-284 to S-296; I-285 to S-296; S-286 to
S-296; G-287 to S-296; S-288 to S-296; V-289 to S-296; Q-290 to
S-296; L-291 to S-296; of SEQ ID NO:27. Polynucleotides encoding
such polypeptides are also provided.
[0165] Further embodiments of the invention are directed to
C-terminal deletions of the galectin 11 polypeptide described by
the general formula I to n, where n is an integer from 6 to 295
corresponding to the position of amino acid residue identified in
SEQ ID NO:27 and preferably, corresponds to one of the C-terminal
amino acid residues identified in the C-terminal deletions
specified herein. In specific embodiments, C terminal deletions of
the galectin 11 polypeptide of the invention comprise, or
alternatively, consist of, amino acid residues: M-1 to H-295; M-1
to V-294; M-1 to C-293; M-1 to Y-292; M-1 to L-291; M-1 to Q-290;
M-1 to V-289; M-1 to S-288; M-1 to G-287; M-1 to S-286; M-1 to
I-285; M-1 to R-284; M-1 to L-283; M-1 to E-282; M-1 to R-281; M-1
to L-280; M-1 to Q-279; M-1 to E-278; M-1 to L-277; M-1 to A-276;
M-1 to Q-275; M-1 to Q-274; M-1 to N-273; M-1 to M-272; M-1 to
S-271; M-1 to T-270; M-1 to A-269; M-1 to G-268; M-1 to L-267; M-1
to G-266; M-1 to Q-265; M-1 to G-264; M-1 to N-263; M-1 to L 262;
M-1 to A-261; M-1 to L-260; M-1 to K-259; M-1 to L-258; M-1 to
G-257; M-1 to G-256; M-1 to E-255; M-1 to Q-254; M-1 to F-253; M-1
to L-252; M-1 to L-251; M-1 to L-250; M-1 to V-249; M-1 to E-248;
M-1 to F-247; M-1 to F-246; M-1 to R-245; M-1 to Q-244; M-1 to
P-243; M-1 to Y-242; M-1 to F-241; M-1 to L-240; M-1 to F-239; M-1
to P-238; M-1 to A-237; M-1 to S-236; M-1 to 1-235; M-1 to L-234;
M-1 to K-233; M-1 to K-232; M-1 to Q-231; M-1 to G-230; M-1 to
W-229; M-1 to R-228; M-1 to S-227; M-1 to I-226; M-1 to W-225; M-1
to A-224; M-1 to L-223; M-1 to T-222; M-1 to R-221; M-1 to D-220;
M-1 to A-219; M-1 to F-218; M-1 to S-217; M-1 to A-216; M-1 to
R-215; M-1 to L-214; M-1 to T-213; M-1 to V-212; M-1 to P-211; M-1
to A-210; M-1 to H-209; M-1 to A-208; M-1 to A-207; M-1 to Q-206;
M-1 to D-205; M-1 to R-204; M-1 to L-203; M-1 to S-202; M-1 to
V-201; M-1 to T-200; M-1 to F-199; M-1 to H-198; M-1 to K-197; M-1
to P-196; M-1 to E-195; M-1 to Q-194; M-1 to L-193; M-1 to V-192;
M-1 to L-191; M-1 to G-190; M-1 to R-189; M-1 to V-188; M-1 to
I-187; M-1 to I-186; M-1 to V-185; M-1 to Q-184; M-1 to G-183; M-1
to P-182; M-1 to S-181; M-1 to L-180; M-1 to G-179; M-1 to Q-178;
M-1 to P-177; M-1 to L-176; M-1 to A-175; M-1 to H-174; M-1 to
S-173; M-1 to C-172; M-1 to P-171; M-1 to V-170; M-1 to E-169; M-1
to L-168; M-1 to R-167; M-1 to P-166; M-1 to S-165; M-1 to M-164;
M-1 to L-163; M-1 to L-162; M-1 to F-161; M-1 to P-160; M-1 to
H-159; M-1 to G-158; M-1 to A-157; M-1 to P-156; M-1 to Y-155; M-1
to E-154; M-1 to R-153; M-1 to S-152; M-1 to G-151; M-1 to E-150;
M-1 to V-149; M-1 to F-148; M-1 to P-147; M-1 to N-146; M-1 to
I-145; M-1 to N-144; M-1 to L-143; M-1 to F-142; M-1 to G-141; M-1
to V-140; M-1 to A-139; M-1 to E-138; M-1 to V-137; M-1 to L-136;
M-1 to 1-135; M-1 to D-134; M-1 to G-133; M-1 to F-132; M-1 to
I-131; M-1 to G-130; M-1 to L-129; M-1 to T-128; M-1 to D-127; M-1
to V-126; M-1 to H-125; M-1 to S-124; M-1 to L-123; M-1 to P-122;
M-1 to L-121; M-1 to R-120; M-1 to Y-119; M-1 to R-118; M-1 to
F-117; M-1 to H-116; M-1 to L-115; M-1 to F-114; M-1 to H-113; M-1
to Q-112; M-1 to G-111; M-1 to N-110; M-1 to V-109; M-1 to S-108;
M-1 to V-107; M-1 to K-106; M-1 to V-105; M-1 to E-104; M-1 to
E-103; M-1 to N-102; M-1 to G-101; M-1 to F-100; M-1 to L-99; M-1
to F-98; M-1 to L-97; M-1 to I-96; M-1 to L-95; M-1 to F-94; M-1 to
S-93; M-1 to S-92; M-1 to G-91; M-1 to R-90; M-1 to R-89; M-1 to
L-88; M-1 to A-87; M-1 to L-86; M-1 to H-85; M-1 to P-84; M-1 to
W-83; M-1 to R-82; M-1 to A-81; M-1 to E-80; M-1 to R-79; M-1 to
Q-78; M-1 to W-77; M-1 to R-76; M-1 to G-75; M-1 to G-74; M-1 to
H-73; M-1 to L-72; M-1 to T-71; M-1 to N-70; M-1 to C-69; M-1 to
I-68; M-1 to V-67; M-1 to H-66; M-1 to P-65; M-1 to K-64; M-1 to
T-63; M-1 to T-62; M-1 to H-61; M-1 to F-60; M-1 to R-59; M-1 to
P-58; M-1 to N-57; M-1 to F-56; M-1 to H-55; M-1 to F-54; M-1 to
A-53; M-1 to 1-52; M-1 to D-51; M-1 to P-50; M-1 to R-49; M-1 to
P-48; M-1 to C-47; M-1 to L-46; M-1 to S-45; M-1 to C-44; M-1 to
G-43; M-1 to C-42; M-1 to Q-41; M-1 to F-40; M-1 to D-39; M-1 to
V-38; M-1 to Q-37; M-1 to T-36; M-1 to C-35; M-1 to R-34; M-1 to
S-33; M-1 to P-32; M-1 to G-31; M-1 to S-30; M-1 to R-29; M-1 to
A-28; M-1 to A-27; M-1 to H-26; M-1 to G-25; M-1 to D-24; M-1 to
Q-23; M-1 to R-22; M-1 to C-21; M-1 to A-20; M-1 to P-19; M-1 to
R-18; M-1 to W-17; M-1 to F-16; M-1 to D-15; M-1 to D-14; M-1 to
H-13; M-1 to C-12; M-1 to L-11; M-1 to S-10; M-1 to G-9; M-1 to
G-8; M-1 to S-7; M-1 to C-6, all of SEQ ID NO:27. Polynucleotides
encoding such polypeptides are also encompassed by the
invention.
[0166] Further embodiments of the invention are directed to
polypeptide fragments comprising, or alternatively, consisting of,
amino acids described by the general formula m to n, where m and n
correspond to any one of the amino acid residues specified above
for these symbols, respectively. Polynucleotides encoding these
polypeptides are also encompassed by the invention.
[0167] FIG. 2 provides a comparison of the galectin 11 polypeptide
with other galectins. Identical amino acids shared between the
galectins are shaded, while conservative amino acid changes are
boxed. By examining the regions of amino acids shaded and/or boxed,
the skilled artisan can readily identify conserved domains between
the two polypeptides. The amino acid sequences falling within these
conserved, shaded and/or boxed domains are contained in the
preferred polypeptide fragments of the invention. Similar analyses
for the full-length galectin-11.alpha. and .beta. is deemed to be
within the skill of the ordinary artisan given the teachings
provided herein.
[0168] Representative examples of polypeptide residue fragments of
the invention including, for example, fragments from about amino
acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40-108, 41-60, 47-108,
47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120;
114-128; and 101 to the end of the galectin 11 polypeptide depicted
in FIG. 1 (SEQ ID NO:2). In this context, "about" includes the
particularly recited ranges larger or smaller by several, 5, 4, 3,
2, or 1 amino acid at either end or at both extremes.
Representative examples of polypeptide residue fragments of the
invention including, for example, fragments from about amino acid
number 1-20, 1-66, 5-108, 5-128, 21-40, 40-108, 41-60, 47-108,
47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120;
114-128; 129-150; 145-175; 170-200; 195-225; 220-250; and 245-275
of SEQ ID NO:25. Additional representative examples of polypeptide
residue fragments of the invention including, for example,
fragments from about amino acid number 1-20, 1-66, 5-108, 5-128,
21-40, 40-108, 41-60, 47-108, 47-128, 61-80, 65-108, 65-128,
81-100, 88-108, 88-128, 108-120; 114-128; 129-150; 145-175;
170-200; 195-225; 220-250; 245-275; and 270-296 of SEQ ID NO:27.
Polypeptides comprising such amino acid sequences are provided as
well as polynucleotides encoding such polypeptides.
[0169] Preferably, the polynucleotide fragments of the invention
encode a polypeptide which demonstrates a galectin 11 functional
activity. By a polypeptide demonstrating a galectin 11 "functional
activity" is meant, a polypeptide capable of displaying one or more
known functional activities associated with a full-length
(complete) galectin 11 protein. Such functional activities include,
but are not limited to, biological activity, antigenicity [ability
to bind (or compete with a galectin 11 polypeptide for binding) to
an anti-galectin 11 antibody], immunogenicity (ability to generate
antibody which binds to a galectin 11 polypeptide), ability to form
multimers with galectin 11 polypeptides of the invention.
[0170] Among the especially preferred fragments of the invention
are fragments characterized by structural or functional attributes
of galectin 11. 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., having an antigenic region of three or more
contiguous amino acid residues each of which having an antigenic
index of greater than or equal to 1.5) of galectin 11. Certain
preferred regions are those set out in FIG. 3 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, turn-regions,
and coil-regions; Kyte-Doolittle predicted hydrophilic and
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.
[0171] In additional embodiments, the polynucleotides of the
invention encode functional attributes of galectin 11. 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
high antigenic index regions of galectin 11.
[0172] The data representing the structural or functional
attributes of galectin 11 set forth in FIG. 1 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, XII, and
XIII of Table I can be used to determine regions of galectin 11
which exhibit a high degree of potential for antigenicity. Regions
of high antigenicity are determined from the data presented in
columns VIII, XII, and/or XIII 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.
[0173] Certain preferred regions in these regards are set out in
FIG. 3, but may, as shown in Table I, be represented or identified
by using tabular representations of the data presented in FIG. 3.
The DNA*STAR computer algorithm used to generate FIG. 3 (set on the
original default parameters) was used to present the data in FIG. 3
in a tabular format (See Table I). The tabular format of the data
in FIG. 3 may be used to easily determine specific boundaries of a
preferred region.
[0174] The above-mentioned preferred regions set out in FIG. 3 and
in Table I include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIG. 1. As set out in FIG. 3 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, Eisenberg alpha- and beta-amphipathic regions,
Karplus-Schulz flexible regions, Emini surface-forming regions and
Jameson-Wolf regions of high antigenic index. TABLE-US-00002 TABLE
I Res Position I II III IV V VI VII VIII IX X XI XII XIII Met 1 . .
B . . . . 0.13 . * . 0.65 1.50 Ser 2 . . . . . T C 0.52 . * . 0.90
0.97 Pro 3 . . B . . T . 0.06 . * . 0.85 1.32 Arg 4 . . . . T T .
0.23 . * . 1.10 0.99 Leu 5 A . . . . T . -0.04 . * . 0.85 1.14 Glu
6 . . B . . . . 0.26 . * . 0.50 0.39 Val 7 . . B . . T . 0.52 * .
0.70 0.27 Pro 8 . . B . . T . 0.14 * * . 0.10 0.45 Cys 9 . . . . T
T . -0.78 * * . 0.50 0.26 Ser 10 . . B . . T . -0.18 . . . -0.20
0.29 His 11 . . B . . . . -0.18 . . . -0.40 0.29 Ala 12 . . B . . .
. 0.33 . . . -0.40 0.93 Leu 13 . . B . . . . -0.27 * . . -0.10 0.69
Pro 14 . . . . T T . 0.10 * . F 0.35 0.42 Gln 15 . . . . T T . 0.19
* . F 0.35 0.55 Gly 16 . . . . T T . -0.12 * . F 0.50 1.04 Leu 17 .
. . . . T C 0.47 . . F 0.45 0.66 Ser 18 . . . . . T C 0.42 * . F
0.45 0.66 Pro 19 . . . . . T C -0.26 * . F 0.45 0.50 Gly 20 . . B .
. T . -1.14 . . F -0.05 0.42 Gln 21 . . B . . T . -1.66 * . F -0.05
0.22 Val 22 . . B B . . . -0.73 * . . -0.60 0.11 Ile 23 . . B B . .
. -0.78 * . . -0.60 0.21 Ile 24 . . B B . . . -1.38 . . . -0.60
0.12 Val 25 . . B B . . . -1.89 . . . -0.60 0.13 Arg 26 . . B B . .
. -2.70 . . . -0.60 0.14 Gly 27 . . B B . . . -1.84 . . . -0.60
0.17 Leu 28 . . B B . . . -0.96 . . . -0.60 0.39 Val 29 . . B B . .
. -0.28 * . . 0.30 0.34 Leu 30 A . . B . . . 0.62 * * . -0.30 0.54
Gln 31 A . . B . . . 0.48 * . F 0.60 1.30 Glu 32 . . B B . . . 0.12
* . F 0.60 2.39 Pro 33 A . . . . . . 0.62 . . F 0.80 2.51 Lys 34 A
. . B . . . 0.62 * . F 0.60 2.09 His 35 A . . B . . . 1.13 * . .
0.30 0.89 Phe 36 . . B B . . . 0.32 * * . -0.30 0.78 Thr 37 . . B B
. . . 0.43 * * . -0.60 0.32 Val 38 . . B B . . . 0.64 * * . -0.60
0.46 Ser 39 A A . . . . . 0.60 * * . 0.30 0.89 Leu 40 A A . . . . .
0.04 . * . 0.45 1.07 Arg 41 A A . . . . . 0.16 . * . 0.45 1.45 Asp
42 A A . . . . . 0.43 . * . 0.75 1.09 Gln 43 A A . . . . . 0.70 . *
. 0.45 1.80 Ala 44 A A . . . . . 0.79 . * . 0.60 0.93 Ala 45 A A .
. . . . 0.74 . * . 0.30 0.86 His 46 A A . . . . . 0.32 . * . -0.60
0.37 Ala 47 . A B . . . . -0.49 . * . -0.60 0.53 Pro 48 . A B . . .
. -0.38 . * . -0.60 0.43 Val 49 A A . . . . . -0.38 . * . -0.30
0.62 Thr 50 A A . . . . . -0.09 . * . -0.30 0.62 Leu 51 . A B . . .
. -0.76 . * . -0.30 0.54 Arg 52 . A B . . . . -0.76 . * . -0.60
0.63 Ala 53 A A . . . . . -0.54 . * . -0.30 0.44 Ser 54 A A . . . .
. 0.42 . * . 0.30 0.89 Phe 55 A A . . . . . 0.42 . * . 0.60 0.89
Ala 56 A A . . . . . 0.42 . * . 0.45 1.27 Asp 57 A A . . . . .
-0.28 . * F 0.45 0.78 Arg 58 A A . . . . . 0.02 * . F 0.45 0.91 Thr
59 A A . . . . . -0.57 * . . -0.30 0.95 Leu 60 A A . . . . . -0.17
* . . -0.30 0.40 Ala 61 A A . . . . . 0.53 * . . -0.60 0.27 Trp 62
A A . . . . . 0.24 * . . -0.60 0.37 Ile 63 A A . . . . . -0.21 * .
. -0.26 0.47 Ser 64 A . . . . T . 0.10 * . . 0.48 0.46 Arg 65 . . .
. T T . 0.96 * . . 1.22 0.76 Trp 66 . . . . T T . 1.59 * . F 2.76
2.17 Gly 67 . . . . T T . 1.07 * . F 3.40 3.24 Gln 68 . A . . T . .
1.07 * . F 2.66 1.36 Lys 69 . A . . T . . 1.07 * . F 1.27 0.91 Lys
70 . A B . . . . 0.37 * . F 1.28 1.23 Leu 71 . A B . . . . 0.44 . .
F 0.79 0.72 Ile 72 . A B . . . . 0.09 . . . 0.30 0.55 Ser 73 . . B
. . . . -0.72 . . . -0.40 0.24 Ala 74 . . B . . . . -1.47 * . .
-0.40 0.24 Pro 75 . . B B . . . -1.76 . . . -0.60 0.30 Phe 76 . . B
B . . . -1.16 . . . -0.60 0.35 Leu 77 . . B B . . . -0.27 . * .
-0.60 0.53 Phe 78 . . B B . . . 0.14 * . . -0.60 0.59 Tyr 79 . . B
. . T . 0.03 * . . -0.05 1.35 Pro 80 . . . . . T C -0.46 * . F 0.30
1.41 Gln 81 . . . . T T . 0.24 * . F 0.50 1.41 Arg 82 A . . . . T .
0.20 * . F 0.40 1.56 Phe 83 A A . . . . . 0.09 * . . -0.30 0.75 Phe
84 A A . . . . . -0.48 * . . -0.30 0.36 Glu 85 A A . . . . . -1.08
* . . -0.60 0.15 Val 86 A A . . . . . -1.78 * . . -0.60 0.14 Leu 87
A A . . . . . -1.89 * . . -0.60 0.14 Leu 88 A A . . . . . -1.19 * .
. -0.60 0.14 Leu 89 A A . . . . . -0.83 . . . -0.60 0.33 Phe 90 A A
. . . . . -1.18 . . . -0.60 0.40 Gln 91 A . . . . T . -1.13 * . F
-0.05 0.48 Glu 92 A . . . . T . -0.28 * * F -0.05 0.48 Gly 93 A . .
. . T . -0.28 . . F 1.00 1.11 Gly 94 A . . . . T . -0.06 . * F 0.85
0.53 Leu 95 A A . . . . . -0.17 * * F 0.45 0.31 Lys 96 A A . . . .
. -0.17 * * . -0.60 0.26 Leu 97 . A B . . . . -0.51 * * . -0.30
0.42 Ala 98 . A B . . . . -0.17 . * . -0.30 0.50 Leu 99 . A B . . .
. -0.17 . * . -0.30 0.43 Asn 100 . A B . . . . -0.17 . * F -0.45
0.52 Gly 101 . . B . . T . -0.56 . * F -0.05 0.43 Gln 102 . . B . .
T C -0.33 . * F 0.15 0.51 Gly 103 . . . . . T C -0.06 . . F 0.45
0.32 Leu 104 . . . . . T C 0.46 . . F 0.45 0.47 Gly 105 . . . . . .
C -0.14 . . F 0.25 0.36 Ala 106 . . B . . . . 0.20 . . F -0.25 0.36
Thr 107 . . B . . . . 0.20 . . F -0.25 0.71 Ser 108 . . B . . T .
0.54 . . F 0.40 1.23 Met 109 . . B . . T . 0.77 . . F 0.40 2.12 Asn
110 A . . . . T . 0.30 * . F 0.40 1.48 Gln 111 A . . . . T . 0.89 *
. F 0.25 0.91 Gln 112 A A . . . . . 1.20 * . F 0.60 1.60 Ala 113 A
A . . . . . 0.69 * * F 0.60 1.72 Leu 114 A A . . . . . 1.40 * . F
-0.15 0.82 Glu 115 A A . . . . . 1.40 * . . 0.30 0.93 Gln 116 A A .
. . . . 0.59 * . F 0.90 1.59 Leu 117 A A . . . . . 0.70 * * F 0.90
1.59 Arg 118 A A . . . . . 0.40 * * F 1.15 1.79 Glu 119 A A . . . .
. 0.91 * * . 1.10 0.73 Leu 120 A A . . . . . 0.57 * * . 1.50 1.18
Arg 121 . A . . T . . 0.27 * * . 2.00 0.60 Ile 122 . . . . T T .
0.22 * * F 2.50 0.46 Ser 123 . . . . T T . 0.11 * * F 1.65 0.42 Gly
124 . . . . T T . -0.70 . * F 2.00 0.37 Ser 125 . . . . T T . -0.13
. * F 0.85 0.43 Val 126 . . B B . . . -0.91 . * . -0.35 0.50 Gln
127 . . B B . . . -0.88 . * . -0.60 0.27 Leu 128 . . B B . . .
-0.61 . . . -0.60 0.15 Tyr 129 . . B B . . . -0.57 . . . -0.60 0.28
Cys 130 . . B B . . . -0.66 . . . -0.60 0.21 Val 131 . . B B . . .
-0.19 . . . -0.60 0.33 His 132 . . B B . . . -0.58 . . . -0.60 0.27
Ser 133 . . B B . . . -0.16 . . . -0.30 0.65
[0175] FIG. 2 provides a comparison of the galectin 11 polypeptide
with other galectins. Identical amino acids shared between the
galectins are shaded, while conservative amino acid changes are
boxed. By examining the regions of amino acids shaded and/or boxed,
the skilled artisan can readily identify conserved domains between
the two polypeptides. The amino acid sequences falling within these
conserved, shaded and/or boxed domains are contained in the
preferred polypeptide fragments of the invention.
[0176] Among highly preferred fragments in this regard are those
that comprise regions of galectin 11 that combine several
structural features, such as several of the features set out
above.
[0177] Other preferred polypeptide fragments are biologically
active galectin 11 fragments. Biologically active fragments are
those exhibiting activity similar, but not necessarily identical,
to an activity of the galectin 11 polypeptide. The biological
activity of the fragments may include an improved desired activity,
or a decreased undesirable activity. Polynucleotides encoding these
polypeptide fragments are also encompassed by the invention.
[0178] Representative examples of polypeptide residue fragments of
the invention including, for example, fragments from about amino
acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40-108, 41-60, 47-108,
47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120;
114-128; and 101 to the end of the galectin 11 polypeptide depicted
in FIG. 1 (SEQ ID NO:2). In this context, "about" includes the
particularly recited ranges larger or smaller by several, 5, 4, 3,
2, or 1 amino acid at either end or at both extremes.
[0179] As one of skill in the art will appreciate, galectin 11
polypeptides of the present invention such as, for example,
epitope-bearing fragments of galectin 11, can be combined with
parts of the constant domain of immunoglobulins (IgG), resulting in
chimeric polypeptides. Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG part can also be more efficient in
binding and neutralizing other molecules than the monomeric
galectin 11 protein or protein fragment alone (Fountoulakis et al.,
J. Biochem. 270:3958-3964 (1995)).
[0180] 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) resulting in chimeric polypeptides.
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
desulfide 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., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-897). 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.
[0181] 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. 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 polynucleotides corresponding to
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 the
encoded 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 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.
[0182] Polypeptides of the invention include polypeptides encoded
by polynucleotides that hybridize (e.g., under stringent
hybridization conditions) to the polynucleotide sequence depicted
in FIG. 1 (SEQ ID NO:1), the complementary strand thereto, and/or
the nucleotide sequence contained in the deposited clone. In
specific embodiments, the polypeptides of the invention have
galectin 11 functional and/or biological activity.
Assays for Galectin 11 Functional Activity
[0183] The functional and/or biological activity of galectin 11
polypeptides, fragments, variants, derivatives and analogs of the
invention can be assayed by various methods.
[0184] For example, in one embodiment, where one is assaying for
the ability to bind or compete with galectin 11 polypeptide for
binding to anti-galectin 11 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, "sandwich" immunoassays,
immunoradiometric assays, and diffusion precipitin 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. Many means are known in the art
for detecting binding in an immunoassay and are within the scope of
the present invention.
[0185] In another embodiment, the ability of galectin 11
polypeptides, fragments, variants, derivatives and analogs of the
invention to bind .beta.-galactoside sugars may be determined
using, or routinely modifying, assays known in the art. For
example, lactose binding activity of the expressed galectin 11
polypeptides and fragments, variants, derivatives, or analogs
thereof, may be assayed by immunodectection of in situ binding
activity to asialofetuin (Sigma) immobilized on nitrocellulose
(Amersham) (Madsen et al., J. Biol. Chem. 270(11):5823-5829
(1995)). For example, in one assay, thirty .mu.g of asialofetuin
dissolved in 3 .mu.l of water is spotted on a 1-cm2 strip of
nitrocellulose. The nitrocellulose pieces are then placed in a
24-well tissue culture plant and incubated overnight in buffer B
(58 mM NA.sub.2HPO.sub.4, 18 mM KH.sub.2PO.sub.4, 75 mM NaCl, 2 mM
EDTA, and 3% BSA, pH 7.2) with constant agitation at 22.degree. C.
Following incubation, the blocking medium is aspirated and the
nitrocellulose pieces are washed three times in buffer A (58 mM
Na.sub.2HPO.sub.4 18 mM KH.sub.22PO.sub.4, 75 mM NaCl, 2 mM EDTA, 4
mM .beta.-mercaptoethanol and 0.2% BSA, pH 7.2). Cell extracts
(preferably, COS cells) are prepared containing 1% BSA and either
with or without 150 mM lactose (105 .mu.l of primary extract, 15
.mu.l of 10% BSA in buffer A and either 30 .mu.l of 0.75 M lactose
in buffer A or 30 .mu.l of buffer A). The immobilized asialofetuin
is incubated with the extracts for 2 h and washed 5 times in buffer
A. The nitrocellulose pieces are then fixed in 2% formalin in PBS
(58 mM Na.sub.2HPO.sub.4, 18 mM KH.sub.2PO.sub.4, 75 mM NaCl, 2 mM
EDTA pH 7.2) for 1 h to prevent loss of bound galectin 11.
Following extensive washing in PBS the pieces are incubated with a
rabbit anti-galectin 11. Polyclonal serum (generated using
techniques known in the art) diluted 1:100 in PBS for 2 h at
22.degree. C. The pieces are then washed in PBS and incubated with
peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following
incubation for 2 h at 22.degree. C., the pieces are washed in PBS
and the substrate is added. Nitrocellulose pieces are incubated
until the color developed and the reaction is stopped by washing in
distilled water.
[0186] The ability of galectin 11 polypeptides, fragments,
variants, derivatives and analogs of the invention to agglutinate
trypsin-treated rabbit erythrocytes can routinely be assayed using
techniques known in the art.
[0187] The ability of the galectin 11 polypeptides, fragments,
variants, derivatives and analogs of the invention to induce
apoptosis of T-cells may be determined using, or routinely
modifying, techniques described herein (see e.g., Example 5) or
otherwise known in the art. See e.g., Perillo et al., Nature
378:736-739 (1995); Chinnaiyan et al., Cell 81:505-512 (1995);
Boldin et al., J. Biol. Chem. 270:7795-7798 (1995); Kischkel et
al., EMBO J. 14:5579-5585 (1995); Chinnaiyan et al., J. Biol. Chem.
271:4961-4965 (1996); the contents of each of which is herein
incorporated by reference in its entirety).
[0188] The galectin 11 polynucleotides and polypeptides, and
fragments, variants derivatives and analogs thereof; and
antibodies, agonists and antagonists thereto; can be tested in vivo
for the desired therapeutic or prophylactic activity. For example,
such compounds can be tested in suitable animal model systems prior
to testing in humans. Such animal models include, but are not
limited to, rats, mice, chickens, cows, monkeys, rabbits, etc. Such
testing may also be utilized to routinely determine dosage for
delivery to human patients. For in vivo testing prior to
administration to human, any animal model system known in the art
may be used (see, for example, Levi et al., Eur. J. Imun.
13:500-507 (1983); and Offner et al., J. Neuroimmunol 28:177-184
(1990)). For example, an animal model useful for the study of the
treatment of human MS is experimental allergic encephalomyelitis
(EAE). EAE is an experimentally induced disease that shares many of
the same clinical and pathological symptoms of MS (Martin et al.,
Ann. Rev. Immunol. 10:153-187 (1992); Hafler et al., Immunology
Today 10:104-107 (1989)). Several studies in rodents have shown
that, similar to MS, CD4.sup.+ T cells participate in the
pathophysiology of EAE, Traugott et al., Cellular Immunology
91:240-254 (1985); Ben-Nun, Eur. J. Immunol. 11:195-199 (1981);
Pettinel et al., J. Immunol. 127:1420-1423 (1981). EAE can be
induced in certain strains of mice by immunization with myelin in
an adjuvant. The immunization activates CD4.sup.+ T cells specific
for myelin basic protein (MBP) and proteolipid (PLP), Bernard et
al., J. Immunol. 114:1537-1540 (1975); Chou et al., J. Immunol.
130:2183-2186 (1983); Kurchroo et al., J. Immunol. 148:3776-3782
(1992). The activated T cells enter the central nervous system and
their local action causes both the anatomic pathology and clinical
signs, e.g., ascending hind limb paresis leading to paralysis, of
the disease. As discussed above, the galectin 1 has been
demonstrated to suppress clinical and histological signs of
experimental autoimmune encephalomyelitis in rats (Offner et al.,
J. Neuroimmunol. 28:177-184 (1990)).
[0189] Another model system that may be utilized to both study the
role of the polypeptides, variants, derivatives and analogs of the
invention as a suppresser of immune responses, and to determine
effective dosages for doing so, is experimental autoimmune
myasthenia gravis (EAMG) in rabbits. EAMG is an autoimmune disease
induced by immunization with the purified acetylcholine receptor
protein (AChR) and is considered to be a good model for the human
disease myasthenia gravis. As further discussed above, galectin 1
has been demonstrated to have a prophylactic and therapeutic action
on experimental autoimmune myasthenia gravis in rabbits (Levi et
al., Eur. J. Immunol. 13:500-507 (1983)).
[0190] Other art known model assays that may be used to determine
the desired therapeutic or prophylactic activity of compounds of
the invention (e.g., as a suppresser of immune responses) include,
but are not limited to, T-cell proliferation in mixed lymphocyte
reaction assays (an art-accepted model for allogeneic graft
rejection), and murine allograft models known in the art.
[0191] Assays described herein or otherwise known in the art may be
applied to routinely determine which galectin 11 polypeptides,
fragments, variants, derivatives and analogs of the invention
demonstrate galectin 11 functional activity and the optimal
concentration at which these compounds demonstrate this activity.
These assays may additionally be utilized to identify molecules
which enhance (agonists) or suppress (antagonists) galectin 11
functional activity.
Epitopes
[0192] The present invention encompasses polypeptides comprising,
or alternatively consisting of, an epitope of the polypeptide
having an amino acid sequence of SEQ ID NO:2, 25, or 27, or an
epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in ATCC Deposit No: 209053 or encoded by a
polynucleotide that hybridizes to the complement of the sequence of
SEQ ID NO:1, 24, or 26 or contained in ATCC Deposit No: 209053
under stringent hybridization conditions or lower stringency
hybridization conditions as defined supra. The present invention
further encompasses polynucleotide sequences encoding an epitope of
a polypeptide sequence of the invention (such as, for example, the
sequence disclosed in SEQ ID NO:1, 24, or 26), polynucleotide
sequences of the complementary strand of a polynucleotide sequence
encoding an epitope of the invention, and polynucleotide sequences
which hybridize to the complementary strand under stringent
hybridization conditions or lower stringency hybridization
conditions defined supra.
[0193] 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 infra. (See, for
example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002
(1983)). 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.
[0194] 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).
[0195] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful, for example, to raise antibodies,
including monoclonal antibodies, that bind specifically to a
polypeptide of the invention. 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, for instance, Wilson et al., Cell 37:767-778
(1984); Sutcliffe et al., Science 219:660-666 (1983)).
[0196] 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 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).
[0197] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least 7, 9, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 110 or 120 contiguous amino acid
residues of the amino acid sequence depicted in FIG. 1 or 6A-B (SEQ
ID NO:2, 25, or 27). In the present invention, antigenic epitopes
preferably contain a sequence of 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.
Additional non-exclusive preferred antigenic epitopes include the
antigenic epitopes disclosed herein, as well as portions
thereof.
[0198] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate galectin 11-specific antibodies
include: a polypeptide comprising amino acid residues from about
65-70 and 118-124 in FIG. 1 (SEQ ID NO:2). As indicated above, the
inventors have determined that the above polypeptide fragments are
antigenic regions of the galectin 11 protein.
[0199] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. See generally,
Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985).
This Simultaneous Multiple Peptide Synthesis (SMPS) process is
further described in U.S. Pat. No. 4,631,211 to Houghten et al.
(1986).
[0200] 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 hemacyanin (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.
Antibodies
[0201] Further polypeptides of the invention relate to antibodies
and T-cell antigen receptors (TCR) which immunospecifically bind a
polypeptide, polypeptide fragment, or variant of SEQ ID NO:2,
and/or an epitope, of the present invention (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.
[0202] 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. 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).
[0203] 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. Preferred epitopes of the invention include: amino acids
65-70 and 118-124 of SEQ ID NO:2, as well as polynucleotides that
encode these epitopes. 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.
[0204] 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,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10M,
10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
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.-15 M.
[0205] 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%.
[0206] 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. Preferrably, antibodies of the
present invention bind an antigenic epitope disclosed herein, or a
portion thereof. The invention features both receptor-specific
antibodies and ligand-specific antibodies. The invention also
features receptor-specific antibodies which 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 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.
[0207] 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
dimerization of the receptor. 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).
[0208] 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).
[0209] 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.
[0210] 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, phosphylation, 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.
[0211] 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.
[0212] 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.
[0213] 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. 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. 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.
[0214] 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.
[0215] 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.
[0216] 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 which 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. Phage 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.
[0217] 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).
[0218] 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).
[0219] 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.
[0220] 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; and 5,939,598, 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.
[0221] 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)).
[0222] 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. 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.
[0223] The polypeptides of the invention and their fragments,
variants, derivatives or analogs, or cells expressing them, can
also be used as immunogens to produce antibodies immunospecific for
galectin 11 polypeptide of the invention. The term "immunospecific"
means that the antibodies have substantially greater affinity for
the polypeptides of the invention than their affinity for other
related polypeptides in the prior art.
[0224] The term "antibody" (Ab) or "monoclonal antibody" (mAb) as
used herein is meant to include intact molecules as well as
fragments thereof (such as, for example, Fab, and F(ab').sub.2
fragments) which are capable of binding an antigen. Fab, Fab', and
F (ab').sub.2 fragments lack the Fc fragment of intact antibody,
clear more rapidly from the circulation, and may have less
non-specific tissue binding of an intact antibody (Wahl et al., J.
Nucl. Med. 24:316-325 (1983)).
[0225] Antibodies according to the present invention may be
prepared by any of a variety of standard methods using galectin 11
immunogens of the present invention. For example, antibodies
generated against full-length galectin 11 polypeptides can be
obtained by administering the polypeptides or epitope-bearing
fragments, variants, derivatives, analogs, or cells, to an animal,
preferably a nonhuman, using routine protocols. For preparation of
monoclonal antibodies, any technique which provides antibodies
produced by continuous cell line cultures can be used. Examples
include the hybridoma technique (Kohler et al., Nature 256:495-497
(1975)), the trioma technique, the human B-cell hybridoma technique
(Kozbor et al., Immunology Today 4:72 (1983)) and the EBV-hybridoma
technique (Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY,
pp. 77-96, Alan R. Liss, Inc., 1985). Additionally, techniques for
the production of single chain antibodies (U.S. Pat. No. 4,946,778)
can also be adapted to produce single chain antibodies to
polypeptides of the invention. Also, transgenic mice, or other
organisms including other mammals, may be used to express humanized
antibodies.
[0226] Antibodies of the invention can be used in methods known in
the art relating to the localization and activity of the
polypeptide sequences of the invention, e.g., for imaging these
polypeptides, measuring levels thereof in appropriate physiological
samples, etc. The antibodies also have use in immunoassays and in
therapeutics as agonists and antagonists of galectin 11.
Additionally, the antibodies of the invention may be employed to
isolate or to identify clones expressing the polypeptide or to
purify the polypeptides by affinity chromatography.
Polynucleotides Encoding Antibodies
[0227] 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 polypeptide
of the invention, preferably, an antibody that binds to a
polypeptide having the amino acid sequence of SEQ ID NO:2.
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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 Antibodies
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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)).
[0238] 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.
[0239] 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).
[0240] 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. (e.g., see 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)).
[0241] 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, W138, 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.
[0242] 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.
[0243] 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.
[0244] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentscbel, 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)).
[0245] 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.
[0246] 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.
[0247] 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.
[0248] 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 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 can be made by fusing the 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).
[0249] As discussed, supra, the polypeptides corresponding to a
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, 25, or 27 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).
[0250] 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" tag.
[0251] 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
materials include 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.201Ti), gallium (.sup.68 Ga, .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, and .sup.97Ru.
[0252] 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, 213Bi. 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).
[0253] 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), 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.
[0254] 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.
[0255] 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).
[0256] 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.
[0257] 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
[0258] 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)).
[0259] 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 Antibody Binding
[0260] 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).
[0261] 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% Trasylol) 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.
[0262] 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 enzymatic substrate (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.
[0263] 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 enzymatic
substrate (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.
[0264] 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. Other suitable radioactive
materials include, but are not limited to, radioisotopes such as
iodine (.sup.131I, .sup.125I, .sup.123I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.115In, .sup.113In, .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,
and .sup.97Ru.
Therapeutic Uses
[0265] 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. 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. Antibodies of the
invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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, 10.sup.-8 M,
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, and
10.sup.-15 M.
Gene Therapy
[0270] 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.
[0271] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0272] 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).
[0273] 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 the heavy and light chains, or fragments thereof, of
the antibody.
[0274] 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.
[0275] 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)).
[0276] In a specific embodiment, viral vectors that contains
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 facilitates 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).
[0277] 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.
[0278] 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).
[0279] 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.
[0280] 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 cell
progeny.
[0281] 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.
[0282] 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 Tlymphocytes, Blymphocytes,
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.
[0283] In a preferred embodiment, the cell used for gene therapy is
autologous to the patient.
[0284] 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)).
[0285] 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
[0286] 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.
Diagnosis and Prognosis
[0287] It is believed that certain tissues in mammals with certain
diseases (e.g., autoimmune diseases which include, but are not
limited to, lupus erythematosus (SLE), rheumatoid arthritis (RA),
insulin-dependent diabetes, multiple sclerosis (MS), giant cell
arteritis, polyarteritis nodosa, myasthenia gravis, scleroderma,
and graft versus host disease; graft rejection; mammalian cancers
which include, but are not limited, to, melanoma, renal
astrocytoma, Hodgkin's disease, breast, ovarian, prostate, bone,
liver, lung, pancreatic, and spleenic cancers; inflammatory
diseases; asthma; and allergeic diseases) express significantly
altered (e.g., enhanced or decreased) levels of the galectin 11
polypeptide and mRNA encoding the galectin 11 polypeptide when
compared to a corresponding "standard" mammal, i.e., a mammal of
the same species not having the disease. Further, it is believed
that altered levels of the galectin 11 polypeptide can be detected
in certain body fluids (e.g., sera, plasma, urine, and spinal
fluid) from mammals with the disorder when compared to sera from
mammals of the same species not having the disorder. Thus, the
invention provides a diagnostic method useful during diagnosis,
which involves assaying the expression level of the gene encoding
the galectin 11 polypeptide in mammalian cells or body fluid and
comparing the gene expression level with a standard galectin 11
gene expression level, whereby an increase or decrease in the gene
expression level over the standard is indicative of the
disease.
[0288] Where a diagnosis has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting altered galectin
11 gene expression will experience a worse clinical outcome
relative to patients expressing the gene at a normal level.
[0289] By "assaying" the expression level of the gene encoding the
galectin 11 polypeptide" is intended qualitatively or
quantitatively measuring or estimating the level of the galectin 11
polypeptide or the level of the mRNA encoding the galectin 11
polypeptide in a first biological sample either directly (e.g., by
determining or estimating absolute polypeptide or mRNA level) or
relatively (e.g., by comparing to the galectin 11 polypeptide level
or mRNA level in a second biological sample).
[0290] Nucleic acids for diagnosis may be obtained from a
biological sample of a subject, such as from blood, urine, saliva,
tissue biopsy or autopsy material, using techniques known in the
art. The genomic DNA may be used directly for detection or may be
amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. 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 labeled galectin 11 nucleotide sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in electrophoretic mobility of DNA fragments in gels,
with or without denaturing agents, or by direct DNA sequencing
(see, e.g., Myers et al., Science 230:1242 (1985)). Sequence
changes at specific locations may also be revealed by nuclease
protection assays, such as RNase and S1 protection or the chemical
cleavage method (see Cotton et al., Proc. Natl. Acad. Sci. USA
85:4397-4401 (1985)). In another embodiment, an array of
oligonucleotides probes comprising galectin 11 polynucleotide
sequences or fragments thereof, can be constructed to conduct
efficient screening of e.g., genetic mutations. Array technology
methods are well known and have general applicability and can be
used to address a variety of questions in molecular genetics
including gene expression, genetic linkage, and genetic variability
(see for example, Chee et al., Science 274:610-613 (1996)).
[0291] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to specific diseases through detection
of mutation in the galectin 11 gene by the methods described.
[0292] In addition, specific diseases can be diagnosed by methods
comprising determining from a sample derived from a subject an
abnormally decreased or increased level of galectin 11 polypeptide
or mRNA. Decreased or increased expression can be measured at the
RNA level using any of the methods well known in the art, which
include, but are not limited to, Northern blot analysis, (Harada et
al., Cell 63:303-312 (1990), S1 nuclease mapping (Fijita et al.,
Cell 49:357-367 (1987)), RNAse protection, the polymerase chain
reaction (PCR), reverse transcription in combination with the
polymerase chain reaction (RT-PCR) (Makino et al., Technique
2:295-301 (1990), reverse transcription in combination with the
ligase chain reaction (RT-LCR) and other hybridization methods.
[0293] Assaying galectin 11 polypeptide levels in a biological
sample can be by any techniques known in the art, which include,
but are not limited to, radioimmunoassays, competitive-binding
assays, Western Blot analysis and enzyme linked immunosorbent
assays (ELISAs) and other antibody-based techniques. For example,
galectin 11 polypeptide expression in tissues can be studied with
classical immunohistological methods (Jalkanen et al., J. Cell.
Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol.
105:3087-3096 (1987)).
[0294] Suitable 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.115In,
.sup.113In, .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.133
Xe), fluorine (.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd,
.sup.149 Pm, .sup.140La, 175Yb, .sup.166Ho, .sup.90Y, .sup.47SC,
.sup.186Re, .sup.188Re, .sup.142Pr, 105 Rh, .sup.97Ru; luminescent
labels, such as luminol; and fluorescent labels, such as
fluorescein and rhodamine, and biotin.
[0295] Thus in another aspect, the present invention relates to a
diagnostic kit for a disease or susceptibility to a disease which
comprises:
[0296] (a) a galectin 11 polynucleotide, preferably the nucleotide
sequence of SEQ ID NO:1, 24, or 26, or a fragment thereof;
[0297] (b) a nucleotide sequence complementary to that of (a);
[0298] (c) a galectin 11 polypeptide of the invention, preferably
the polypeptide of SEQ ID NO:2, 25, or 27 or a fragment thereof;
or
[0299] (d) an antibody to a galectin 11 polypeptide of the
invention, preferably to the polypeptide of SEQ ID NO: 2, 25, or
27.
[0300] It will be appreciated that in any such kit, (a), (b), (c)
or (d) may comprise a substantial component.
[0301] Thus, the invention also provides a diagnostic method useful
during diagnosis of a disorder, involving measuring the expression
level of polynucleotides of the present invention in cells or body
fluid from an individual and comparing the measured gene expression
level with a standard level of polynucleotide expression level,
whereby an increase or decrease in the gene expression level
compared to the standard is indicative of a disorder.
[0302] In still another embodiment, the invention includes a kit
for analyzing samples for the presence of proliferative and/or
cancerous polynucleotides derived from a test subject. In a general
embodiment, the kit includes at least one polynucleotide probe
containing a nucleotide sequence that will specifically hybridize
with a polynucleotide of the present invention and a suitable
container. In a specific embodiment, the kit includes two
polynucleotide probes defining an internal region of the
polynucleotide of the present invention, where each probe has one
strand containing a 31 'mer-end internal to the region. In a
further embodiment, the probes may be useful as primers for
polymerase chain reaction amplification.
[0303] Where a diagnosis of a disorder, has already been made
according to conventional methods, the present invention is useful
as a prognostic indicator, whereby patients exhibiting enhanced or
depressed polynucleotide of the present invention expression will
experience a worse clinical outcome relative to patients expressing
the gene at a level nearer the standard level.
[0304] By "measuring the expression level of polynucleotide of the
present invention" is intended qualitatively or quantitatively
measuring or estimating the level of the polypeptide of the present
invention or the level of the mRNA encoding the 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 polypeptide level or mRNA level in a
second biological sample). Preferably, the polypeptide level or
mRNA level in the first biological sample is measured or estimated
and compared to a standard 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. As will be appreciated in the art, once a standard
polypeptide level or mRNA level is known, it can be used repeatedly
as a standard for comparison.
[0305] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains the polypeptide of the present
invention or mRNA. As indicated, biological samples include body
fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and spinal fluid) which contain the polypeptide of the present
invention, and other tissue sources found to express the
polypeptide of the present invention. 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.
[0306] The method(s) provided above may preferrably be applied in a
diagnostic method and/or kits in which polynucleotides and/or
polypeptides are attached to a solid support. In one exemplary
method, the support may be a "gene chip" or a "biological chip" as
described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174.
Further, such a gene chip with polynucleotides of the present
invention attached may be used to identify polymorphisms between
the polynucleotide sequences, with polynucleotides isolated from a
test subject. The knowledge of such polymorphisms (i.e. their
location, as well as, their existence) would be beneficial in
identifying disease loci for many disorders, including cancerous
diseases and conditions. Such a method is described in U.S. Pat.
Nos. 5,858,659 and 5,856,104. The US patents referenced supra are
hereby incorporated by reference in their entirety herein.
Screening Assays for Galectin 11 Agonists or Antagonists
[0307] Aberrancies in galectin 11 expression are responsible for
many biological functions, including many pathologies. Accordingly,
it is desirous to find compounds and drugs which enhance galectin
11 activity or, alternatively, suppress galectin 11 activity. The
invention also provides a method of screening compounds to identify
those which enhance or suppress galectin 11 activity. An agonist is
a compound which increases the natural biological functions of
galectin 11 or which functions in a manner similar to galectin 11,
while antagonists decrease or eliminate such functions.
[0308] Thus, embodiments of the invention are directed to assays
designed to identify compounds that interact with (e.g., bind to)
galectin 11 polypeptides of the invention, compounds that interfere
or enhance the interaction of galectin 11 with its cognate ligands,
and to compounds which modulate the galectin 11 gene (i.e.,
modulate the level of galectin 11 gene expression) or modulate the
level of galectin 11 functional or biological activity. Assays may
also be used to identify compounds which bind galectin 11 gene
regulatory sequences (e.g., promoter sequences) and which may
modulate galectin 11 gene expression. See e.g., Platt, J. Biol.
Chem. 269:28558-28562 (1994), which is incorporated herein by
reference in its entirety.
[0309] Thus, polypeptides of the invention may be used to assess
the binding of small molecule substrates and ligands in, for
example, cells, cell-free preparations, chemical libraries, and
natural product mixtures. These substrates and ligands may be
natural substrates and ligands or may be structural or functional
mimetics. See Coligan et al., Current Protocols in Immunology
1(2):Chapter 5 (1991). Further examples of compounds that may be
screened include, but are not limited to, peptides such as, for
example soluble peptides, including but not limited to, those
found: in random peptide libraries (see, e.g., Lam et al., Nature
354:84-86 (1991)), and combinatorial chemistry-derived molecular
libraries made of D- and L-configuration amino acid;
phosphopeptides (including, but not limited to, members of random
or partially degenerate, directed phosphopeptide libraries (see
e.g., Songyang et al., Cell 72:767-778 (1993)); antibodies
(including but not limited to, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and Fab,
F(ab')2, and FAB expression library fragments, and epitope-binding
fragments thereof); and small organic or inorganic molecules.
[0310] Numerous experimental methods may be used to select and
detect compounds that bind galectin 11 polypeptides of the
invention and thereby modulate galectin 11 expression or activity,
including, but not limited to, protein affinity chromotography,
affinity blotting, immunoprecipitation, cross-linking, and library
based methods such as protein probing, phage display, the
two-hybrid system (Fields and Song, Nature 340:245-246 (1989)), and
modified versions of the two-hybrid system (Gyuris et al., Cell
75:791-803 (1993); Zervos et al., Cell 72:223-232 (1993)). See
generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995).
[0311] The principle behind assays that identify compounds that
bind to galectin 11 polypeptides of the invention involves
preparing a reaction mixture of galectin 11 polypeptide and test
compound under conditions that allow the two components to interact
and bind, thus forming a complex which can be detected in the
reaction mixture and purified using techniques known in the art.
Accordingly, the assays may simply test binding of a candidate
compound to galectin 11.
[0312] Further, the assays may simply comprise the steps of
combining a candidate compound with a solution containing a
galectin 11 polypeptide to form a mixture, and determining the
ability of galectin 11 contained in this mixture to bind galectin
11 cognate ligands (e.g., compounds containing a .beta. galactoside
sugar and/or molecules expressed on the surface of T-cells), to
agglutinate trypsin-treated rabbit erythrocytes, or to induce
apoptosis of T-cells, and comparing this ability with that observed
for the galectin 11 polypeptide in the same or similar solution
under the same or similar conditions, but absent the candidate
compound. The ability of the candidate molecule to interfere with
binding of galectin 11 to the cognate ligand is reflected in
decreased binding of the labeled galectin 11 to the cognate ligand
relative to that in the absence of candidate molecule. Molecules
which interfere with the ability of galectin 11 to elicit cellular
responses (e.g., apoptosis) resulting from galectin 11 binding to
its cognate ligand are antagonists. Molecules that enhance galectin
11 induced cellular responses when mixed with galectin 11, or which
are able to induce a similar cellular response in the absence of
galectin 11, are agonists.
[0313] The galectin 11 polynucleotides, polypeptides, and
antibodies of the invention may also be used to configure assays
for detecting the effect of added compounds on the production of
galectin 11 mRNA and protein in cells. For example, an ELISA may be
constructed for measuring secreted or cell associated levels of
galectin 11 protein using monoclonal and polyclonal antibodies by
standard methods known in the art, and this can be used to discover
agents which may inhibit or enhance the production of galectin 11
(also called antagonist or agonist, respectively) from suitably
manipulated cells or tissues. Standard methods for conducting
screening assays are well understood in the art.
[0314] Examples of potential galectin 11 antagonists include
antibodies or, in some cases, oligonucleotides or proteins which
are closely related to the galectin 11 or its cognate ligand, e.g.,
a fragment of galectin 11 or galectin 11 ligand, or small molecules
which bind to the cognate ligand, but do not elicit a response, so
that the activity of the galectin 11 is prevented.
[0315] Thus in another aspect, the present invention relates to a
screening kit for identifying agonists, antagonists, ligands,
receptors, substrates, enzymes, etc. for galectin 11 polypeptides;
or compounds which decrease or enhance the production of galectin
11, which comprises:
[0316] (a) a galectin 11 polypeptide of the invention, such as, for
example, that of SEQ ID NO:2, 25, or 27;
[0317] (b) a cell expressing a galectin 11 ligand, such as, for
example, a T-cell;
[0318] (c) a cell membrane expressing a galectin 11 ligand,
preferably a membrane of a T-cell;
[0319] (d) a compound containing a .beta. galactoside sugar; or
[0320] (e) antibody to a galectin 11 polypeptide of the invention,
preferably that of SEQ ID NO: 2, 25, or 27.
[0321] It will be appreciated that in any such kit, (a), (b), (c),
(d), or (e) may comprise a substantial component.
[0322] Compounds identified via assays such as those described
herein, may be useful, for example, in elaborating the biological
function of the galectin 11 gene product and for regulating cell
growth, cell proliferation and differentiation, and apoptosis. For
example, antibodies against galectin 11 and galectin 11
polypeptides, fragments, derivatives, variants or analogs of the
invention may be employed to suppress galectin 11 activity to treat
abnormalities resulting from elevated galectin 11. The combination
of these identified compounds with a pharmaceutically acceptable
carrier (e.g., as described herein) and their administration to
treat or prevent growth regulatory and immunomodulatory disorders,
including, but not limited to, autoimmune diseases, cancer, and
inflammatory diseases, are also encompassed by the invention.
Prophylactic and Therapeutic Methods
[0323] It is to be understood that although the following
discussion is specifically directed to human patients, the
teachings are also applicable to any animal that expresses galectin
11.
[0324] As noted above, galectin 11 shares significant homology with
other galectins. Additionally, as disclosed herein, galectin 11,
like galectin 1 induces apoptosis of T-cell lines. Further, as
discussed above, galectin 1 has been demonstrated to play a role in
regulating cell proliferation and some immune functions (e.g.,
therapeutic activity against autoimmune diseases in experimental
myasthenia gravis and experimental autoimmune encephalomyelitis
animal model systems). Thus, it is likely that galectin 11, like
galectin 1, is active in modulating growth regulatory activities
(e.g., cell differentiation and/or cell proliferation),
immunomodulatory activity, cell-cell and cell-substrate
interactions, and apoptosis.
[0325] Apoptosis, or 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, or the inhibition of apoptosis, that could be
treated or detected by galectin 11 polynucleotides or polypeptides,
as well as antagonists or agonists of galectin 11, 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,
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) and viral infections (such as herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection. In
preferred embodiments, galectin 11 polynucleotides, polypeptides,
and/or antagonists of the invention are used to inhibit growth,
progression, and/or metasis of cancers, in particular those listed
above.
[0326] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by galectin 11
polynucleotides or polypeptides, or agonists or antagonists of
galectin 11, include, but are not limited to, 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)) 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.
[0327] Diseases associated with increased apoptosis that could be
treated or detected by galectin 11 polynucleotides or polypeptides,
as well as agonists or antagonists of galectin 11, include 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 (e.g., 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.
[0328] Any method which neutralizes or enhances galectin 11
activity can be used to modulate growth regulatory activities
(e.g., cell proliferation), immunomodulatory activity, cell-cell
and cell-substrate interactions, and apoptosis.
[0329] Galectin 11 polypeptides or polynucleotides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists and antagonists as described herein) may be
useful in treating deficiencies or disorders of the immune system,
by activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
The etiology of these immune deficiencies or disorders may be
genetic, somatic, such as cancer or some autoimmune disorders,
acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover, galectin 11 polynucleotides or polypeptides can be used
as a marker or detector of a particular immune system disease or
disorder.
[0330] Galectin 11 polynucleotides or polypeptides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists and antagonists as described herein) may be
useful in treating or detecting deficiencies or disorders of
hematopoietic cells. As further discussed below, galectin 11
polypeptides or polynucleotides or agonists or antagonists of
galectin 11, could be used to increase differentiation and
proliferation of hematopoietic cells, including the pluripotent
stem cells, in an effort to treat those disorders associated with a
decrease in certain (or many) types hematopoietic cells. Examples
of immunologic deficiency syndromes include, but are not limited
to: blood protein disorders (e.g. agammaglobulinemia,
dysgammaglobulinemia), ataxia telangiectasia, common variable
immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV
infection, leukocyte adhesion deficiency syndrome, lymphopenia,
phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia, or hemoglobinuria.
[0331] Moreover, galectin 11 polypeptides or polynucleotides
(including galectin 11 fragments, variants, derivatives, and
anaologs, and galectin 11 agonists and antagonists as described
herein) can also be used to modulate hemostatic (the stopping of
bleeding) or thrombolytic activity (clot formation). For example,
by increasing hemostatic or thrombolytic activity, galectin 11
polynucleotides or polypeptides could be used to treat blood
coagulation disorders (e.g., afibrinogenemia, factor deficiencies),
blood platelet disorders (e.g. thrombocytopenia), or wounds
resulting from trauma, surgery, or other causes. Alternatively,
galectin 11 polynucleotides or polypeptides, or agonists or
antagonists of galectin 11, that can decrease hemostatic or
thrombolytic activity could be used to inhibit or dissolve
clotting. These molecules could be important in the treatment of
heart attacks (infarction), strokes, or scarring.
[0332] Galectin 11 polynucleotides or polypeptides (including
galectin 11 fragments, variants, derivatives, and anaologs), and
galectin 11 agonists or antagonists (as described herein) may also
be useful in treating or detecting autoimmune disorders. As
disclosed herein, galectin 11 induces apoptosis of T-cell lines
(see Example 5, FIGS. 5A and 5B). 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 galectin 11 polypeptides or polynucleotides
that can inhibit an immune response, particularly the
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing autoimmune disorders.
[0333] Examples of autoimmune disorders that can be treated or
detected include, but are not limited to: Addison's Disease,
hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,
dermatitis, allergic encephalomyelitis, glomerulonephritis,
Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis,
Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease,
Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus
Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre
Syndrome, insulin dependent diabetes mellitis, and autoimmune
inflammatory eye disease.
[0334] Similarly, allergic reactions and conditions, such as asthma
particularly allergic asthma) or other respiratory problems, may
also be treated by galectin 11 polypeptides or polynucleotides, or
agonists or antagonists of galectin 11. Moreover, these molecules
can be used to treat anaphylaxis, hypersensitivity to an antigenic
molecule, or blood group incompatibility.
[0335] Galectin 11 polynucleotides or polypeptides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists or antagonists as described herein) may also
be used to treat and/or prevent organ rejection or
graft-versus-host disease (GVHD). 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 galectin 11
polypeptides or polynucleotides, or agonists or antagonists of
galectin 11, 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.
[0336] Similarly, galectin 11 polypeptides or polynucleotides
(including galectin 11 fragments, variants, derivatives, and
anaologs, and galectin 11 agonists or antagonists as described
herein) may also be used to modulate inflammation. For example,
galectin 11 polypeptides or polynucleotides, or agonists or
antagonists of galectin 11, may inhibit the proliferation and
differentiation of cells involved in an inflammatory response.
These molecules can be used to treat inflammatory conditions, both
chronic and acute conditions, including 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 resulting
from over production of cytokines (e.g., TNF or IL-1).
[0337] Galectin 11 polypeptides or polynucleotides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists and antagonists as described herein) can be
used to treat or detect hyperproliferative disorders, including
neoplasms. Galectin 11 polypeptides or polynucleotides, or agonists
or antagonists of galectin 11, may inhibit the proliferation of the
disorder through direct or indirect interactions. Alternatively,
galectin 11 polypeptides or polynucleotides, or agonists or
antagonists of galectin 11, may proliferate other cells which can
inhibit the hyperproliferative disorder.
[0338] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[0339] Examples of hyperproliferative disorders that can be treated
or detected by galectin 11 polynucleotides or polypeptides include,
but are not limited to, neoplasms located in the: colon, abdomen,
bone, breast, digestive system, liver, pancreas, prostate,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and peripheral), lymphatic system, pelvic, skin, soft
tissue, spleen, thoracic, and urogenital.
[0340] Similarly, other hyperproliferative disorders can also be
treated or detected by galectin 11 polynucleotides or polypeptides,
or agonists or antagonists of galectin 11. Examples of such
hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis,
and any other hyperproliferative disease, besides neoplasia,
located in an organ system listed above.
[0341] One preferred embodiment utilizes polynucleotides of the
present invention to inhibit aberrant cellular division, by gene
therapy using the present invention, and/or protein fusions or
fragments thereof.
[0342] Thus, the present invention provides a method for treating
cell proliferative disorders by inserting into an abnormally
proliferating cell a polynucleotide of the present invention,
wherein said polynucleotide represses said expression.
[0343] Another embodiment of the present invention provides a
method of treating cell-proliferative disorders in individuals
comprising administration of one or more active gene copies of the
present invention to an abnormally proliferating cell or cells. In
a preferred embodiment, polynucleotides of the present invention is
a DNA construct comprising a recombinant expression vector
effective in expressing a DNA sequence encoding said
polynucleotides. In another preferred embodiment of the present
invention, the DNA construct encoding the polynucleotides of the
present invention is inserted into cells to be treated utilizing a
retrovirus, or more preferrably an adenoviral vector (See G J.
Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated
by reference). In a most preferred embodiment, the viral vector is
defective and will not transform non-proliferating cells, only
proliferating cells. Moreover, in a preferred embodiment, the
polynucleotides of the present invention inserted into
proliferating cells either alone, or in combination with or fused
to other polynucleotides, can then be modulated via an external
stimulus (i.e. magnetic, specific small molecule, chemical, or drug
administration, etc.), which acts upon the promoter upstream of
said polynucleotides to induce expression of the encoded protein
product. As such the beneficial therapeutic affect of the present
invention may be expressly modulated (i.e. to increase, decrease,
or inhibit expression of the present invention) based upon said
external stimulus.
[0344] Polynucleotides of the present invention may be useful in
repressing expression of oncogenic genes or antigens. By
"repressing expression of the oncogenic genes" is intended the
suppression of the transcription of the gene, the degradation of
the gene transcript (pre-message RNA), the inhibition of splicing,
the destruction of the messenger RNA, the prevention of the
post-translational modifications of the protein, the destruction of
the protein, or the inhibition of the normal function of the
protein.
[0345] For local administration to abnormally proliferating cells,
polynucleotides of the present invention may be administered by any
method known to those of skill in the art including, but not
limited to transfection, electroporation, microinjection of cells,
or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any other method described throughout the
specification. The polynucleotide of the present invention may be
delivered by known gene delivery systems such as, but not limited
to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci.
U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems
(Yates et al., Nature 313:812 (1985)) known to those skilled in the
art. These references are exemplary only and are hereby
incorporated by reference. In order to specifically deliver or
transfect cells which are abnormally proliferating and spare
non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as described in the art and elsewhere herein) delivery
system known to those of skill in the art. Since host DNA
replication is required for retroviral DNA to integrate and the
retrovirus will be unable to self replicate due to the lack of the
retrovirus genes needed for its life cycle. Utilizing such a
retroviral delivery system for polynucleotides of the present
invention will target said gene and constructs to abnormally
proliferating cells and will spare the non-dividing normal
cells.
[0346] The polynucleotides of the present invention may be
delivered directly to cell proliferative disorder/disease sites in
internal organs, body cavities and the like by use of imaging
devices used to guide an injecting needle directly to the disease
site. The polynucleotides of the present invention may also be
administered to disease sites at the time of surgical
intervention.
[0347] By "cell proliferative disease" is meant any human or animal
disease or disorder, affecting any one or any combination of
organs, cavities, or body parts, which is characterized by single
or multiple local abnormal proliferations of cells, groups of
cells, or tissues, whether benign or malignant.
[0348] Any amount of the polynucleotides of the present invention
may be administered as long as it has a biologically inhibiting
effect on the proliferation of the treated cells. Moreover, it is
possible to administer more than one of the polynucleotide of the
present invention simultaneously to the same site. By "biologically
inhibiting" is meant partial or total growth inhibition as well as
decreases in the rate of proliferation or growth of the cells. The
biologically inhibitory dose may be determined by assessing the
effects of the polynucleotides of the present invention on target
malignant or abnormally proliferating cell growth in tissue
culture, tumor growth in animals and cell cultures, or any other
method known to one of ordinary skill in the art.
[0349] The present invention is further directed to antibody-based
therapies which involve administering of anti-polypeptides and
anti-polynucleotide antibodies to a mammalian, preferably human,
patient for treating one or more of the described disorders.
Methods for producing anti-polypeptides and anti-polynucleotide
antibodies polyclonal and monoclonal antibodies are described in
detail elsewhere herein. Such antibodies may be provided in
pharmaceutically acceptable compositions as known in the art or as
described herein.
[0350] 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.
[0351] In particular, the antibodies, fragments and derivatives of
the present invention are useful for treating a subject having or
developing cell proliferative and/or differentiation disorders as
described herein. Such treatment comprises administering a single
or multiple doses of the antibody, or a fragment, derivative, or a
conjugate thereof.
[0352] The antibodies of this invention may be advantageously
utilized in combination with other monoclonal or chimeric
antibodies, or with lymphokines or hematopoietic growth factors,
for example, which serve to increase the number or activity of
effector cells which interact with the antibodies.
[0353] 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, including fragments thereof. Preferred binding
affinities include those with a dissociation constant or Kd less
than 5.times.10.sup.-6M, 10.sup.-6M, 5.times.10.sup.-7M,
10.sup.-7M, 5.times.10.sup.-8M, 10.sup.-8M, 5.times.10.sup.-9M,
10.sup.-9M, 5.times.10.sup.-10M, 10.sup.-10M, 5.times.10.sup.-11M,
10.sup.-11M, 5.times.10.sup.-12M, 10.sup.-12M, 5.times.10.sup.-13M,
10.sup.-13M, 5.times.10.sup.-14M, 10.sup.-14M, 5.times.10.sup.-15M,
and 10.sup.15M.
[0354] Moreover, polypeptides of the present invention are useful
in inhibiting the angiogenesis of proliferative cells or tissues,
either alone, as a protein fusion, or in combination with other
polypeptides directly or indirectly, as described elsewhere herein.
In a most preferred embodiment, said anti-angiogenesis effect may
be achieved indirectly, for example, through the inhibition of
hematopoietic, tumor-specific cells, such as tumor-associated
macrophages (See Joseph I B, et al. J Natl Cancer Inst,
90(21):1648-53 (1998), which is hereby incorporated by reference).
Antibodies directed to polypeptides or polynucleotides of the
present invention may also result in inhibition of angiogenesis
directly, or indirectly (See Witte L, et al., Cancer Metastasis
Rev. 17(2):155-61 (1998), which is hereby incorporated by
reference)).
[0355] Polypeptides, including protein fusions, of the present
invention, or fragments thereof may be useful in inhibiting
proliferative cells or tissues through the induction of apoptosis.
Said polypeptides may act either directly, or indirectly to induce
apoptosis of proliferative cells and tissues, for example in the
activation of a death-domain receptor, such as tumor necrosis
factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related
apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See
Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998),
which is hereby incorporated by reference). Moreover, in another
preferred embodiment of the present invention, said polypeptides
may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or
through stimulating the expression of said proteins, either alone
or in combination with small molecule drugs or adjuviants, such as
apoptonin, galectins, thioredoxins, antiinflammatory proteins (See
for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.
50(5):423-33 (1998), Chem Biol Interact. Apr 24; 111-112:23-34
(1998), J Mol. Med. 76(6):402-12 (1998), Int J Tissue React;
20(1):3-15 (1998), which are all hereby incorporated by
reference).
[0356] Polypeptides, including protein fusions to, or fragments
thereof, of the present invention are useful in inhibiting the
metastasis of proliferative cells or tissues. Inhibition may occur
as a direct result of administering polypeptides, or antibodies
directed to said polypeptides as described elsewhere herein, or
indirectly, such as activating the expression of proteins known to
inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr
Top Microbiol Immunol 1998; 231:125-41, which is hereby
incorporated by reference). Such therapeutic affects of the present
invention may be achieved either alone, or in combination with
small molecule drugs or adjuvants.
[0357] In another embodiment, the invention provides a method of
delivering compositions containing the polypeptides of the
invention (e.g., compositions containing polypeptides or
polypeptide antibodies associated with heterologous polypeptides,
heterologous nucleic acids, toxins, or prodrugs) to targeted cells
expressing the polypeptide of the present invention. Polypeptides
or polypeptide antibodies of the invention may be associated with
heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent
interactions.
[0358] Polypeptides, protein fusions to, or fragments thereof, of
the present invention are useful in enhancing the immunogenicity
and/or antigenicity of proliferating cells or tissues, either
directly, such as would occur if the polypeptides of the present
invention `vaccinated` the immune response to respond to
proliferative antigens and immunogens, or indirectly, such as in
activating the expression of proteins known to enhance the immune
response (e.g. chemokines), to said antigens and immunogens.
[0359] Galectin 11 polypeptides or polynucleotides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists and antagonists as described herein) can be
used to treat or detect infectious agents. For example, by
increasing the immune response, particularly increasing the
proliferation and differentiation of B and/or T 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, galectin 11 polypeptides or
polynucleotides may also directly inhibit the infectious agent,
without necessarily eliciting an immune response.
[0360] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by
galectin 11 polynucleotides or polypeptides. Examples of viruses,
include, but are not limited to, the following DNA and RNA viral
families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Birnaviridae, 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, Picornaviridae, 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. Galectin 11 polypeptides or polynucleotides,
or agonists or antagonists of the invention, can be used to treat
or detect any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat: meningitis, Dengue,
EBV, and/or hepatitis (e.g., hepatitis B). In an additional
specific embodiment polynucleotides, polypeptides, or agonists or
antagonists of the invention are used to treat patients
nonresponsive to one or more other commercially available hepatitis
vaccines. In a further specific embodiment polynucleotides,
polypeptides, or agonists or antagonists of the invention are used
to treat AIDS.
[0361] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated or detected by galectin 11
polynucleotides or polypeptides and/or agonist or antagonists of
the present invention, include, but are not limited to, the
following Gram-Negative and Gram-positive bacterial families and
fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,
Norcardia), 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, Mycoplasmatales, Mycobacterium leprae,
Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea,
Menigococcal), Meisseria meningitidis, Pasteurellacea Infections
(e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza
type B), Pasteurella), Pseudomonas, Rickeftsiaceae, Chlamydiaceae,
Syphilis, Shigella spp., Staphylococcal, Meningiococcal,
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.,
mengitis 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. Galectin 11
polypeptides or polynucleotides, or agonists or antagonists of the
invention, can be used to treat or detect any of these symptoms or
diseases. In specific embodiments, polynucleotides, polypeptides,
agonists or antagonists of the invention are used to treat:
tetanus, Diptheria, botulism, and/or meningitis type B.
[0362] Moreover, parasitic agents causing disease or symptoms that
can be treated or detected by galectin 11 polynucleotides or
polypeptides, agonists or antagonists, include, but not limited to,
the following families: 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. Galectin 11 polypeptides or polynucleotides, or
agonists or antagonists, can be used to treat or detect any of
these symptoms or diseases. In specific embodiments,
polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat malaria.
[0363] Preferably, treatment using a polypeptide or polynucleotide
and/or agonist or antagonist of the present invention could either
be by administering an effective amount of a polypeptide to the
patient, or by removing cells from the patient, supplying the cells
with a polynucleotide of the present invention, and returning the
engineered cells to the patient (ex vivo therapy). Moreover, the
polypeptide or polynucleotide of the present invention can be used
as an antigen in a vaccine to raise an immune response against
infectious disease.
[0364] Galectin 11 polynucleotides or polypeptides (including
galectin 11 fragments, variants, derivatives, and anaologs, and
galectin 11 agonists and antagonists as described herein) can be
used to differentiate, proliferate, and attract cells, leading to
the regeneration of tissues. (See, Science 276:59-87 (1997).) The
regeneration of tissues could be used to repair, replace, or
protect tissue damaged by congenital defects, trauma (wounds,
burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery,
including cosmetic plastic surgery, fibrosis, reperfusion injury,
or systemic cytokine damage.
[0365] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac), vascular
(including vascular endothelium), nervous, hematopoietic, and
skeletal (bone, cartilage, tendon, and ligament) tissue.
Preferably, regeneration occurs without or decreased scarring.
Regeneration also may include angiogenesis.
[0366] Moreover, galectin 11 polynucleotides or polypeptides
(including galectin 11 fragments, variants, derivatives, and
anaologs, and galectin 11 agonists and antagonists as described
herein) may increase regeneration of tissues difficult to heal. For
example, increased tendon/ligament regeneration would quicken
recovery time after damage. Galectin 11 polynucleotides or
polypeptides, or agonists or antagonists of the present invention
could also be used prophylactically in an effort to avoid damage.
Specific diseases that could be treated include of tendinitis,
carpal tunnel syndrome, and other tendon or ligament defects. A
further example of tissue regeneration of non-healing wounds
includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
[0367] Similarly, nerve and brain tissue could also be regenerated
by using galectin 11 polynucleotides or polypeptides, or agonists
or antagonists of the present invention, to proliferate and
differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the galectin 11
polynucleotides or polypeptides or agonists or antagonists of
galectin 11.
[0368] Galectin 11 polynucleotides or polypeptides, or agonists or
antagonists of the present invention, may have chemotaxis activity.
A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or endothelial cells) to a particular site in the
body, such as inflammation, infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or
heal the particular trauma or abnormality.
[0369] Galectin 11 polynucleotides or polypeptides, or agonists or
antagonists of the present invention, may increase chemotaxic
activity of particular cells. These chemotactic molecules can then
be used to treat inflammation, infection, hyperproliferative
disorders, or any immune system disorder by increasing the number
of cells targeted to a particular location in the body. For
example, chemotaxic molecules can be used to treat wounds and other
trauma to tissues by attracting immune cells to the injured
location. Chemotactic molecules of the present invention can also
attract fibroblasts, which can be used to treat wounds.
[0370] It is also contemplated that galectin 11 polynucleotides or
polypeptides, or agonists or antagonists of the present invention,
may inhibit chemotactic activity. These molecules could also be
used to treat disorders. Thus, galectin 11 polynucleotides or
polypeptides, or agonists or antagonists of the present invention,
could be used as an inhibitor of chemotaxis.
[0371] Nervous system disorders, which can be treated with the
galectin 11 compositions of the invention (e.g., galectin 11
polypeptides, polynucleotides, and/or agonists or antagonists),
include, but are not limited to, nervous system injuries, and
diseases or disorders which result in either a disconnection of
axons, a diminution or degeneration of neurons, or demyelination.
Nervous system lesions which may be treated in a patient (including
human and non-human mammalian patients) according to the invention,
include but are not limited to, the following lesions of either the
central (including spinal cord, brain) or peripheral nervous
systems: (1) ischemic lesions, in which a lack of oxygen in a
portion of the nervous system results in neuronal injury or death,
including cerebral infarction or ischemia, or spinal cord
infarction or ischemia; (2) traumatic lesions, including lesions
caused by physical injury or associated with surgery, for example,
lesions which sever a portion of the nervous system, or compression
injuries; (3) malignant lesions, in which a portion of the nervous
system is destroyed or injured by malignant tissue which is either
a nervous system associated malignancy or a malignancy derived from
non-nervous system tissue; (4) infectious lesions, in which a
portion of the nervous system is destroyed or injured as a result
of infection, for example, by an abscess or associated with
infection by human immunodeficiency virus, herpes zoster, or herpes
simplex virus or with Lyme disease, tuberculosis, syphilis; (5)
degenerative lesions, in which a portion of the nervous system is
destroyed or injured as a result of a degenerative process
including but not limited to degeneration associated with
Parkinson's disease, Alzheimer's disease, Huntington's chorea, or
amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional diseases or disorders, in which a portion of the
nervous system is destroyed or injured by a nutritional disorder or
disorder of metabolism including but not limited to, vitamin B12
deficiency, folic acid deficiency, Wernicke disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary
degeneration of the corpus callosum), and alcoholic cerebellar
degeneration; (7) neurological lesions associated with systemic
diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma,
or sarcoidosis; (8) lesions caused by toxic substances including
alcohol, lead, or particular neurotoxins; and (9) demyelinated
lesions in which a portion of the nervous system is destroyed or
injured by a demyelinating disease including, but not limited to,
multiple sclerosis, human immunodeficiency virus-associated
myelopathy, transverse myelopathy or various etiologies,
progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[0372] In a preferred embodiment, the galectin 11 polypeptides,
polynucleotides, or agonists or antagonists of the present
invention are used to protect neural cells from the damaging
effects of cerebral hypoxia. According to this embodiment, the
galectin 11 compositions of the invention are used to treat or
prevent neural cell injury associated with cerebral hypoxia. In one
aspect of this embodiment, the galectin 11 polypeptides,
polynucleotides, or agonists or antagonists of the invention are
used to treat or prevent neural cell injury associated with
cerebral ischemia. In another aspect of this embodiment, the
galectin 11 polypeptides, polynucleotides, or agonists or
antagonists of the present invention are used to treat or prevent
neural cell injury associated with cerebral infarction. In another
aspect of this embodiment, the galectin 11 polypeptides,
polynucleotides, or agonists or antagonists of the present
invention are used to treat or prevent neural cell injury
associated with a stroke. In a further aspect of this embodiment,
the galectin 11 polypeptides, polynucleotides, or agonists or
antagonists of the present invention are used to treat or prevent
neural cell injury associated with a heart attack.
[0373] The compositions of the invention which are useful for
treating or preventing a nervous system disorder may be selected by
testing for biological activity in promoting the survival or
differentiation of neurons. For example, and not by way of
limitation, galectin 11 compositions of the invention which elicit
any of the following effects may be useful according to the
invention: (1) increased survival time of neurons in culture; (2)
increased sprouting of neurons in culture or in vivo; (3) increased
production of a neuron-associated molecule in culture or in vivo,
e.g., choline acetyltransferase or acetylcholinesterase with
respect to motor neurons; or (4) decreased symptoms of neuron
dysfunction in vivo. Such effects may be measured by any method
known in the art. In preferred, non-limiting embodiments, increased
survival of neurons may routinely be measured using a method set
forth herein or otherwise known in the art, such as, for example,
the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515
(1990)); increased sprouting of neurons may be detected by methods
known in the art, such as, for example, the methods set forth in
Pestronk et al. (Exp. Neurol. 70:65-82 (1980)) or Brown et al.
(Ann. Rev. Neurosci. 4:17-42 (1981)); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic
assay, antibody binding, Northern blot assay, etc., using
techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing
the physical manifestation of motor neuron disorder, e.g.,
weakness, motor neuron conduction velocity, or functional
disability.
[0374] In specific embodiments, motor neuron disorders that may be
treated according to the invention include, but are not limited to,
disorders such as infarction, infection, exposure to toxin, trauma,
surgical damage, degenerative disease or malignancy that may affect
motor neurons as well as other components of the nervous system, as
well as disorders that selectively affect neurons such as
amyotrophic lateral sclerosis, and including, but not limited to,
progressive spinal muscular atrophy, progressive bulbar palsy,
primary lateral sclerosis, infantile and juvenile muscular atrophy,
progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary
Motorsensory Neuropathy (Charcot-Marie-Tooth Disease). Additional
examples of neurologic diseases which can be treated or detected
with polynucleotides, polypeptides, agonists, and/or antagonists of
the present invention include brain diseases, such as metabolic
brain diseases which includes phenylketonuria such as maternal
phenylketonuria, pyruvate carboxylase deficiency, pyruvate
dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain
edema, brain neoplasms such as cerebellar neoplasms which include
infratentorial neoplasms, cerebral ventricle neoplasms such as
choroid plexus neoplasms, hypothalamic neoplasms, supratentorial
neoplasms, canavan disease, cerebellar diseases such as cerebellar
ataxia which include spinocerebellar degeneration such as ataxia
telangiectasia, cerebellar dyssynergia, Friederich's Ataxia,
Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar
neoplasms such as infratentorial neoplasms, diffuse cerebral
sclerosis such as encephalitis periaxialis, globoid cell
leukodystrophy, metachromatic leukodystrophy and subacute
sclerosing panencephalitis, cerebrovascular disorders (such as
carotid artery diseases which include carotid artery thrombosis,
carotid stenosis and Moyamoya Disease, cerebral amyloid angiopathy,
cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral arteriovenous malformations, cerebral artery diseases,
cerebral embolism and thrombosis such as carotid artery thrombosis,
sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage
such as epidural hematoma, subdural hematoma and subarachnoid
hemorrhage, cerebral infarction, cerebral ischemia such as
transient cerebral ischemia, Subclavian Steal Syndrome and
vertebrobasilar insufficiency, vascular dementia such as
multi-infarct dementia, periventricular leukomalacia, vascular
headache such as cluster headache, migraine, dementia such as AIDS
Dementia Complex, presenile dementia such as Alzheimer's Disease
and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's
Disease and progressive supranuclear palsy, vascular dementia such
as multi-infarct dementia, encephalitis which include encephalitis
periaxialis, viral encephalitis such as epidemic encephalitis,
Japanese Encephalitis, St. Louis Encephalitis, tick-borne
encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meningoencephalitis such as
uveomeningoencephalitic syndrome, Postencephalitic Parkinson
Disease and subacute sclerosing panencephalitis, encephalomalacia
such as periventricular leukomalacia, epilepsy such as generalized
epilepsy which includes infantile spasms, absence epilepsy,
myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic
epilepsy, partial epilepsy such as complex partial epilepsy,
frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua,
Hallervorden-Spatz Syndrome, hydrocephalus such as Dandy-Walker
Syndrome and normal pressure hydrocephalus, hypothalamic diseases
such as hypothalamic neoplasms, cerebral malaria, narcolepsy which
includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett
Syndrome, Reye's Syndrome, thalamic diseases, cerebral
toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome,
central nervous system infections such as AIDS Dementia Complex,
Brain Abscess, subdural empyema, encephalomyelitis such as Equine
Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing
Hemorrhagic Encephalomyelitis, Visna, cerebral malaria, meningitis
such as arachnoiditis, aseptic meningtitis such as viral
meningtitis which includes lymphocytic choriomeningitis. Bacterial
meningtitis which includes Haemophilus Meningtitis, Listeria
Meningtitis, Meningococcal Meningtitis such as
Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and
meningeal tuberculosis, fungal meningitis such as Cryptococcal
Meningtitis, subdural effusion, meningoencephalitis such as
uvemeningoencephalitic syndrome, myelitis such as transverse
myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion
diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform
Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie)
cerebral toxoplasmosis, central nervous system neoplasms such as
brain neoplasms that include cerebellear neoplasms such as
infratentorial neoplasms, cerebral ventricle neoplasms such as
choroid plexus neoplasms, hypothalamic neoplasms and supratentorial
neoplasms, meningeal neoplasms, spinal cord neoplasms which include
epidural neoplasms, demyelinating diseases such as Canavan
Diseases, diffuse cerebral sceloris which includes
adrenoleukodystrophy, encephalitis periaxialis, globoid cell
leukodystrophy, diffuse cerebral sclerosis such as metachromatic
leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic
encephalomyelitis, progressive multifocal leukoencephalopathy,
multiple sclerosis, central pontine myelinolysis, transverse
myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue
Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal
cord diseases such as amyotonia congenita, amyotrophic lateral
sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann
Disease, spinal cord compression, spinal cord neoplasms such as
epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man
Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat
Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such
as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease,
Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome,
Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such
as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal
syndrome, phenylketonuria such as maternal phenylketonuria,
Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome,
Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities
such as holoprosencephaly, neural tube defects such as anencephaly
which includes hydrangencephaly, Arnold-Chairi Deformity,
encephalocele, meningocele, meningomyelocele, spinal dysraphism
such as spina bifida cystica and spina bifida occulta, hereditary
motor and sensory neuropathies which include Charcot-Marie Disease,
Hereditary optic atrophy, Refsum's Disease, hereditary spastic
paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and
Autonomic Neuropathies such as Congenital Analgesia and Familial
Dysautonomia, Neurologic manifestations (such as agnosia that
include Gerstmann's Syndrome, Amnesia such as retrograde amnesia,
apraxia, neurogenic bladder, cataplexy, communicative disorders
such as hearing disorders that includes deafness, partial hearing
loss, loudness recruitment and tinnitus, language disorders such as
aphasia which include agraphia, anomia, broca aphasia, and Wernicke
Aphasia, Dyslexia such as Acquired Dyslexia, language development
disorders, speech disorders such as aphasia which includes anomia,
broca aphasia and Wernicke Aphasia, articulation disorders,
communicative disorders such as speech disorders which include
dysarthria, echolalia, mutism and stuttering, voice disorders such
as aphonia and hoarseness, decerebrate state, delirium,
fasciculation, hallucinations, meningism, movement disorders such
as angelman syndrome, ataxia, athetosis, chorea, dystonia,
hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and
tremor, muscle hypertonia such as muscle rigidity such as stiff-man
syndrome, muscle spasticity, paralysis such as facial paralysis
which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia,
opthalmoplegia such as diplopia, Duane's Syndrome, Horner's
Syndrome, Chronic progressive external opthalmoplegia such as
Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis,
Paraplegia such as Brown-Sequard Syndrome, quadriplegia,
respiratory paralysis and vocal cord paralysis, paresis, phantom
limb, taste disorders such as ageusia and dysgeusia, vision
disorders such as amblyopia, blindness, color vision defects,
diplopia, hemianopsia, scotoma and subnormal vision, sleep
disorders such as hypersomnia which includes Kleine-Levin Syndrome,
insomnia, and somnambulism, spasm such as trismus, unconsciousness
such as coma, persistent vegetative state and syncope and vertigo,
neuromuscular diseases such as amyotonia congenita, amyotrophic
lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron
disease, muscular atrophy such as spinal muscular atrophy,
Charcot-Marie Disease and Werdnig-Hoffmann Disease,
Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis,
Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial
Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic
Paraparesis and Stiff-Man Syndrome, peripheral nervous system
diseases such as acrodynia, amyloid neuropathies, autonomic nervous
system diseases such as Adie's Syndrome, Barre-Lieou Syndrome,
Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic
Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as
Acoustic Nerve Diseases such as Acoustic Neuroma which includes
Neurofibromatosis 2, Facial Nerve Diseases such as Facial
Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders
which includes amblyopia, nystagmus, oculomotor nerve paralysis,
opthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic
Progressive External Opthalmoplegia which includes Kearns Syndrome,
Strabismus such as Esotropia and Exotropia, Oculomotor Nerve
Paralysis, Optic Nerve Diseases such as Optic Atrophy which
includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic
Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal
Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as
Neuromyelitis Optica and Swayback, Diabetic neuropathies such as
diabetic foot, nerve compression syndromes such as carpal tunnel
syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as
cervical rib syndrome, ulnar nerve compression syndrome, neuralgia
such as causalgia, cervico-brachial neuralgia, facial neuralgia and
trigeminal neuralgia, neuritis such as experimental allergic
neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and
radiculities such as polyradiculitis, hereditary motor and sensory
neuropathies such as Charcot-Marie Disease, Hereditary Optic
Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic
Neuropathies which include Congenital Analgesia and Familial
Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and
Tetany).
[0375] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing galectin 11
polynucleotides or polypeptides, as well as agonists or antagonists
of galectin 11, for therapeutic purposes, for example, to stimulate
epithelial cell proliferation and basal keratinocytes for the
purpose of wound healing, and to stimulate hair follicle production
and healing of dermal wounds. Galectin 11 polynucleotides or
polypeptides, as well as agonists or antagonists of galectin 11,
may be clinically useful in stimulating wound healing including
surgical wounds, excisional wounds, deep wounds involving damage of
the dermis and epidermis, eye tissue wounds, dental tissue wounds,
oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers,
arterial ulcers, venous stasis ulcers, burns resulting from heat
exposure or chemicals, and other abnormal wound healing conditions
such as uremia, malnutrition, vitamin deficiencies and
complications associated with systemic treatment with steroids,
radiation therapy and antineoplastic drugs and antimetabolites.
Galectin 11 polynucleotides or polypeptides, as well as agonists or
antagonists of galectin 11, could be used to promote dermal
reestablishment subsequent to dermal loss
[0376] Galectin 11 polynucleotides or polypeptides, as well as
agonists or antagonists of galectin 11, could be used to increase
the adherence of skin grafts to a wound bed and to stimulate
re-epithelialization from the wound bed. The following are types of
grafts that galectin 11 polynucleotides or polypeptides, agonists
or antagonists of galectin 11, could be used to increase adherence
to a wound bed: autografts, artificial skin, allografts, autodermic
graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts,
bone graft, brephoplastic grafts, cutis graft, delayed graft,
dermic graft, epidermic graft, fascia graft, full thickness graft,
heterologous graft, xenograft, homologous graft, hyperplastic
graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch
graft, omenpal graft, patch graft, pedicle graft, penetrating
graft, split skin graft, thick split graft. Galectin 11
polynucleotides or polypeptides, as well as agonists or antagonists
of galectin 11, can be used to promote skin strength and to improve
the appearance of aged skin.
[0377] It is believed that galectin 11 polynucleotides or
polypeptides, as well as agonists or antagonists of galectin 11,
will also produce changes in hepatocyte proliferation, and
epithelial cell proliferation in the lung, breast, pancreas,
stomach, small intestine, and large intestine. Galectin 11
polynucleotides or polypeptides, as well as agonists or antagonists
of galectin 11, could promote proliferation of epithelial cells
such as sebocytes, hair follicles, hepatocytes, type H pneumocytes,
mucin-producing goblet cells, and other epithelial cells and their
progenitors contained within the skin, lung, liver, and
gastrointestinal tract. Galectin 11 polynucleotides or
polypeptides, agonists or antagonists of galectin 11, may promote
proliferation of endothelial cells, keratinocytes, and basal
keratinocytes.
[0378] Galectin 11 polynucleotides or polypeptides, as well as
agonists or antagonists of galectin 11, could also be used to
reduce the side effects of gut toxicity that result from radiation,
chemotherapy treatments or viral infections. Galectin 11
polynucleotides or polypeptides, as well as agonists or antagonists
of galectin 11, may have a cytoprotective effect on the small
intestine mucosa. Galectin 11 polynucleotides or polypeptides, as
well as agonists or antagonists of galectin 11, may also stimulate
healing of mucositis (mouth ulcers) that result from chemotherapy
and viral infections.
[0379] Galectin 11 polynucleotides or polypeptides, as well as
agonists or antagonists of galectin 11, could further be used in
full regeneration of skin in full and partial thickness skin
defects, including burns, (i.e., repopulation of hair follicles,
sweat glands, and sebaceous glands), treatment of other skin
defects such as psoriasis. Galectin 11 polynucleotides or
polypeptides, as well as agonists or antagonists of galectin 11,
could be used to treat epidermolysis bullosa, a defect in adherence
of the epidermis to the underlying dermis which results in
frequent, open and painful blisters by accelerating
reepithelialization of these lesions. Galectin 11 polynucleotides
or polypeptides, as well as agonists or antagonists of galectin 11,
could also be used to treat gastric and doudenal ulcers and help
heal by scar formation of the mucosal lining and regeneration of
glandular mucosa and duodenal mucosal lining more rapidly.
Inflammatory bowel diseases, such as Crohn's disease and ulcerative
colitis, are diseases which result in destruction of the mucosal
surface of the small or large intestine, respectively. Thus,
galectin 11 polynucleotides or polypeptides, as well as agonists or
antagonists of galectin 11, could be used to promote the
resurfacing of the mucosal surface to aid more rapid healing and to
prevent progression of inflammatory bowel disease. Treatment with
galectin 11 polynucleotides or polypeptides, agonists or
antagonists of galectin 11, is expected to have a significant
effect on the production of mucus throughout the gastrointestinal
tract and could be used to protect the intestinal mucosa from
injurious substances that are ingested or following surgery.
Galectin 11 polynucleotides or polypeptides, as well as agonists or
antagonists of galectin 11, could be used to treat diseases
associate with the under expression of galectin 11.
[0380] Moreover, galectin 11 polynucleotides or polypeptides, as
well as agonists or antagonists of galectin 11, could be used to
prevent and heal damage to the lungs due to various pathological
states. A growth factor such as galectin 11 polynucleotides or
polypeptides, as well as agonists or antagonists of galectin 11,
which could stimulate proliferation and differentiation and promote
the repair of alveoli and bronchiolar epithelium to prevent or
treat acute or chronic lung damage. For example, emphysema, which
results in the progressive loss of aveoli, and inhalation injuries,
i.e., resulting from smoke inhalation and burns, that cause
necrosis of the bronchiolar epithelium and alveoli could be
effectively treated using galectin 11 polynucleotides or
polypeptides, agonists or antagonists of galectin 11. Also,
galectin 11 polynucleotides or polypeptides, as well as agonists or
antagonists of galectin 11, could be used to stimulate the
proliferation of and differentiation of type II pneumocytes, which
may help treat or prevent disease such as hyaline membrane
diseases, such as infant respiratory distress syndrome and
bronchopulmonary displasia, in premature infants.
[0381] Galectin 11 polynucleotides or polypeptides, as well as
agonists or antagonists of galectin 11, could stimulate the
proliferation and differentiation of hepatocytes and, thus, could
be used to alleviate or treat liver diseases and pathologies such
as fulminant liver failure caused by cirrhosis, liver damage caused
by viral hepatitis and toxic substances (i.e., acetaminophen,
carbon tetraholoride and other hepatotoxins known in the art).
[0382] In addition, galectin 11 polynucleotides or polypeptides, as
well as agonists or antagonists of galectin 11, could be used treat
or prevent the onset of diabetes mellitus. In patients with newly
diagnosed Types I and II diabetes, where some islet cell function
remains, galectin 11 polynucleotides or polypeptides, as well as
agonists or antagonists of galectin 11, could be used to maintain
the islet function so as to alleviate, delay or prevent permanent
manifestation of the disease. Also, galectin 11 polynucleotides or
polypeptides, as well as agonists or antagonists of galectin 11,
could be used as an auxiliary in islet cell transplantation to
improve or promote islet cell function.
[0383] Galectin 11 polynucleotides or polypeptides, or agonists or
antagonists of galectin 11, encoding galectin 11 may be used to
treat cardiovascular disorders, including peripheral artery
disease, such as limb ischemia.
[0384] 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.
[0385] 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.
[0386] 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.
[0387] Heart valve disease 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.
[0388] 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.
[0389] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0390] 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, and venous insufficiency.
[0391] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0392] 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.
[0393] 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,
subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar insufficiency.
[0394] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[0395] 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.
[0396] Galectin 11 polynucleotides or polypeptides, or agonists or
antagonists of galectin 11, are especially effective for the
treatment of critical limb ischemia and coronary disease. Galectin
11 polypeptides may be administered using any method known in the
art, including, but not limited to, direct needle injection at the
delivery site, intravenous injection, topical administration,
catheter infusion, biolistic injectors, particle accelerators,
gelfoam sponge depots, other commercially available depot
materials, osmotic pumps, oral or suppositorial solid
pharmaceutical formulations, decanting or topical applications
during surgery, aerosol delivery. Such methods are known in the
art. Galectin 11 polypeptides may be administered as part of a
Therapeutic, described in more detail below. Methods of delivering
galectin 11 polynucleotides are described in more detail
herein.
[0397] The naturally occurring balance between endogenous
stimulators and inhibitors of angiogenesis is one in which
inhibitory influences predominate. 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).
[0398] The present invention provides for treatment of diseases or
disorders associated with neovascularization by administration of
the polynucleotides and/or polypeptides of the invention, as well
as agonists or antagonists of the present invention. Malignant and
metastatic conditions which can be treated with the polynucleotides
and polypeptides, or agonists or antagonists of the invention
include, but are not limited to, 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)). Thus, the present
invention provides a method of treating an angiogenesis-related
disease and/or disorder, comprising administering to an individual
in need thereof a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist of the
invention. For example, polynucleotides, polypeptides, antagonists
and/or agonists of the present invention may be utilized in a
variety of additional methods in order to therapeutically treat a
cancer or tumor. Cancers which may be treated with polynucleotides,
polypeptides, antagonists and/or agonists of the present invention
include, but are not limited to solid tumors, including prostate,
lung, breast, ovarian, stomach, pancreas, larynx, esophagus,
testes, liver, parotid, biliary tract, colon, rectum, cervix,
uterus, endometrium, kidney, bladder, thyroid cancer; primary
tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma;
leiomyosarcoma; non-small cell lung cancer; colorectal cancer;
advanced malignancies; and blood born tumors such as leukemias. For
example, polynucleotides, polypeptides, antagonists and/or agonists
of the present invention may be delivered topically, in order to
treat cancers such as skin cancer, head and neck tumors, breast
tumors, and Kaposi's sarcoma.
[0399] Within yet other aspects, polynucleotides, polypeptides,
antagonists and/or agonists of the present invention may be
utilized to treat superficial forms of bladder cancer by, for
example, intravesical administration. Polynucleotides,
polypeptides, antagonists and/or agonists of the present invention
may be delivered directly into the tumor, or near the tumor site,
via injection or a catheter. Of course, as the artisan of ordinary
skill will appreciate, the appropriate mode of administration will
vary according to the cancer to be treated. Other modes of delivery
are discussed herein.
[0400] Polynucleotides, polypeptides, antagonists and/or agonists
of the present invention may be useful in treating other disorders,
besides cancers, which involve angiogenesis. These disorders
include, but are not limited to: benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; artheroscleric plaques; ocular angiogenic
diseases, for example, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye; rheumatoid arthritis; psoriasis; delayed wound healing;
endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular
adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and
atherosclerosis.
[0401] For example, within one aspect of the present invention,
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering a polynucleotide, polypeptide,
antagonist and/or agonist of the invention to a hypertrophic scar
or keloid.
[0402] Within one embodiment of the polynucleotides, polypeptides,
antagonists and/or agonists of the present invention are injected
directly into a hypertrophic scar or keloid, in order to prevent
the progression of these lesions. This therapy is of particular
value in the prophylactic treatment of conditions which are known
to result in the development of hypertrophic scars and keloids
(e.g., burns), and is preferably initiated after the proliferative
phase has had time to progress (approximately 14 days after the
initial injury), but before hypertrophic scar or keloid
development. As noted above, the present invention also provides
methods for treating neovascular diseases of the eye, including for
example, corneal neovascularization, neovascular glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and
macular degeneration.
[0403] Moreover, Ocular disorders associated with
neovascularization which can be treated with the polynucleotides
and polypeptides of the present invention (including agonists
and/or 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).
[0404] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a compound (as
described above) to the cornea, such that the formation of blood
vessels is inhibited. Briefly, the cornea is a tissue which
normally lacks blood vessels. In certain pathological conditions
however, capillaries may extend into the cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also becomes clouded, resulting in a decline in
the patient's visual acuity. Visual loss may become complete if the
cornea completely opacitates. A wide variety of disorders can
result in corneal neovascularization, including for example,
corneal infections (e.g., trachoma, herpes simplex keratitis,
leishmaniasis and onchocerciasis), immunological processes (e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns,
trauma, inflammation (of any cause), toxic and nutritional
deficiency states, and as a complication of wearing contact
lenses.
[0405] Within particularly preferred embodiments of the invention,
may be prepared for topical administration in saline (combined with
any of the preservatives and antimicrobial agents commonly used in
ocular preparations), and administered in eyedrop form. The
solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic
compositions, prepared as described above, may also be administered
directly to the cornea. Within preferred embodiments, the
anti-angiogenic composition is prepared with a muco-adhesive
polymer which binds to cornea. Within further embodiments, the
anti-angiogenic factors or anti-angiogenic compositions may be
utilized as an adjunct to conventional steroid therapy. Topical
therapy may also be useful prophylactically in corneal lesions
which are known to have a high probability of inducing an
angiogenic response (such as chemical burns). In these instances
the treatment, likely in combination with steroids, may be
instituted immediately to help prevent subsequent
complications.
[0406] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself.
[0407] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of a
polynucleotide, polypeptide, antagonist and/or agonist of the
present invention to the eye, such that the formation of blood
vessels is inhibited. In one embodiment, the compound may be
administered topically to the eye in order to treat early forms of
neovascular glaucoma. Within other embodiments, the compound may be
implanted by injection into the region of the anterior chamber
angle. Within other embodiments, the compound may also be placed in
any location such that the compound is continuously released into
the aqueous humor. Within another aspect of the present invention,
methods are provided for treating proliferative diabetic
retinopathy, comprising the step of administering to a patient a
therapeutically effective amount of a polynucleotide, polypeptide,
antagonist and/or agonist to the eyes, such that the formation of
blood vessels is inhibited.
[0408] Within particularly preferred embodiments of the invention,
proliferative diabetic retinopathy may be treated by injection into
the aqueous humor or the vitreous, in order to increase the local
concentration of the polynucleotide, polypeptide, antagonist and/or
agonist in the retina. Preferably, this treatment should be
initiated prior to the acquisition of severe disease requiring
photocoagulation.
[0409] Within another aspect of the present invention, methods are
provided for treating retrolental fibroplasia, comprising the step
of administering to a patient a therapeutically effective amount of
a polynucleotide, polypeptide, antagonist and/or agonist of the
present invention to the eye, such that the formation of blood
vessels is inhibited. The compound may be administered topically,
via intravitreous injection and/or via intraocular implants.
[0410] Additionally, disorders which can be treated with the
polynucleotides, polypeptides, agonists and/or agonists of the
present invention 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.
[0411] Moreover, disorders and/or states, which can be treated with
be treated with the polynucleotides, polypeptides, agonists and/or
agonists of the present invention include, but are not limited to,
solid tumors, blood born tumors such as leukemias, tumor
metastasis, Kaposi's sarcoma, benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular
angiogenic diseases, for example, diabetic retinopathy, retinopathy
of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and uvietis, delayed wound healing, endometriosis,
vascluogenesis, granulations, hypertrophic scars (keloids),
nonunion fractures, scleroderma, trachoma, vascular adhesions,
myocardial angiogenesis, coronary collaterals, cerebral
collaterals, arteriovenous malformations, ischemic limb
angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia, hemophiliac joints, angiofibroma fibromuscular
dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control agent by preventing vascularization required for
embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch
disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and bacillary angiomatosis.
[0412] In one aspect of the birth control method, an amount of the
compound sufficient to block embryo implantation is administered
before or after intercourse and fertilization have occurred, thus
providing an effective method of birth control, possibly a "morning
after" method. Polynucleotides, polypeptides, agonists and/or
agonists of the present invention may also be used in controlling
menstruation or administered as either a peritoneal lavage fluid or
for peritoneal implantation in the treatment of endometriosis.
[0413] Polynucleotides, polypeptides, agonists and/or agonists of
the present invention may be incorporated into surgical sutures in
order to prevent stitch granulomas.
[0414] Polynucleotides, polypeptides, agonists and/or agonists of
the present invention may be utilized in a wide variety of surgical
procedures. For example, within one aspect of the present invention
a compositions (in the form of, for example, a spray or film) may
be utilized to coat or spray an area prior to removal of a tumor,
in order to isolate normal surrounding tissues from malignant
tissue, and/or to prevent the spread of disease to surrounding
tissues. Within other aspects of the present invention,
compositions (e.g., in the form of a spray) may be delivered via
endoscopic procedures in order to coat tumors, or inhibit
angiogenesis in a desired locale. Within yet other aspects of the
present invention, surgical meshes which have been coated with
anti-angiogenic compositions of the present invention may be
utilized in any procedure wherein a surgical mesh might be
utilized. For example, within one embodiment of the invention a
surgical mesh laden with an anti-angiogenic composition may be
utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[0415] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering a polynucleotide, polypeptide, agonist and/or agonist
of the present invention to the resection margins of a tumor
subsequent to excision, such that the local recurrence of cancer
and the formation of new blood vessels at the site is inhibited.
Within one embodiment of the invention, the anti-angiogenic
compound is administered directly to the tumor excision site (e.g.,
applied by swabbing, brushing or otherwise coating the resection
margins of the tumor with the anti-angiogenic compound).
Alternatively, the anti-angiogenic compounds may be incorporated
into known surgical pastes prior to administration. Within
particularly preferred embodiments of the invention, the
anti-angiogenic compounds are applied after hepatic resections for
malignancy, and after neurosurgical operations.
[0416] Within one aspect of the present invention, polynucleotides,
polypeptides, agonists and/or agonists of the present invention may
be administered to the resection margin of a wide variety of
tumors, including for example, breast, colon, brain and hepatic
tumors. For example, within one embodiment of the invention,
anti-angiogenic compounds may be administered to the site of a
neurological tumor subsequent to excision, such that the formation
of new blood vessels at the site are inhibited.
[0417] The polynucleotides, polypeptides, agonists and/or agonists
of the present invention may also be administered along with other
anti-angiogenic factors. Representative examples of other
anti-angiogenic factors include: Anti-Invasive Factor, retinoic
acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor
of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,
Plasminogen Activator Inhibitor-1, Plasminogen Activator
Inhibitor-2, and various forms of the lighter "d group" transition
metals.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include 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
(Tomkinson 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); Thalidomide;
Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
[0422] Thus, in one aspect, the present invention is directed to a
method for enhancing apoptosis, cell proliferation, cell
differentiation, or other cell growth activity regulated by
galectin 11, which involves administering to an individual in need
of an increased level of galectin 11 functional or biological
activity, a therapeutically effective amount of galectin 11
polypeptide, fragment, variant, derivative, or analog, or an
agonist capable of increasing galectin 11 mediated cellular
responses. In specific embodiments, galectin 11 mediated signaling
is increased to treat a disease wherein decreased apoptosis is
exhibited.
[0423] Given the activities modulated by galectin 11, it is readily
apparent that a substantially altered (increased or decreased)
level of expression of galectin 11 in an individual compared to the
standard or "normal" level produces pathological conditions such as
those described above. It will also be appreciated by one of
ordinary skill that the galectin 11 polypeptides of the invention
will exert its modulating activities on any of its target cells.
Therefore, it will be appreciated that conditions caused by a
decrease in the standard or normal level of galectin 11 activity in
an individual, can be treated by administration of galectin 11
protein or an agonist thereof.
[0424] In addition to treating diseases associated with elevated or
decreased levels of galectin 11 activity, the invention encompasses
methods of administering galectin 11 polypeptides or
polynucleotides (including fragments, variants, derivatives and
analogs, and agonists and antagonists as described herein) to
elevate galectin 11 associated biological activity.
[0425] For example, any method which elevates galectin 11
concentration and/or activity can be used to stimulate
hematopoiesis. Using these methods, the galectin 11 polypeptide and
nucleotide sequences described herein may be used to stimulate
hematopoiesis. In a specific embodiment, galectin 11 polypeptides
and polynucleotides are used in erythropoietin therapy, which is
directed toward supplementing the oxygen carrying capacity of
blood. Galectin 11 treatment within the scope of the invention
includes, but is not limited, to 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 hemophilia, cystic
fibrosis, pregnancy, menstrual disorders, early anemia of
prematurity, spinal cord injury, space flight, 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.
[0426] The invention also encompasses combining the galectin 11
polypeptides and polynucleotides described herein with other
proposed or conventional hematopoietic therapies. Thus, for
example, galectin 11 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 with compounds
generally used to treat aplastic anemia, such as methenolene,
stanozolol, and nandrolone; to treat iron-deficiency anemia, such
as iron preparations; to treat malignant anemia, such as vitamin
B12 and/or folic acid; and to treat hemolytic anemia, such as
adrenocortical steroids, e.g., corticoids. See e.g., Resegofti et
al., 1981, Pamminerva Medica, 23:243-248; Kurtz, 1982, FEBS
Letters, 14a:105-108; McGonigle et al., 1984, Kidney Int.,
25:437-444; and Pavlovic-Kantera, 1980, Expt. Hematol., 8(supp. 8)
283-291.
[0427] 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);
Weiland et al., 1982, Blut, 44:173-175; Kalmani, 1982, Kidney Int.,
22:383-391; Shahidi, 1973, New Eng. J. Med., 289:72-80; Urabe et
al., 1979, J. Exp. Med., 149:1314-1325; Billat et al., 1982, Expt.
Hematol., 10:133-140; Naughton et al., 1983, Acta Haemat,
69:171-179; 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.
[0428] 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 galectin 11 to a patient. The galectin 11 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 B12, folic
acid and/or adrenocortical steroids. The galectin 11 and
cotreatment drug(s) are suitably delivered by separate or by the
same administration route, and at the same or at different times,
depending, e.g., on dosing, the clinical condition of the patient,
etc.
[0429] For treating abnormal conditions related to an
under-expression of galectin 11 and its activity, or in which
elevated or decreased levels of galectin 11 are desired, several
approaches are available. One approach comprises administering to
an individual in need of an increased level of galectin 11 in the
body, a therapeutically effective amount of an isolated galectin 11
polypeptide, fragment, variant, derivative or analog of the
invention, or a compound which activates galectin 11, i.e., an
agonist as described above, optionally in combination with a
pharmaceutically acceptable carrier. Alternatively, gene therapy
may be employed to effect the endogenous production of galectin 11
by the relevant cells in the subject. For example, a polynucleotide
of the invention may be engineered for expression in a replication
defective retroviral vector using techniques known in the art. The
retroviral expression construct may then be isolated and introduced
into a packaging cell transduced with a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention such
that the packaging cell now produces infectious viral particles
containing the gene of interest. These producer cells may be
administered to a subject for engineering cells in vivo and
expression of the polypeptide in vivo. For a overview of gene
therapy, see Chapter 20, Gene Therapy and other Molecular
Genetic-based Therapeutic Approaches, (and references cited
therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS
Scientific Publishers Ltd (1996).
[0430] Further, treatment can be administered, for example, in the
form of gene replacement therapy. Specifically, one or more copies
of a galectin 11 nucleotide sequence of the invention that directs
the production of a galectin 11 gene product exhibiting normal
function, may be inserted into the appropriate cells within a
patient or animal subject, using vectors which include, but are not
limited to, adenovirus, adeno-associated virus, retrovirus and
herpesvirus vectors, in addition to other particles that introduce
DNA into cells, such as liposomes and gene activated matrices.
Because the galectin 11 gene is expressed in neutrophils, such gene
replacement techniques should be capable of delivering galectin 11
gene sequence to these cells within patients, or, alternatively,
should involve direct administration of such galectin 11
polynucleotide sequences to the site of the cells in which the
galectin 11 gene sequences are to be expressed. Alternatively,
targeted homologous recombination can be utilized to correct the
defective endogenous galectin 11 gene and/or regulatory sequences
thereof (e.g., promoter and enhancer sequences), or alternatively,
to "turn on" other dormant galectin 11 activity in the appropriate
tissue or cell type.
[0431] Additional methods which may be utilized to increase the
overall level of galectin 11 expression and/or galectin 11 activity
include the introduction of appropriate galectin 11-expressing
cells, preferably autologous cells, into a patient at positions and
in numbers which are sufficient to ameliorate the symptoms of
abnormalities in cells growth regulation. Such cells may be either
recombinant or non-recombinant. Among the cells which can be
administered to increase the overall level of galectin 11 gene
expression in a patient are normal cells, which express the
galectin 11 gene. Cell-based gene therapy techniques are well known
to those skilled in the art, see, e.g., Anderson et al., U.S. Pat.
No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No.
5,460,959.
[0432] If the activity of galectin 11 is in excess, several
approaches are available to reduce or inhibit galectin 11 activity
using molecules derived from the polypeptide and polynucleotide
sequences described above. Accordingly, a further aspect of the
invention is related to a method for treating an individual in need
of a decreased level of galectin 11 activity in the body
comprising, administering to such an individual a composition
comprising a therapeutically effective amount of a galectin 11
polypeptide, fragment, variant, derivative or analog of the
invention which acts as a galectin 11 antagonist, optionally, in
combination with a pharmaceutically acceptable carrier. Preferably,
galectin 11 activity is decreased to treat a disease wherein
increased apoptosis or other cell growth activity regulated by
galectin 11 is exhibited. Polypeptides, derivatives, variants and
analogs of the invention which function as antagonists of galectin
11 can routinely be identified using the assays described infra and
other techniques known in the art. Preferred antagonists for use in
the present invention are galectin 1-specific antibodies.
[0433] Thus, one embodiment of the invention comprises
administering to a subject an inhibitor compound (antagonist), such
as for example, an antibody or fragment, variant, derivative or
analog of the invention, along with a pharmaceutically acceptable
carrier in an amount effective to suppress (i.e. lower) galectin 11
activity.
[0434] In another approach, galectin 11 activity can be reduced or
inhibited by decreasing the level of galectin 11 gene expression.
In specific embodiments, antagonists according to the present
invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:1, or the complementary strand thereof,
and/or to nucleotide sequences contained in the deposited clone
209053. In one embodiment, this is accomplished through the use of
antisense sequences, either internally generated, by the organism,
or separately administered (see, for example, O'Connor, J.
Neurochem. (1991) 56:560 in 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.
[0435] For example, the use of c-myc and c-myb antisense RNA
constructs to inhibit the growth of the non-lymphocytic leukemia
cell line HL-60 and other cell lines was previously described.
(Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments
were performed in vitro by incubating cells with the
oligoribonucleotide. A similar procedure for in vivo use is
described in WO 91/15580. Briefly, a pair of oligonucleotides for a
given antisense RNA is produced as follows: A sequence
complimentary to the first 15 bases of the open reading frame is
flanked by an EcoR1 site on the 5 end and a HindIII site on the 3
end. Next, the pair of oligonucleotides is heated at 90.degree. C.
for one minute and then annealed in 2.times. ligation buffer (20 mM
TRIS HCl pH 7.5, 10 mM MgCl.sub.2, 10 mM dithiothreitol (DTT) and
0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the
retroviral vector PMV7 (WO 91/15580).
[0436] For example, the 5' coding portion of a polynucleotide that
encodes galectin 11 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 the galectin
11 polypeptide. The antisense RNA oligonucleotide hybridizes to the
mRNA in vivo and blocks translation of the mRNA molecule into
polypeptide.
[0437] In one embodiment, the galectin 11 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
galectin 11 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 galectin 11, 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 (Bernoist 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.
[0438] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a galectin 11 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 galectin 11
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 galectin
11 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.
[0439] 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 galectin
11 shown in FIG. 1 could be used in an antisense approach to
inhibit translation of endogenous galectin 11 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 galectin 11 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.
[0440] The polynucleotides of the present 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., 1987, Proc. Natl. Acad. Sci.
84:648-652; 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., 1988, BioTechniques
6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm.
Res. 5:539-549). To this end, the oligonucleotide may be conjugated
to another molecule, e.g., a peptide, hybridization triggered
cross-lining agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0441] 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, xantine, 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-N-6-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-N2-carboxypropyl)uracil, (acp3)w, and
2,6-diaminopurine.
[0442] 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.
[0443] 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.
[0444] In yet another embodiment, the antisense oligonucleotide is
an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual b-units, the strands run parallel to each
other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The
oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987,
Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue
(Inoue et al., 1987, FEBS Lett. 215:327-330).
[0445] 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.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0446] While antisense nucleotides complementary to the galectin 11
coding region sequence could be used, those complementary to the
transcribed untranslated region are most preferred.
[0447] Potential galectin 11 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 sequences can be used to
destroy galectin 11 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 of galectin 11 (FIG. 1; SEQ ID
NO:1). Preferably, the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the galectin 11
mRNA; i.e., to increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts. 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.
[0448] 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 galectin 11 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 galectin 11 messages and inhibit translation. Since
ribozymes, unlike antisense molecules are catalytic, a lower
intracellular concentration is required for efficiency.
[0449] Endogenous galectin 11 gene expression can also be reduced
by inactivating or "knocking out" the galectin 11 gene or its
promoter using targeted homologous recombination (e.g., see
Smithies et al., Nature 317:330-234 (1985); Thomas et al., Cell
51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of
which is incorporated by reference herein in its entirety). Such
approach can be 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.
[0450] Alternatively, endogenous galectin 11 gene expression can be
reduced by targeted deoxyribonucleotide sequences complementary to
the regulatory region of the galectin 11 gene (i.e., the galectin
11 promoter and/or enhancers) to form triple helical structures
that prevent transcription of the galectin 11 gene in target cells
in the body, see generally, Helene et al., Ann, N.Y. Acad. Sci.
660:27-36 (1992); Helene, C., Anticancer Drug Des., 6(6):569-584
(1991); and Maher, L. J., Bioassays 14(12):807-815 (1992)).
[0451] In yet another embodiment of the invention, the activity of
galectin 11 can be reduced using a "dominant negative". To this
end, constructs which encode defective galectin 11, such as, for
example, mutants lacking all or a portion of region of galectin 11
that binds .beta.-galactosides, can be used in gene therapy
approaches to diminish the activity of galectin 11 on appropriate
target cells. For example, nucleotide sequences that direct host
cell expression of galectin 11 in which all or a portion of the
region of galectin 11 that binds .beta.-galactoside is altered or
missing can be introduced into neutrophil cells, or other cells or
tissue which express galectin 11 (either by in vivo or ex vivo gene
therapy methods as for example, described herein). Alternatively,
targeted homologous recombination can be utilized to introduce such
deletions or mutations into the subjects endogenous galectin 11
gene in neutrophils or other cells expressing galectin 11.
[0452] Antagonist/agonist compounds may be employed to inhibit the
cell growth and proliferation effects of the polypeptides of the
present invention on neoplastic cells and tissues, i.e. stimulation
of angiogenesis of tumors, and, therefore, retard or prevent
abnormal cellular growth and proliferation, for example, in tumor
formation or growth.
[0453] The antagonist/agonist may also be employed to prevent
hyper-vascular diseases, and prevent the proliferation of
epithelial lens cells after extracapsular cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the
present invention may also be desirous in cases such as restenosis
after balloon angioplasty.
[0454] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[0455] The antagonist/agonist may also be employed to treat the
diseases described herein.
[0456] Thus, the invention provides a method of treating disorders
or diseases, including but not limited to the disorders or diseases
listed throughout this application, associated with overexpression
of a polynucleotide of the present invention by administering to a
patient (a) an antisense molecule directed to the polynucleotide of
the present invention, and/or (b) a ribozyme directed to the
polynucleotide of the present invention.
Formulation and Administration
[0457] It will be appreciated that conditions caused by a decrease
in the standard or normal level of galectin 11 activity in an
individual, can be treated by administration of galectin 11
polypeptide or fragment, variant, derivative, or analog of the
invention or an agonist thereof. Thus, the invention further
provides a method of treating an individual in need of an increased
level of galectin 11 activity comprising administering to such an
individual a pharmaceutical composition comprising an effective
amount of an isolated galectin 11 polypeptide or fragment, variant,
derivative, or analog of the invention, such as for example, the
full length form of the galectin 11, effective to increase the
galectin 11 activity level in such an individual.
[0458] 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,
preferably 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.
[0459] The dosage range required depends on the choice of peptide,
the route of administration, the nature of the formulation, the
nature of the subject's condition, and the judgment of the
attending practitioner. As a general proposition, the total
pharmaceutically effective amount of galectin 11 polypeptide
administered parenterally per dose will be in the range of about 1
.mu.g/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 this dose is in the range of 0.1-100 mg/kg of
subject, or between about 0.01 and 1 mg/kg/day. If given
continuously, the galectin 11 polypeptide is typically administered
at a dose rate of about 1 .mu.g/kg/hour to about 50 .mu.g/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. Wide variations in the needed
dosage, however, are to be expected in view of the variety of
compounds available and the differing efficiencies of various
routes of administration. For example, oral administration would be
expected to require higher dosages than administration by
intravenous injection. Variations in these dosage levels can be
adjusted using standard empirical routines for optimization, as is
well understood in the art.
[0460] Pharmaceutical compositions containing the galectin 11
polypeptides and polynucleotides of the invention (including
fragments, variants, derivatives or analogs), and galectin 11
agonists and antagonists may be routinely formulated in combination
with a pharmaceutically acceptable carrier. By "pharmaceutically
acceptable carrier" is meant 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. Pharmacopeia or other generally
recognized pharmacopeia 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, saline, buffered saline, glycerol, ethanol,
and oils, including those of petroleum, animal, vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Formulation should suit the mode of
administration, and is well within the skill of the art. For
example, 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 invention
additionally relates to pharmaceutical packs and kits comprising
one or more containers filled with one or more of the ingredients
of the aforementioned compositions of the invention.
[0461] Polypeptides and other compounds of the present invention
may be administered alone or in conjunction with other compounds,
such as therapeutic compounds. The pharmaceutical composition of
the invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. Preferred forms of systemic administration
of the pharmaceutical compositions include parenteral injection,
typically by intravenous injection. Other injection routes, such as
subcutaneous, intramuscular, intrasternal, intraarticular or
intraperitoneal, can be used. Alternative means for systemic
administration include transmucosal and transdermal administration
using penetrants such as bile salts or fusidic acids or other
detergents. In addition, if properly formulated in enteric or
encapsulated formulations, oral administration may also be
possible. Administration of these compounds may also be topical
and/or localized, in the form of salves, pastes, gels and the
like.
[0462] Polypeptides used in treatment can also be generated
endogenously in the subject, in treatment modalities often referred
to as "gene therapy" as described above. Thus, for example, cells
from a subject may be engineered with a polynucleotide, such as a
DNA or RNA, to encode a polypeptide ex vivo, and for example, by
the use of a retroviral plasmid vector. The cells are then
introduced into the subject.
[0463] 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.
[0464] 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.
[0465] 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
absorb.
[0466] 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.)
[0467] 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)).
[0468] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0469] 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.
[0470] 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. Pharmacopeia or other
generally recognized pharmacopeia 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.
[0471] 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.
[0472] 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.
[0473] 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.
[0474] 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.
[0475] 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
[0476] 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.
[0477] 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.
[0478] Assaying galectin 11 polypeptide levels in a biological
sample can occur using antibody-based techniques. For example,
galectin 11 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 galectin 11 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.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.149 Pm,
.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.
[0479] 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).
[0480] 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.
[0481] A galectin 11 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.149 Pm,
.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 cell proliferation 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 galectin 11 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)).
[0482] Additionally, to detect galectin 11 ligand, any galectin 11
polypeptide whose presence can be detected, can be administered.
For example, galectin 11 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
galectin 11 polypeptides can be utilized for in vitro diagnostic
procedures.
[0483] 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.
[0484] 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.
[0485] 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.
[0486] 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).
[0487] 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).
[0488] 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.
[0489] 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.
[0490] 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.
[0491] 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, St.
Louis, Mo.).
[0492] 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).
[0493] 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.
Chromosome Assays
[0494] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on human chromosome 11. The mapping of DNAs to chromosomes
according to the present invention is an important first step in
correlating those sequences with genes associated with disease.
[0495] Since the galectin 11 gene 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).
[0496] 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.
[0497] 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.
EXAMPLES
Example 1
Expression and Purification of Galectin 11 in E. coli
[0498] The DNA sequence encoding the galectin 11 protein in the
deposited cDNA clone is amplified using PCR oligonucleotide primers
specific to the amino terminal sequences of the galectin 11 protein
and to vector sequences 3' to the gene. Additional nucleotides
containing restriction sites to facilitate cloning are added to the
5' and 3' sequences respectively.
[0499] The 5' galectin 11 oligonucleotide primer has the sequence
5' cgc CCATGG ATGAGCCCCAGGCTGGAGGTG 3' (SEQ ID NO:23) containing
the underlined NcoI restriction site and nucleotides 49 to 69 of
the galectin 11 nucleotide sequence depicted in FIG. 1 (SEQ ID
NO:1).
[0500] The 3' galectin 11 primer has the sequence 5' cgc AAGCTT
TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:6) containing the underlined
HindIII restriction site followed by nucleotides complementary to
position 431 to 451 of the galectin 11 nucleotide sequence depicted
in FIG. 1 (SEQ ID NO:1).
[0501] The restriction sites are convenient to restriction enzyme
sites in the bacterial expression vector pQE60 which are used for
bacterial expression in these examples. (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, Calif., 91311). pQE60 encodes ampicillin
antibiotic resistance ("Ampr") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, a ribosome binding
site ("RBS"), a 6-His tag and restriction enzyme sites.
[0502] The amplified galectin 11 DNA and the pQE60 vector is
digested with NcoI and HindIII and the digested DNAs are then
ligated together. Insertion of the galectin 11 polypeptide DNA into
the restricted pQE60 vector places the galectin 11 polypeptide
coding region downstream of and operably linked to the vector's
IPTG-inducible promoter and in-frame with an initiating AUG
appropriately positioned for translation of galectin 11.
[0503] The ligation mixture is transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kanr"), is used in carrying out the example described
herein. This strain, which is only one of many that are suitable
for expressing galectin 11 protein, is available commercially from
Qiagen.
[0504] Transformants are identified by their ability to grow on LB
plates in the presence of ampicillin and kanamycin. Plasmid DNA is
isolated from resistant colonies and the identity of the cloned DNA
confirmed by restriction analysis.
[0505] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml).
[0506] The O/N culture is used to inoculate a large culture, at a
dilution of approximately 1:100 to 1:250. The cells are grown to an
optical density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalactopyranoside (IPTG) is then added to a final
concentration of 1 mM to induce transcription from lac repressor
sensitive promoters, by inactivating the lacI repressor. Cells
subsequently are incubated further for 3 to 4 hours. Cells then are
harvested by centrifugation and disrupted, by standard methods.
Inclusion bodies are purified from the disrupted cells using
routine collection techniques, and protein is solubilized from the
inclusion bodies into 8M urea. The 8M urea solution containing the
solubilized polypeptide is passed over a PD-10 column in 2.times.
phosphate-buffered saline ("PBS"), thereby removing the urea,
exchanging the buffer and refolding the protein. The polypeptide is
purified by a further step of chromatography to remove endotoxin.
Then, it is sterile filtered. The sterile filtered protein
preparation was stored in 2.times.PBS at a concentration of 95
.mu./ml.
Example 2
Cloning and Expression of Galectin 11 Protein in a Baculovirus
Expression System
[0507] The cDNA sequence encoding the full length galectin 11
protein in the deposited clone is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of
the gene:
[0508] The 5' galectin 11 oligonucleotide primer has the sequence
5' cgc CCC GGG GCCT ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:7) containing
the underlined SmaI restriction site and nucleotides 49 to 66 of
the galectin 11 nucleotide sequence depicted in FIG. 1 (SEQ ID
NO:1).
[0509] The 3' Galectin 11 primer has the sequence 5' cgc GGT ACC
TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the underlined
Asp718 restriction site followed by nucleotides complementary to
position 432 to 450 of the galectin 11 nucleotide sequence depicted
in FIG. 1 (SEQ ID NO:1).
[0510] An efficient signal for initiation of translation in
eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:
947-950 (1987) is appropriately located in the vector portion of
the construct.
[0511] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with XbaI and again
is purified on a 1% agarose gel. This fragment is designated herein
F2.
[0512] The vector pA2-GP is used to express the galectin 11 protein
in the baculovirus expression system, using standard methods, as
described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS
VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural
Experimental Station Bulletin No. 1555 (1987). This expression
vector contains the strong polyhedrin promoter of the Autographa
californica nuclear polyhedrosis virus (AcMNPV) followed by
convenient restriction sites. The signal peptide of AcMNPV gp67,
including the N-terminal methionine, is located just upstream of a
BamHI site. The polyadenylation site of the simian virus 40
("SV40") is used for efficient polyadenylation. For an easy
selection of recombinant virus the beta-galactosidase gene from E.
coli is inserted in the same orientation as the polyhedrin promoter
and is followed by the polyadenylation signal of the polyhedrin
gene. The polyhedrin sequences are flanked at both sides by viral
sequences for cell-mediated homologous recombination with wild-type
viral DNA to generate viable virus that express the cloned
polynucleotide.
[0513] Many other baculovirus vectors could be used in place of
pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of
skill readily will appreciate, that construction provides
appropriately located signals for transcription, translation,
trafficking and the like, such as an in-frame AUG and a signal
peptide, as required. Such vectors are described in Luckow et al.,
Virology 170: 31-39, among others.
[0514] The plasmid is digested with the restriction enzyme SmaI and
Asp718 and then is dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein V2.
[0515] Fragment F2 and the dephosphorylated plasmid V2 are ligated
together with T4 DNA ligase. E. coli HB101 cells are transformed
with ligation mix and spread on culture plates. Bacteria are
identified that contain the plasmid with the human galectin 11 gene
by digesting DNA from individual colonies using XbaI and then
analyzing the digestion product by gel electrophoresis. The
sequence of the cloned fragment is confirmed by DNA sequencing.
This plasmid is designated herein pBacgalectin 11.
[0516] 5 .mu.g of the plasmid pBacgalectin 11 is co-transfected
with 1.0 .mu.g of a commercially available linearized baculovirus
DNA ("BaculoGold baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofection method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 .mu.g of BaculoGold
virus DNA and 5 .mu.g of the plasmid pBacgalectin 11 are mixed in a
sterile well of a microtiter plate containing 50 .mu.l of
serum-free Grace's medium (Life Technologies Inc., Gaithersburg,
Md.). Afterwards 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium
are added, mixed and incubated for 15 minutes at room temperature.
Then the transfection mixture is added drop-wise to Sf9 insect
cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate is rocked back and forth
to mix the newly added solution. The plate is then incubated for 5
hours at 27.degree. C. After 5 hours the transfection solution is
removed from the plate and 1 ml of Grace's insect medium
supplemented with 10% fetal calf serum is added. The plate is put
back into an incubator and cultivation is continued at 27.degree.
C. for four days.
[0517] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with "Blue Gal" (Life Technologies Inc.,
Gaithersburg) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0518] Four days after serial dilution, the virus is added to the
cells. After appropriate incubation, blue stained plaques are
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses is then resuspended in an Eppendorf tube
containing 200 .mu.l of Grace's medium. The agar is removed by a
brief centrifugation and the supernatant containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4.degree. C. A clone
containing properly inserted hESSB I, II and III is identified by
DNA analysis including restriction mapping and sequencing. This is
designated herein as V-galectin 11.
[0519] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-galectin 11 at a multiplicity of infection ("MOI") of
about 2 (about 1 to about 3). Six hours later the medium is removed
and is replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Gaithersburg). 42 hours
later, 5 .mu.Ci of 35S-methionine and 5 .mu.Ci 35S-cysteine
(available from Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by
centrifugation, lysed and the labeled proteins are visualized by
SDS-PAGE and autoradiography.
Example 3
Cloning and Expression in Mammalian Cells
[0520] Most of the vectors used for the transient expression of the
galectin 11 polypeptide gene sequence in mammalian cells should
carry the SV40 origin of replication. This allows the replication
of the vector to high copy numbers in cells (e.g. COS cells) which
express the T antigen required for the initiation of viral DNA
synthesis. Any other mammalian cell line can also be utilized for
this purpose.
[0521] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular signals
can also be used (e.g. human actin promoter). Suitable expression
vectors for use in practicing the present invention include, for
example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,
Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI
(ATCC 67109). Mammalian host cells that could be used include,
human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3
cells, mouse L cells and Chinese hamster ovary cells.
[0522] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0523] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
is a useful marker to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) cells are often used for the production of
proteins.
[0524] The expression vectors pCl and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology, 438-4470 (March, 1985)) plus a fragment of
the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, e.g. with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of the gene of
interest. The vectors contain in addition the 3 intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a)
Cloning and Expression in COS Cells
[0525] The expression plasmid, pgalectin 11, is made by cloning a
cDNA encoding galectin 11 into the expression vector pcDNAI/Amp
(which can be obtained from Invitrogen, Inc.).
[0526] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron, and a polyadenylation
signal arranged so that a cDNA conveniently can be placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
[0527] A DNA fragment encoding the galectin 11 protein and an HA
tag fused in frame to its 3' end is cloned into the polylinker
region of the vector so that recombinant protein expression is
directed by the CMV promoter. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection of the recombinant protein
with an antibody that recognizes the HA epitope.
[0528] The plasmid construction strategy is as follows. The
galectin 11 cDNA of the deposited clone is amplified using primers
that contain convenient restriction sites, much as described above
regarding the construction of expression vectors for expression of
galectin 11 in E. coli. To facilitate detection, purification and
characterization of the expressed galectin 11, one of the primers
contains a hemagglutinin tag ("HA tag") as described above.
[0529] Suitable primers include the following, which are used in
this example. The 5' galectin 11 primer has the sequence 5' cgc CCC
GGG gcc atc ATG GCCTATC ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:9)
containing the underlined SmaI restriction enzyme site followed by
nucleotide sequence 49 to 66 of FIG. 1 (SEQ ID NO:1).
[0530] The 3' galectin 11 primer has the sequence 5' cgc GGT ACC
TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the Asp718
restriction followed by nucleotides complementary to nucleotides
432 to 450 of the galectin 11 nucleotide sequence depicted in FIG.
1 (SEQ ID NO:1).
[0531] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with HindIII and XhoI and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037), and the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis and gel
sizing for the presence of the galectin 11-encoding fragment.
[0532] For expression of recombinant galectin 11, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of galectin 11 by the vector.
[0533] Expression of the galectin 11 HA fusion protein is detected
by radiolabelling and immunoprecipitation, using methods described
in, for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing 35S-cysteine for 8 hours.
The cells and the media are collected, and the cells are washed and
the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1%
NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as
described by Wilson et al. cited above. Proteins are precipitated
from the cell lysate and from the culture media using an
HA-specific monoclonal antibody. The precipitated proteins then are
analyzed by SDS-PAGE gels and autoradiography. An expression
product of the expected size is seen in the cell lysate, which is
not seen in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0534] The vector pC1 is used for the expression of galectin 11
protein. Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC
Accession No. 37146]. Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary- or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J.
R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin,
J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenham, M. A. 1991, Biotechnology Vol. 9:64-68).
Cells grown in increasing concentrations of MTX develop resistance
to the drug by overproducing the target enzyme, DHFR, as a result
of amplification of the DHFR gene. If a second gene is linked to
the DHFR gene it is usually co-amplified and over-expressed. It is
state of the art to develop cell lines carrying more than 1,000
copies of the genes. Subsequently, when the methotrexate is
withdrawn, cell lines contain the amplified gene integrated into
the chromosome(s).
[0535] Plasmid pCl contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular
Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer of the immediate early gene of human cytomegalovirus (CMV)
(Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter
are the following single restriction enzyme cleavage sites that
allow the integration of the genes: BamHI, Pvull, and Nrul. Behind
these cloning sites the plasmid contains translational stop codons
in all three reading frames followed by the 3 intron and the
polyadenylation site of the rat preproinsulin gene. Other high
efficient promoters can also be used for the expression, e.g., the
human-actin promoter, the SV40 early or late promoters or the long
terminal repeats from other retroviruses, e.g., HIV and HTLVI. For
the polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well.
[0536] Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0537] The plasmid pCl is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphatase
by procedures known in the art. The vector is then isolated from a
1% agarose gel.
[0538] The DNA sequence encoding galectin 11, ATCC Deposit No.
209053 is amplified using PCR oligonucleotide primers corresponding
to the 5' and 3' sequences of the gene:
[0539] The 5' Galectin 11 primer has the sequence 5' cgc CCC GGG
gcc atc ATG GCCTATC ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:9) containing
the underlined SmaI restriction enzyme site followed by nucleotide
sequence 49-66 of FIG. 1 (SEQ ID NO:1). Inserted into an expression
vector, as described below, the 5' end of the amplified fragment
encoding human galectin 11 provides an efficient signal peptide. An
efficient signal for initiation of translation in eukaryotic cells,
as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is
appropriately located in the vector portion of the construct.
[0540] The 3' Galectin 11 primer has the sequence 5' cgc GGT ACC
TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the Asp718
restriction followed by nucleotides complementary to nucleotides
432-450 of the galectin 11 nucleotide sequence depicted in FIG. 1
(SEQ ID NO:1).
[0541] The amplified fragments are isolated from a 1% agarose gel
as described above and then digested with the endonucleases SmaI
and Asp718 and then purified again on a 1% agarose gel.
[0542] The isolated fragment and the dephosphorylated vector are
then ligated with T4 DNA ligase. E. coli HB101 cells are then
transformed and bacteria identified that contained the plasmid pCl
inserted in the correct orientation using the restriction enzyme
SmaI. The sequence of the inserted gene is confirmed by DNA
sequencing.
Transfection of CHO-DHFR-Cells
[0543] Chinese hamster ovary cells lacking an active DHFR enzyme
are used for transfection. 5 .mu.g of the expression plasmid C1 are
cotransfected with 0.5 .mu.g of the plasmid pSVneo using the
lipofecting method (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the gene neo from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) and cultivated from 10-14 days. After this period, single
clones are trypsinized and then seeded in 6-well petri dishes using
different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200
nM, 400 DM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing
even higher concentrations of methotrexate (500 nM, 1 .mu.M, 2
.mu.M, 5 .mu.M). The same procedure is repeated until clones grow
at a concentration of 100 .mu.M.
[0544] The expression of the desired gene product is analyzed by
Western blot analysis and SDS-PAGE.
Example 4
Tissue Distribution of Protein Expression
[0545] Northern blot analysis is carried out to examine galectin 11
gene expression in human tissues, using methods described by, among
others, Sambrook et al., cited above. A cDNA probe containing the
entire nucleotide sequence encoding galectin 11 protein (SEQ ID
NO:1) is labeled with 32P using the rediprime DNA labeling system
(Amersham Life Science), according to manufacturer's instructions.
After labeling, the probe is purified using a CHROMA SPIN-100
column (Clontech Laboratories, Inc.), according to manufacturer's
protocol number PT1200-1. The purified labeled probe is then used
to examine various human tissues for galectin 11 mRNA.
[0546] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from Clontech and are examined with labeled probe using ExpressHyb
hybridization solution (Clontech) according to manufacturer's
protocol number PT1190-1. Following hybridization and washing, the
blots are mounted and exposed to film at -70.degree. C. overnight,
and films developed according to standard procedures.
[0547] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
Example 5
Galectin 11 Induced Apoptosis in Transfected Cells
[0548] This example presents data demonstrating that transfection
of a constitutive galectin 11 expression construct into human
Jurkat T-cells induces apoptosis of the transfected cells.
[0549] A T cell is a type of lymphocyte, or "white blood cell",
that mediates the cellular immune response to foreign
macromolecule, termed antigens. While T cells are necessary for
normal mammalian immune responses, in some instances it is
desirable to inhibit their activation: for example, in some
autoimmune diseases, the T cells of a subject respond to
"self-antigens", i.e., macromolecule produced by the subject,
rather than foreign-made macromolecule, and damage the cells and
tissues of the subject. Autoimmune T cell responses are found in
subjects having systemic lupus erythematosus (SLE), rheumatoid
arthritis (RA), insulin-dependent diabetes, myasthenia gravis, and
multiple sclerosis (MS) and contribute to the pathophysiology of
each. T cells also cause graft rejection and graft versus host
disease (GVHD). Graft rejection is caused by an immune response
against the transplanted tissues (the graft), which are recognized
as "foreign" by T cells of the recipient (host). Graft versus host
disease is caused by engrafted T cells, which recognize host-made
macromolecule as "foreign."
Methods
[0550] The DNA sequence encoding the galectin 11 protein in the
deposited cDNA clone was amplified using PCR oligonucleotide
primers specific to the amino terminal sequences of the galectin 11
protein and to vector sequences 3' to the gene. The 5' galectin 11
oligonucleotide primer had the sequence 5' CGCCGCCACCATGAGCCCCAGGC
3' (SEQ ID NO:10) containing nucleotides 49 to 61 of the galectin
11 nucleotide sequence in FIG. 1 (SEQ ID NO:1). The 3' galectin 11
primer has the sequence 5' GGAATCTAGATCAGGAGTGGAC 3' (SEQ ID NO:1)
containing the underlined XbaI restriction site followed by
nucleotide sequence complementary to position 439 to 450 of the
galectin 11 nucleotide sequence in FIG. 1 (SEQ ID NO:1).
[0551] The amplified galectin 11 fragments were isolated from a 1%
agarose gel as described above, digested with the endonuclease
XbaI, purified again on a 1% agarose gel, and ligated into the
multiple cloning site of restricted pEF1 using T4 DNA ligase.
[0552] The pEF1 vector was generated by replacing the CMV promoter
on pIRES1neo (Clontech) with the human elongation factor 1 a
constitutive promoter from pEF-BOS. The EF1a promoter has been
shown to be highly active in a variety of cell types (data not
shown). This vector also contains a bovine growth hormone poly A
signal and a ampicilin resistance gene, a ribosome binding site
("RBS"), a 6-His tag and restriction enzyme sites. This vector was
digested with EcoRI, BamHI, and phosphatase using techniques known
in the art.
[0553] Insertion of the isolated galectin 11 fragment into the
restricted pEF1 vector placed the galectin 11 polypeptide coding
region downstream of and operably associated with the vector's
constitutive elongation factor-1 promoter and in-frame with an
initiating AUG appropriately positioned for translation of galectin
11. E. coli cells were then transformed with the ligation reaction
and those cells containing the desired construct (pEFLeg11) were
identified using techniques known in the art. Cells containing the
pEFLeg11 expression construct were then cultured under known
conditions favoring high yield and the expression construct was
isolated from the bacterial cell culture using techniques known in
the art.
[0554] For detection of apoptosis, techniques known in the art were
used to cotransfect human Jurkat T-cells with the pEFLeg11
expression construct together with a marker plasmid encoding green
fluorescent protein (GFP). The transfected cells were then stained
with MitoTracker Red (Molecular Probes) to determine the
transmembrane potentials of mitochondria, and analyzed by two-color
flow cytometry. Transfected populations were identified by emission
of green fluorescence due to the expression of GFP. Apoptotic cells
exhibit disrupted mitochondrial transmembrane potential and thus
have lower red fluorescence emission because of their reduced
ability to sequester the dye MitoTracker Red.
Results
[0555] Jurkat cells transfected with the constitutive expression
plasmid for "galectin 11" underwent significant apoptosis 24 h
after transfection. Approximately 30% of "galectin 11" transfected
cells showed reduced mitochondrial transmembrane potential compared
to less than 10% in cells transfected with the control vector with
no insert (pEF1) (FIG. 5A).
[0556] We also followed the number of GFP positive cells during a
4-day culture period after co-transfection with either the control
vector pEF1 or the "galectin 11" expression vector pEF1-Leg11.
There were about 4 times more surviving GFP positive cells after
transfection with pEF1 than with pEF1-Leg11 (FIG. 5B).
Example 6
Construction of N-Terminal and/or C-Terminal Deletion Mutants
[0557] The following general approach may be used to clone a
N-terminal or C-terminal deletion galectin 11 deletion mutant.
Generally, two oligonucleotide primers of about 15-25 nucleotides
are derived from the desired 5' and 3' positions of a
polynucleotide of SEQ ID NO:1. The 5' and 3' positions of the
primers are determined based on the desired galectin 11
polynucleotide fragment. An initiation and stop codon are added to
the 5' and 3' primers respectively, if necessary, to express the
galectin 11 polypeptide fragment encoded by the polynucleotide
fragment. Preferred galectin 11 polynucleotide fragments are those
encoding the N-terminal and C-terminal deletion mutants disclosed
above in the "Galectin 11 Polypeptide and Fragments" section of the
Specification.
[0558] Additional nucleotides containing restriction sites to
facilitate cloning of the galectin 11 polynucleotide fragment in a
desired vector may also be added to the 5' and 3' primer sequences.
The galectin 11 polynucleotide fragment is amplified from genomic
DNA or from the deposited cDNA clone using the appropriate PCR
oligonucleotide primers and conditions discussed herein or known in
the art. The galectin 11 polypeptide fragments encoded by the
galectin 11 polynucleotide fragments of the present invention may
be expressed and purified in the same general manner as the full
length polypeptides, although routine modifications may be
necessary due to the differences in chemical and physical
properties between a particular fragment and full length
polypeptide.
[0559] As a means of exemplifying but not limiting the present
invention, the polynucleotide encoding the galectin 11 polypeptide
fragment L-5 to L-128 is amplified and cloned as follows: A 5'
primer is generated comprising a restriction enzyme site followed
by an initiation codon in frame with the polynucleotide sequence
encoding the N-terminal portion of the polypeptide fragment
beginning with L-5. A complementary 3' primer is generated
comprising a restriction enzyme site followed by a stop codon in
frame with the polynucleotide sequence encoding C-terminal portion
of the galectin 11 polypeptide fragment ending with L-128.
[0560] The amplified polynucleotide fragment and the expression
vector are digested with restriction enzymes which recognize the
sites in the primers. The digested polynucleotides are then ligated
together. The galectin 11 polynucleotide fragment is inserted into
the restricted expression vector, preferably in a manner which
places the galectin 11 polypeptide fragment coding region
downstream from the promoter. The ligation mixture is transformed
into competent E. coli cells using standard procedures and as
described in the Examples herein. Plasmid DNA is isolated from
resistant colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
Example 7
Protein Fusions of Galectin 11
[0561] Galectin 11 polypeptides are preferably fused to other
proteins. These fusion proteins can be used for a variety of
applications. For example, fusion of galectin 11 polypeptides to
His-tag, HA-tag, protein A, IgG domains, and maltose binding
protein facilitates purification. (See Example 5; see also EP A
394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly,
fusion to IgG-1, IgG-3, and albumin increases the halflife time in
vivo. Nuclear localization signals fused to galectin 11
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 by modifying
the following protocol, which outlines the fusion of a polypeptide
to an IgG molecule, or the protocol described in Example 3.
[0562] 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. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian expression vector.
[0563] For example, if pC4 (Accession No. 209646) 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 galectin 11 polynucleotide, isolated by
the PCR protocol described in Example 1, is ligated into this BamHI
site. Note that the polynucleotide is cloned without a stop codon,
otherwise a fusion protein will not be produced.
[0564] 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.)
[0565] Human IgG Fc Region: TABLE-US-00003 (SEQ ID NO: 13)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT
Example 8
Production of an Antibody
a) Hybridoma Technology
[0566] 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 galectin 11 are
administered to an animal to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of galectin 11 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.
[0567] Monoclonal antibodies specific for galectin 11 protein are
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, an animal (preferably a mouse) is
immunized with galectin 11 polypeptide or, more preferably, with a
secreted galectin 11 polypeptide-expressing cell. Such
polypeptide-expressing cells are cultured in any suitable tissue
culture medium, preferably 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 .mu.g/ml
of streptomycin.
[0568] 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. 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 galectin 11 polypeptide.
[0569] Alternatively, additional antibodies capable of binding to
galectin 11 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 galectin 11 protein-specific
antibody can be blocked by galectin 11. Such antibodies comprise
anti-idiotypic antibodies to the galectin 11 protein-specific
antibody and are used to immunize an animal to induce formation of
further galectin 11 protein-specific antibodies.
[0570] For in vivo use of antibodies in humans, an antibody is
"humanized". Such antibodies can be produced using genetic
constructs derived from hybridoma cells producing the monoclonal
antibodies described above. Methods for producing chimeric and
humanized antibodies are known in the art and are discussed herein.
(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 Galectin 11
Polypeptides from a Library of scFvs
[0571] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against Galectin 11 to which the donor may or may not
have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated
herein by reference in its entirety).
[0572] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100
.mu.g/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
(M13 delta gene III, see PCT publication WO 92/01047) 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 .mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[0573] 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 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100 .mu.g
ampicillin/ml and 25 .mu.g 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 .mu.m filter (Minisart NML; Sartorius) to
give a final concentration of approximately 1013 transducing
units/ml (ampicillin-resistant clones).
[0574] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 .mu.g/ml or 10 .mu.g/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 1013 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 11.0M Tris-HCl,
pH 7.4. Phage 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.
[0575] Characterization of Binders. 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 microtitre 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., PCT publication WO
92/01047) and then by sequencing. These ELISA positive clones may
also be further characterized by techniques known in the art, such
as, for example, epitope mapping, binding affinity, receptor signal
transduction, ability to block or competitively inhibit
antibody/antigen binding, and competitive agonistic or antagonistic
activity.
Example 9
Production of Galectin 11 Protein for High-Throughput Screening
Assays
[0576] The following protocol produces a supernatant containing
galectin 11 polypeptide to be tested. This supernatant can then be
used in the Screening Assays described in Examples 14-21.
[0577] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim)
stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or
magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml.
Add 200 ul of this solution to each well (24 well plates) and
incubate at RT for 20 minutes. Be sure to distribute the solution
over each well (note: a 12-channel pipetter may be used with tips
on every other channel). Aspirate off the Poly-D-Lysine solution
and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should
remain in the well until just prior to plating the cells and plates
may be poly-lysine coated in advance for up to two weeks.
[0578] Plate 293T cells (do not carry cells past P+20) at
2.times.10.sup.5 cells/well in 0.5 ml DMEM (Dulbecco's Modified
Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F
Biowhittaker))/10% heat inactivated FBS (14-503F
Biowhittaker)/1.times. Penstrep (17-602E Biowhittaker). Let the
cells grow overnight.
[0579] The next day, mix together in a sterile solution basin: 300
ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070
Gibco/BRL)/96-well plate. With a small volume multi-channel
pipetter, aliquot approximately 2 ug of an expression vector
containing a polynucleotide insert, produced by the methods
described in Examples 8-10, into an appropriately labeled 96-well
round bottom plate. With a multi-channel pipetter, add 50 ul of the
Lipofectamine/Optimem I mixture to each well. Pipette up and down
gently to mix. Incubate at RT 15-45 minutes. After about 20
minutes, use a multi-channel pipetter to add 150 ul Optimem I to
each well. As a control, one plate of vector DNA lacking an insert
should be transfected with each set of transfections.
[0580] Preferably, the transfection should be performed by
tag-teaming the following tasks. By tag-teaming, hands on time is
cut in half, and the cells do not spend too much time on PBS.
First, person A aspirates off the media from four 24-well plates of
cells, and then person B rinses each well with 0.5-1 ml PBS. Person
A then aspirates off PBS rinse, and person B, using a 12-channel
pipetter with tips on every other channel, adds the 200 ul of
DNA/Lipofectamine/Optimem I complex to the odd wells first, then to
the even wells, to each row on the 24-well plates. Incubate at 37
degree C. for 6 hours.
[0581] While cells are incubating, prepare appropriate media,
either 1% BSA in DMEM with 1.times. penstrep, or HGS CHO-5 media
(116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO4-5H2O; 0.050 mg/L
of Fe(NO3)3-9H2O; 0.417 mg/L of FeSO4-7H2O; 311.80 mg/L of Kcl;
28.64 mg/L of MgCl2; 48.84 mg/L of MgSO4; 6995.50 mg/L of NaCl;
2400.0 mg/L of NaHCO3; 62.50 mg/L of NaH2PO4-H20; 71.02 mg/L of
Na2HPO.sub.4; 0.4320 mg/L of ZnSO4-7H2O; 0.002 mg/L of Arachidonic
Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of
DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010
mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic
Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20
mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of
L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of
L-Asparagine-H20; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of
L-Cystine-2HCL-H20; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of
L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of
Glycine; 52.48 mg/ml of L-Histidine-HCL-H20; 106.97 mg/ml of
L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine
HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine;
40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of
L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of
L-Tryrosine-2Na-2H20; and 99.65 mg/ml of L-Valine; 0.0035 mg/L of
Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline
Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02
mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of
Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine
HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B12; 25 mM of
HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic
Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium
Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;
0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin
complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin
complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin
complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with
2 mm glutamine and 1.times. penstrep. (BSA (81-068-3 Bayer) 100 gm
dissolved in 1 L DMEM for a 10% BSA stock solution). Filter the
media and collect 50 ul for endotoxin assay in 15 ml polystyrene
conical.
[0582] The transfection reaction is terminated, preferably by
tag-teaming, at the end of the incubation period. Person A
aspirates off the transfection media, while person B adds 1.5 ml
appropriate media to each well. Incubate at 37 degree C. for 45 or
72 hours depending on the media used: 1% BSA for 45 hours or CHO-5
for 72 hours.
[0583] On day four, using a 300 ul multichannel pipetter, aliquot
600 ul in one 1 ml deep well plate and the remaining supernatant
into a 2 ml deep well. The supernatants from each well can then be
used in the assays described in Examples 11-17.
[0584] It is specifically understood that when activity is obtained
in any of the assays described below using a supernatant, the
activity originates from either the galectin 11 polypeptide
directly (e.g., as a secreted protein) or by galectin 11 inducing
expression of other proteins, which are then secreted into the
supernatant. Thus, the invention further provides a method of
identifying the protein in the supernatant characterized by an
activity in a particular assay.
Example 10
Construction of GAS Reporter Construct
[0585] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[0586] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATs
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[0587] The STATs are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[0588] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Schidler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor
family, capable of activating Jaks, is divided into two groups: (a)
Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-.alpha., IFN-g, and
IL-10. The Class 1 receptors share a conserved cysteine motif (a
set of four conserved cysteines and one tryptophan) and a WSXWS
motif (a membrane proximal region encoding Trp-Ser-Xxx-Trp-Ser (SEQ
ID NO:5)).
[0589] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway.
[0590] Therefore, activation of the Jaks-STATs pathway, reflected
by the binding of the GAS or the ISRE element, can be used to
indicate proteins involved in the proliferation and differentiation
of cells. For example, growth factors and cytokines are known to
activate the Jaks-STATs pathway. (See Table below.) Thus, by using
GAS elements linked to reporter molecules, activators of the
Jaks-STATs pathway can be identified. TABLE-US-00004 JAKs Ligand
tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN family IFN-a/B
+ + - - 1, 2, 3 ISRE IFN-g + + - 1 GAS (IRF1 > Lys6 > IFP)
Il-10 + ? ? - 1, 3 gp130 family IL-6 (Pleiotrohic) + + + ? 1, 3 GAS
(IRF1 > Lys6 > IFP) Il-11(Pleiotrohic) ? + ? ? 1, 3
OnM(Pleiotrohic) ? + + ? 1, 3 LIF(Pleiotrohic) ? + + ? 1, 3
CNTF(Pleiotrohic) -/+ + + ? 1, 3 G-CSF(Pleiotrohic) ? + ? ? 1, 3
IL-12(Pleiotrohic) + - + + 1, 3 g-C family IL-2 (lymphocytes) - + -
+ 1, 3, 5 GAS IL-4 (lymph/myeloid) - + - + 6 GAS (IRF1 = IFP
>> Ly6)(IgH) IL-7 (lymphocytes) - + - + 5 GAS IL-9
(lymphocytes) - + - + 5 GAS IL-13 (lymphocyte) - + ? ? 6 GAS IL-15
? + ? + 5 GAS gp140 family IL-3 (myeloid) - - + - 5 GAS (IRF1 >
IFP >> Ly6) IL-5 (myeloid) - - + - 5 GAS GM-CSF (myeloid) - -
+ - 5 GAS Growth hormone family GH ? - + - 5 PRL ? +/- + - 1, 3, 5
EPO ? - + - 5 GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor
Tyrosine Kinases EGF ? + + - 1, 3 GAS (IRF1) PDGF ? + + - 1, 3
CSF-1 ? + + - 1, 3 GAS (not IRF1)
[0591] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 11-12,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATs upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00005 (SEQ ID NO: 14)
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCC
CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3'
[0592] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:15)
[0593] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI/Hind III
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence: TABLE-US-00006 (SEQ ID NO: 16)
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA
AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA
TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG
CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA
GGCCTAGGCTTTTGCAAAAAGCTT:3'
[0594] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can be instead of SEAP, in
this or in any of the other Examples. Well known reporter molecules
that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
B-galactosidase, green fluorescent protein (GFP), or any protein
detectable by an antibody.
[0595] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
Clontech using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[0596] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple
cloning site, to create the GAS-SEAP/Neo vector. Once this vector
is transfected into mammalian cells, this vector can then be used
as a reporter molecule for GAS binding as described in Examples
11-12.
[0597] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing NFK-B and EGR
promoter sequences are described in Examples 14 and 13. However,
many other promoters can be substituted using the protocols
described in these Examples. For instance, SRE, IL-2, NFAT, or
Osteocalcin promoters can be substituted, alone or in combination
(e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct
activity, such as HELA (epithelial), HUVEC (endothelial), Reh
(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or
Cardiomyocyte.
Example 11
High-Throughput Screening Assay for T-Cell Activity
[0598] The following protocol is used to assess T-cell activity by
identifying factors, and determining whether supernate containing a
polypeptide of the invention proliferates and/or differentiates
T-cells. T-cell activity is assessed using the GAS/SEAP/Neo
construct produced in Example 10. Thus, factors that increase SEAP
activity indicate the ability to activate the Jaks-STATS signal
transduction pathway. The T-cell used in this assay is Jurkat
T-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCC
Accession No. CRL-1552) and Molt-4 cells (ATCC Accession No.
CRL-1582) cells can also be used.
[0599] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In
order to generate stable cell lines, approximately 2 million Jurkat
cells are transfected with the GAS-SEAP/neo vector using DMRIE-C
(Life Technologies) (transfection procedure described below). The
transfected cells are seeded to a density of approximately 20,000
cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant colonies are expanded and then tested for their
response to increasing concentrations of interferon gamma. The dose
response of a selected clone is demonstrated.
[0600] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml
OPTI-MEM containing 50 ul of DMRIE-C and incubate at room
temperature for 15-45 mins.
[0601] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM to
T25 flask and incubate at 37 degree C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.P
[0602] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/ml Genticin, and 11% Pen-Strep. These cells
are treated with supernatants containing galectin 11 polypeptides
or galectin 11 induced polypeptides, as produced by the protocol
described in Example 9.
[0603] On the day of treatment with the supernatant, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of supernatants being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[0604] Transfer the cells to a triangular reservoir boat, in order
to dispense the cells into a 96 well dish, using a 12 channel
pipette. Using a 12 channel pipette, transfer 200 ul of cells into
each well (therefore adding 100,000 cells per well).
[0605] After all the plates have been seeded, 50 ul of the
supernatants are transferred directly from the 96 well plate
containing the supernatants into each well using a 12 channel
pipette. In addition, a dose of exogenous interferon gamma (0.1,
1.0, 10 ng) is added to wells H9, H10, and H11 to serve as
additional positive controls for the assay.
[0606] The 96 well dishes containing Jurkat cells treated with
supernatants are placed in an incubator for 48 hrs (note: this time
is variable between 48-72 hrs). 35 ul samples from each well are
then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using sellophene
covers) and stored at -20 degree C. until SEAP assays are performed
according to Example 15. The plates containing the remaining
treated cells are placed at 4 degree C. and serve as a source of
material for repeating the assay on a specific well if desired.
[0607] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
[0608] The above protocol may be used in the generation of both
transient, as well as, stable transfected cells, which would be
apparent to those of skill in the art.
Example 12
High-Throughput Screening Assay Identifying Myeloid Activity
[0609] The following protocol is used to assess myeloid activity of
galectin 11 by determining whether galectin 11 proliferates and/or
differentiates myeloid cells. Myeloid cell activity is assessed
using the GAS/SEAP/Neo construct produced in Example 10. Thus,
factors that increase SEAP activity indicate the ability to
activate the Jaks-STATS signal transduction pathway. The myeloid
cell used in this assay is U937, a pre-monocyte cell line, although
TF-1, IL60, or KG1 can be used.
[0610] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 10, a DEAE-Dextran method (Kharbanda
et. al., 1994, Cell Growth & Differentiation, 5:259-265) is
used. First, vest 2.times.10e7 U937 cells and wash with PBS. The
U937 cells are usually grown in RPMI 1640 medium containing 10%
heat-inactivated fetal bovine serum (FBS) supplemented with 100
units/ml penicillin and 100 mg/ml streptomycin.
[0611] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na2BPO4.7H2O, 1 mM MgCl2, and
675 uM CaCl2. Incubate at 37 degrees C. for 45 min.
[0612] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37 degree
C. for 36 hr.
[0613] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[0614] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[0615] Add 50 ul of the supernatant prepared by the protocol
described in Example 9. Incubate at 37 degree C for 48 to 72 hr. As
a positive control, 100 Unit/ml interferon gamma can be used which
is known to activate U937 cells. Over 30 fold induction is
typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 15.
Example 13
High-Throughput Screening Assay Identifying Neuronal Activity
[0616] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR1 is responsible for such induction.
Using the EGR1 promoter linked to reporter molecules, activation of
cells can be assessed by galectin 11.
[0617] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
phenochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells by galectin 11 can be assessed.
[0618] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to +1)
(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified
from human genomic DNA using the following primers: TABLE-US-00007
(SEQ ID NO: 17) 5'GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ ID NO:
18) 5'GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'
[0619] Using the GAS:SEAP/Neo vector produced in Example 10, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR1
promoter.
[0620] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[0621] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #
12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 ug/ml
streptomycin on a precoated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed from the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[0622] Transfect the EGR/SEAP/Neo construct into PC12 using the
Lipofectamine protocol described in Example 11. EGR-SEAP/PC12
stable cells are obtained by growing the cells in 300 ug/ml G418.
The G418-free medium is used for routine growth but every one to
two months, the cells should be re-grown in 300 ug/ml G418 for
couple of passages.
[0623] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[0624] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.105 cells/ml.
[0625] Add 200 ul of the cell suspension to each well of 96-well
plate (equivalent to 1.times.105 cells/well). Add 50 ul supernatant
produced by Example 9, 37 degree C. for 48 to 72 hr. As a positive
control, a growth factor known to activate PC12 cells through EGR
can be used, such as 50 ng/ul of Neuronal Growth Factor (NGF). Over
fifty-fold induction of SEAP is typically seen in the positive
control wells. A SEAP assay of the supernatant is performed
according to Example 15.
Example 14
High-Throughput Screening Assay for T-Cell Activity
[0626] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[0627] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1 MHC.
[0628] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the supernatants produced in Example 9.
Activators or inhibitors of NF-KB would be useful in treating
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[0629] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:19), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00008 (SEQ ID NO: 20)
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGG
ACTTTCCATCCTGCCATCTCAATTAG:3'
[0630] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site: TABLE-US-00009
(SEQ ID NO: 21) 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3'
[0631] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI and Hind
III and subcloned into BLSK2-(Stratagene) Sequencing with the T7
and T3 primers confirms the insert contains the following sequence:
TABLE-US-00010 (SEQ ID NO: 22)
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT
CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG
CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG
CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3'
[0632] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[0633] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),
replacing the GFP gene, after restricting pGFP-1 with SalI and
NotI.
[0634] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 11. Similarly, the method for assaying
supernatants with these stable Jurkat T-cells is also described in
Example 11. As a positive control, exogenous TNF alpha (0.1, 1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 15
Assay for SEAP Activity
[0635] As a reporter molecule for the assays described in Examples
12 and 13, SEAP activity is assayed using the Tropix Phospho-light
Kit (Cat. BP-400) according to the following general procedure. The
Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction
Buffers used below.
[0636] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 ul of 2.5.times. dilution buffer into Optiplates
containing 35 ul of a supernatant. Seal the plates with a plastic
sealer and incubate at 65 degree C. for 30 min. Separate the
Optiplates to avoid uneven heating.
[0637] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the table below). Add 50 ul
Reaction Buffer and incubate at room temperature for 20 minutes.
Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on
luminometer, one should treat 5 plates at each time and start the
second set 10 minutes later.
[0638] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity. TABLE-US-00011 Reaction Buffer
Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3
11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95
4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125
6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155
7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185
9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41
215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12
47 245 12.25 48 250 12.5 49 255 12.75 50 260 13
Example 16
High-Throughput Screening Assay Identifying Changes in Small
Molecule Concentration and Membrane Permeability
[0639] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify
supernatants which bind to receptors of a particular cell. Although
the following protocol describes an assay for calcium, this
protocol can easily be modified to detect changes in potassium,
sodium, pH, membrane potential, or any other small molecule which
is detectable by a fluorescent probe.
[0640] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.;
catalog no. F-14202), used here.
[0641] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with clear bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 ul of
HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer after
the final wash.
[0642] A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic
acid DMSO. To load the cells with fluo-4, 50 ul of 12 ug/ml fluo-4
is added to each well. The plate is incubated at 37 degrees C. in a
CO.sub.2 incubator for 60 min. The plate is washed four times in
the Biotek washer with HBSS leaving 100 ul of buffer.
[0643] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 ul of 1 mg/ml fluo-4 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37 degrees C. water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate, 100
ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate
is then washed once in Denley CellWash with 200 ul, followed by an
aspiration step to 100 ul final volume.
[0644] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-4. The supernatant is added to the well, and
a change in fluorescence is detected.
[0645] To measure the fluorescence of intracellular calcium, the
FLIPR is set for the following parameters: (1) System gain is
300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event caused by the a molecule, either
galectin 11 or a molecule induced by galectin 11, which has
resulted in an increase in the intracellular Ca++
concentration.
Example 17
High-Throughput Screening Assay Identifying Tyrosine Kinase
Activity
[0646] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[0647] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and non-receptor linked and cytosolic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[0648] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, identifying whether galectin
11 or a molecule induced by galectin 11 is capable of activating
tyrosine kinase signal transduction pathways is of interest.
Therefore, the following protocol is designed to identify such
molecules capable of activating the tyrosine kinase signal
transduction pathways.
[0649] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well Loprodyne
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel
purchased from Becton Dickinson (Bedford, Mass.), or calf serum,
rinsed with PBS and stored at 4 degree C. Cell growth on these
plates is assayed by seeding 5,000 cells/well in growth medium and
indirect quantitation of cell number through use of alamarBlue as
described by the manufacturer Alamar Biosciences, Inc. (Sacramento,
Calif.) after 48 hr. Falcon plate covers #3071 from Becton
Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent
Screen Plates. Falcon Microtest III cell culture plates can also be
used in some proliferation experiments.
[0650] To prepare extracts, A431 cells are seeded onto the nylon
membranes of Loprodyne plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example
12, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4,
2 mM Na4P2O7 and a cocktail of protease inhibitors (# 1836170)
obtained from Boeheringer Mannheim (Indianapolis, Ind.) is added to
each well and the plate is shaken on a rotating shaker for 5
minutes at 4.degree. C. The plate is then placed in a vacuum
transfer manifold and the extract filtered through the 0.45 mm
membrane bottoms of each well using house vacuum. Extracts are
collected in a 96-well catch/assay plate in the bottom of the
vacuum manifold and immediately placed on ice. To obtain extracts
clarified by centrifugation, the content of each well, after
detergent solubilization for 5 minutes, is removed and centrifuged
for 15 minutes at 4 degree C. at 16,000.times.g.
[0651] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[0652] Generally, the tyrosine kinase activity of a supernatant is
evaluated by determining its ability to phosphorylate a tyrosine
residue on a specific substrate (a biotinylated peptide).
Biotinylated peptides that can be used for this purpose include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase
cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin).
Both peptides are substrates for a range of tyrosine kinases and
are available from Boehringer Mannheim.
[0653] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 ul of 5 uM
Biotinylated Peptide, then 10 ul ATP/Mg2+ (5 mM ATP/50 mM MgC12),
then 10 ul of 5.times. Assay Buffer (40 mM imidazole hydrochloride,
pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl2, 5 mM
MnCl2, 0.5 mg/ml BSA), then 5 ul of Sodium Vanadate (1 mM), and
then 5 ul of water. Mix the components gently and preincubate the
reaction mix at 30 degree C. for 2 min. Initial the reaction by
adding 10 ul of the control enzyme or the filtered supernatant.
[0654] The tyrosine kinase assay reaction is then terminated by
adding 10 ul of 120 mm EDTA and place the reactions on ice.
[0655] Tyrosine kinase activity is determined by transferring 50 ul
aliquot of reaction mixture to a microtiter plate (MTP) module and
incubating at 37 degree C. for 20 min. This allows the streptavadin
coated 96 well plate to associate with the biotinylated peptide.
Wash the MTP module with 300 ul/well of PBS four times. Next add 75
ul of anti-phosphotyrosine antibody conjugated to horse radish
peroxidase (anti-P-Tyr-POD (0.5 u/ml)) to each well and incubate at
37 degree C. for one hour. Wash the well as above.
[0656] Next add 100 ul of peroxidase substrate solution (Boehringer
Mannheim) and incubate at room temperature for at least 5 mins (up
to 30 min). Measure the absorbance of the sample at 405 nm by using
ELISA reader. The level of bound peroxidase activity is quantitated
using an ELISA reader and reflects the level of tyrosine kinase
activity.
Example 18
High-Throughput Screening Assay Identifying Phosphorylation
Activity
[0657] As a potential alternative and/or compliment to the assay of
protein tyrosine kinase activity described in Example 16, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase
(MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[0658] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr
at room temp, (RT). The plates are then rinsed with PBS and blocked
with 3% BSA/PBS for 1 hr at RT. The protein G plates are then
treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4 degree C. until use.
[0659] A431 cells are seeded at 20,000/well in a 96-well Loprodyne
filterplate and cultured overnight in growth medium. The cells are
then starved for 48 hr in basal medium (DMEM) and then treated with
EGF (6 ng/well) or 50 ul of the supernatants obtained in Example 9
for 5-20 minutes. The cells are then solubilized and extracts
filtered directly into the assay plate.
[0660] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
ug/ml) which specifically recognizes the phosphorylated epitope of
the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation by galectin 11 or a molecule induced by galectin
11.
Example 19
Method of Determining Alterations in the Galectin 11 Gene
[0661] RNA isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease) is be
isolated. cDNA is then generated from these RNA samples using
protocols known in the art. (See, Sambrook.) The cDNA is then used
as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO:1. Suggested PCR conditions consist of 35
cycles at 95 degree C. for 30 seconds; 60-120 seconds at 52-58
degree C.; and 60-120 seconds at 70 degree C., using buffer
solutions described in Sidransky, D., et al., Science 252:706
(1991).
[0662] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase. (Epicentre Technologies). The intron-exon borders of
selected exons of galectin 11 is also determined and genomic PCR
products analyzed to confirm the results. PCR products harboring
suspected mutations in galectin 11 is then cloned and sequenced to
validate the results of the direct sequencing.
[0663] PCR products of galectin 11 are cloned into T-tailed vectors
as described in Holton, T. A. and Graham, M. W., Nucleic Acids
Research, 19:1156 (1991) and sequenced with T7 polymerase (United
States Biochemical). Affected individuals are identified by
mutations in galectin 11 not present in unaffected individuals.
[0664] Genomic rearrangements are also observed as a method of
determining alterations in a gene corresponding to galectin 11. The
full length galectin 11 cDNA amplified according to Example 1 is
nick-translated with digoxigenindeoxy-uridine 5'-triphosphate
(Boehringer Manheim), and FISH performed as described in Johnson,
Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with
the labeled probe is carried out using a vast excess of human cot-1
DNA for specific hybridization to the galectin 11 genomic
locus.
[0665] Chromosomes are counterstained with
4,6-diamino-2-phenylidole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl.,
8:75 (1991).) Image collection, analysis and chromosomal fractional
length measurements are performed using the ISee Graphical Program
System. (Inovision Corporation, Durham, N.C.) Chromosome
alterations of the genomic region of galectin 11 (hybridized by the
probe) are identified as insertions, deletions, and translocations.
These galectin 11 alterations are used as a diagnostic marker for
an associated disease.
Example 20
Method of Detecting Abnormal Levels of Galectin 11 in a Biological
Sample
[0666] Galectin 11 polypeptides can be detected in a biological
sample, and if an increased or decreased level of galectin 11 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.
[0667] For example, antibody-sandwich ELISAs are used to detect
galectin 11 in a sample, preferably a biological sample. Wells of a
microtiter plate are coated with specific antibodies to galectin
11, at a final concentration of 0.2 to 10 ug/ml. The antibodies are
either monoclonal or polyclonal and are produced by the method
described in Example 8. The wells are blocked so that non-specific
binding of galectin 11 to the well is reduced.
[0668] The coated wells are then incubated for >2 hours at RT
with a sample containing galectin 11. 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
unbounded galectin 11.
[0669] 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.
[0670] Add 75 ul of 4-methylumbelliferyl phosphate (NPP) or
p-nitrophenyl phosphate (NPP) substrate solution to each well and
incubate 1 hour at room temperature. Measure the reaction by a
microtiter plate reader. Prepare a standard curve, using serial
dilutions of a control sample, and plot galectin 11 polypeptide
concentration on the X-axis (log scale) and fluorescence or
absorbance of the Y-axis (linear scale). Interpolate the
concentration of the galectin 11 in the sample using the standard
curve.
Example 21
Formulation
[0671] The invention also provides methods of treatment and/or
prevention of diseases or disorders (such as, for example, any one
or more of the diseases or disorders disclosed herein) by
administration to a subject of an effective amount of a
Therapeutic. By therapeutic is meant a polynucleotides or
polypeptides of the invention (including fragments and variants),
agonists or antagonists thereof, and/or antibodies thereto, in
combination with a pharmaceutically acceptable carrier type (e.g.,
a sterile carrier).
[0672] The Therapeutic 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 the Therapeutic alone), the site of
delivery, the method of administration, the scheduling of
administration, and other factors known to practitioners. The
"effective amount" for purposes herein is thus determined by such
considerations.
[0673] As a general proposition, the total pharmaceutically
effective amount of the Therapeutic administered parenterally per
dose will be in the range of about 1 ug/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 for the hormone. If given continuously, the
Therapeutic is typically administered at a dose rate of about 1
ug/kg/hour to about 50 ug/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. 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.
[0674] Therapeutics can be are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any. The term "parenteral" as used herein refers to
modes of administration which include intravenous, intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular
injection and infusion.
[0675] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics are administered orally, rectally,
parenterally, intracistemally, intravaginally, intraperitoneally,
topically (as by powders, ointments, gels, drops or transdermal
patch), bucally, or as an oral or nasal spray. "Pharmaceutically
acceptable carrier" refers to a non-toxic solid, semisolid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type. The term "parenteral" as used herein refers
to modes of administration which include intravenous,
intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular injection and infusion.
[0676] Therapeutics of the invention are also suitably administered
by sustained-release systems. Suitable examples of
sustained-release Therapeutics include suitable polymeric materials
(such as, for example, semi-permeable polymer matrices in the form
of shaped articles, e.g., films, or mirocapsules), 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).
[0677] 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 et al., Biopolymers 22:547-556
(1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J.
Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech.
12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0678] Sustained-release Therapeutics also include liposomally
entrapped Therapeutics 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 the Therapeutic are 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 Therapeutic.
[0679] In yet an additional embodiment, the Therapeutics 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)).
[0680] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0681] For parenteral administration, in one embodiment, the
Therapeutic 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 the Therapeutic.
[0682] Generally, the formulations are prepared by contacting the
Therapeutic 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.
[0683] 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, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0684] The Therapeutic is typically formulated in such vehicles at
a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of polypeptide salts.
[0685] Any pharmaceutical used for therapeutic administration can
be sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutics 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.
[0686] Therapeutics 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 Therapeutic solution, and
the resulting mixture is lyophilized. The infusion solution is
prepared by reconstituting the lyophilized Therapeutic using
bacteriostatic Water-for-Injection.
[0687] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the Therapeutics of the invention. 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. In addition, the Therapeutics may be employed in
conjunction with other therapeutic compounds.
[0688] The Therapeutics of the invention may be administered alone
or in combination with adjuvants. Adjuvants that may be
administered with the Therapeutics 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, Therapeutics of the invention are administered
in combination with alum. In another specific embodiment,
Therapeutics of the invention are administered in combination with
QS-21. Further adjuvants that may be administered with the
Therapeutics 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 Therapeutics of the
invention include, but are not limited to, vaccines directed toward
protection against MMR (measles, mumps, rubella), polio, varicella,
tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,
cholera, yellow fever, Japanese encephalitis, poliomyelitis,
rabies, typhoid fever, and pertussis. 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.
[0689] The Therapeutics of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the Therapeutics of
the invention, include but not limited to, other members of the TNF
family, chemotherapeutic agents, antibiotics, steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic
agents, cytokines and/or growth factors. 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.
[0690] In one embodiment, the Therapeutics of the invention are
administered in combination with members of the TNF family. TNF,
TNF-related or TNF-like molecules that may be administered with the
Therapeutics 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), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), TR6 (International
Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921, 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), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153.
[0691] In certain embodiments, Therapeutics 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 Therapeutics of the invention, include, but
are not limited to, RETROVIR.TM. (zidovudine/AZT), VIDEX.TM.
(didanosine/ddI), HVID.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
Therapeutics 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 Therapeutics 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 Therapeutics of the invention to treat AIDS
and/or to prevent or treat HIV infection.
[0692] In other embodiments, Therapeutics of the invention may be
administered in combination with anti-opportunistic infection
agents. Anti-opportunistic agents that may be administered in
combination with the Therapeutics 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,
Therapeutics of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLE.TM., DAPSONE.TM., PENTAMIDINE.TM.,
and/or ATOVAQUONE.TM. to prophylactically treat or prevent an
opportunistic Pneumocystis carinii pneumonia infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with ISONIAZID.TM., RIFAMPIN.TM., PYRAZINAMIDE.TM.,
and/or ETHAMBUTOL.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium avium complex infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with RIFABUTIN.TM., CLARITHROMYCIN.TM., and/or
AZITHROMYCIN.TM. to prophylactically treat or prevent an
opportunistic Mycobacterium tuberculosis infection. In another
specific embodiment, Therapeutics of the invention are used in any
combination with GANCICLOVIR.TM., FOSCARNET.TM., and/or
CIDOFOVIR.TM. to prophylactically treat or prevent an opportunistic
cytomegalovirus infection. In another specific embodiment,
Therapeutics of the invention are used in any combination with
FLUCONAZOLE.TM., ITRACONAZOLE.TM., and/or KETOCONAZOLE.TM. to
prophylactically treat or prevent an opportunistic fungal
infection. In another specific embodiment, Therapeutics of the
invention are used in any combination with ACYCLOVIR.TM. and/or
FAMCICOLVIR.TM. to prophylactically treat or prevent an
opportunistic herpes simplex virus type I and/or type II infection.
In another specific embodiment, Therapeutics of the invention are
used in any combination with PYRIMETHAMINE.TM. and/or
LEUCOVORIN.TM. to prophylactically treat or prevent an
opportunistic Toxoplasma gondii infection. In another specific
embodiment, Therapeutics of the invention are used in any
combination with LEUCOVORIN.TM. and/or NEUPOGEN.TM. to
prophylactically treat or prevent an opportunistic bacterial
infection.
[0693] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antiviral agent. Antiviral
agents that may be administered with the Therapeutics of the
invention include, but are not limited to, acyclovir, ribavirin,
amantadine, and remantidine.
[0694] In a further embodiment, the Therapeutics of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the Therapeutics of
the invention include, but are not limited to, amoxicillin,
beta-lactamases, aminoglycosides, beta-lactam (glycopeptide),
beta-lactamases, Clindamycin, chloramphenicol, cephalosporins,
ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,
macrolides, metronidazole, penicillins, quinolones, rifampin,
streptomycin, sulfonamide, tetracyclines, trimethoprim,
trimethoprim-sulfamthoxazole, and vancomycin.
[0695] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the Therapeutics of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, cyclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents that act by suppressing the function of
responding T cells.
[0696] In specific embodiments, Therapeutics of the invention are
administered in combination with immunosuppressants.
Immunosuppressants preparations that may be administered with the
Therapeutics of the invention include, but are not limited to,
ORTHOCLONE.TM. (OKT3), SANDIMMUNE.TM./NEORAL.TM./SANGDYA.TM.
(cyclosporin), PROGRAF.TM. (tacrolimus), CELLCEPT.TM.
(mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNE.TM.
(sirolimus). In a specific embodiment, immunosuppressants may be
used to prevent rejection of organ or bone marrow
transplantation.
[0697] In an additional embodiment, Therapeutics 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
Therapeutics 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, Therapeutics of the
invention are administered in combination with intravenous immune
globulin preparations in transplantation therapy (e.g., bone marrow
transplant).
[0698] In an additional embodiment, the Therapeutics of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the Therapeutics 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.
[0699] In another embodiment, compostions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
Therapeutics 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.,
medroxyprogesterone, 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).
[0700] In a specific embodiment, Therapeutics of the invention are
administered in combination with CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) or any combination of the
components of CHOP. In another embodiment, Therapeutics of the
invention are administered in combination with Rituximab. In a
further embodiment, Therapeutics of the invention are administered
with Rituxmab and CHOP, or Rituxmab and any combination of the
components of CHOP.
[0701] In an additional embodiment, the Therapeutics of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the Therapeutics of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha.
In another embodiment, Therapeutics 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, and IL-21.
[0702] In an additional embodiment, the Therapeutics of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the Therapeutics
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PlGF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular
Endothelial Growth Factor B-186 (VEGF-B 186), as disclosed in
International Publication Number WO 96/26736; Vascular Endothelial
Growth Factor-D (VEGF-D), as disclosed in International Publication
Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D),
as disclosed in International Publication Number WO 98/07832; and
Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in
German Patent Number DE19639601. The above mentioned references are
incorporated herein by reference herein.
[0703] In an additional embodiment, the Therapeutics of the
invention are administered in combination with hematopoietic growth
factors. Hematopoietic growth factors that may be administered with
the Therapeutics of the invention include, but are not limited to,
LEUKINE.TM. (SARGRAMOSTIM.TM.) and NEUPOGEN.TM.
(FILGRASTIM.TM.).
[0704] In an additional embodiment, the Therapeutics of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the Therapeutics 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.
[0705] In additional embodiments, the Therapeutics of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
Example 22
Method of Treating Decreased Levels of Galectin 11
[0706] The present invention relates to a method for treating an
individual in need of an increased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an agonist of the invention (including polypeptides of
the invention). Moreover, it will be appreciated that conditions
caused by a decrease in the standard or normal expression level of
galectin 11 in an individual can be treated by administering a
galectin 11 of the present invention, preferably in the secreted
form. Thus, the invention also provides a method of treatment of an
individual in need of an increased level of a galectin 11
polypeptide of the present invention comprising administering to
such an individual a Therapeutic comprising an amount of that
galectin 11 to increase the activity level of galectin 11 in such
an individual.
[0707] For example, a patient with decreased levels of galectin 11
polypeptide receives a daily dose 0.1-100 ug/kg of the polypeptide
for six consecutive days. Preferably, the polypeptide is in the
secreted form. The exact details of the dosing scheme, based on
administration and formulation, are provided in Example 20.
Example 23
Method of Treating Increased Levels of Galectin 11
[0708] The present invention also relates to a method of treating
an individual in need of a decreased level of a polypeptide of the
invention in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an antagonist of the invention (including polypeptides
and antibodies of the invention).
[0709] In one example, antisense technology is used to inhibit
production of galectin 11. This technology is one example of a
method of decreasing levels of galectin 11 polypeptide, preferably
a secreted form, due to a variety of etiologies, such as
cancer.
[0710] For example, a patient diagnosed with abnormally increased
levels of galectin 11 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
treatment was well tolerated. The formulation of the antisense
polynucleotide is provided in Example 20.
Example 24
Method of Treatment Using Gene Therapy
Ex Vivo
[0711] One method of gene therapy transplants fibroblasts, which
are capable of expressing galectin 11 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.
[0712] 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.
[0713] 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.
[0714] A cDNA of the present invention encoding galectin 11 can be
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively as set forth in Example 1. 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
bacteria HB101, which are then plated onto agar containing
kanamycin for the purpose of confirming that the vector contains
properly inserted galectin 11.
[0715] 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 galectin 11 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 galectin 11 gene (the packaging cells are now
referred to as producer cells).
[0716] 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 galectin 11 protein is produced.
[0717] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 25
Gene Therapy Using Endogenous Galectin 11 Gene
[0718] Another method of gene therapy according to the present
invention involves operably associating the endogenous galectin 11
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 No. WO 96/29411, published Sep. 26, 1996;
International Publication No. WO 94/12650, published Aug. 4, 1994;
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.
[0719] Polynucleotide constructs are made which contain a promoter
and targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous galectin 11, flanking the promoter. The
targeting sequence will be sufficiently near the 5' end of galectin
11 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.
[0720] 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.
[0721] 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.
[0722] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous galectin 11 sequence. This results in the
expression of galectin 11 in the cell. Expression may be detected
by immunological staining, or any other method known in the
art.
[0723] 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.
[0724] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the galectin
11 locus, plasmid pUC18 (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 galectin 11
non-coding sequences are amplified via PCR: one galectin 11
non-coding sequence (galectin 11 fragment 1) is amplified with a
HindIII site at the 5' end and an Xba site at the 3' end; the other
galectin 11 non-coding sequence (Galectin 11 fragment 2) is
amplified with a BamHI site at the 5' end and a HindIII site at the
3' end. The CMV promoter and galectin 11 fragments (1 and 2) are
digested with the appropriate enzymes (CMV promoter--XbaI and
BamHI; galectin 11 fragment 1--XbaI; galectin 11 fragment 2--BamHI)
and ligated together. The resulting ligation product is digested
with HindIII, and ligated with the HindIII-digested pUC18
plasmid.
[0725] 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.
[0726] 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.
[0727] 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 26
Method of Treatment Using Gene Therapy
In Vivo
[0728] 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) galectin 11
sequences into an animal to increase or decrease the expression of
the galectin 11 polypeptide. The galectin 11 polynucleotide may be
operatively linked to a promoter or any other genetic elements
necessary for the expression of the galectin 11 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, U.S. Pat. No. 5,705,151, U.S.
Pat. No. 5,580,859; Tabata H. et al. (1997) Cardiovasc. Res.
35(3):470-479, Chao J et al. (1997) Pharmacol. Res. 35(6):517-522,
Wolff J. A. (1997) Neuromuscul. Disord. 7(5):314-318, Schwartz B.
et al. (1996) Gene Ther. 3(5):405-411, Tsurumi Y. et al. (1996)
Circulation 94(12):3281-3290 (incorporated herein by
reference).
[0729] The galectin 11 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
galectin 11 polynucleotide constructs can be delivered in a
pharmaceutically acceptable liquid or aqueous carrier.
[0730] 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 or
precipitating agents and the like. However, the galectin 11
polynucleotides may also be delivered in liposome formulations
(such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad.
Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell
85(1):1-7) which can be prepared by methods well known to those
skilled in the art.
[0731] The galectin 11 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 therapies 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.
[0732] The galectin 11 polynucleotide construct can be delivered to
the interstitial space of tissues within the an 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.
[0733] For the naked galectin 11 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 galectin 11 polynucleotide constructs can
be delivered to arteries during angioplasty by the catheter used in
the procedure.
[0734] The dose response effects of injected galectin 11
polynucleotide in muscle in vivo is determined as follows. Suitable
galectin 11 template DNA for production of mRNA coding for galectin
11 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.
[0735] 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 galectin 11 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.
[0736] 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 galectin 11 protein expression. A time
course for galectin 11 protein expression may be done in a similar
fashion except that quadriceps from different mice are harvested at
different times. Persistence of galectin 11 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 galectin 11 naked
DNA.
Example 27
Galectin 11 Transgenic Animals
[0737] The galectin 11 polypeptides 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.
[0738] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides 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, 1983, 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
pleuripotent 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.
[0739] 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
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[0740] 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. 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.
(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.
[0741] 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. (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.
[0742] Any of the galectin 11 polypeptides disclosed throughout
this application can be used to generate transgenic animals. For
example, the DNA encoding galectin 11 protein can be inserted into
a vector using a primer, such as: A 5' primer containing the
underlined SmaI restriction site shown: 5' cgc CCC GGG GCCT
ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:7) and a 3' primer sequence 5'
cgc GGT ACC TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the
underlined Asp718 restriction site followed by nucleotides
complementary to position 432 to 450 of the galectin 11 nucleotide
sequence depicted in FIG. 1 (SEQ ID NO:1). Besides these two
examples, other fragments of galectin 11 can also be inserted into
a vector to create transgenics having ubiquitous expression.
[0743] Alternatively, polynucleotides of the invention can be
inserted in a vector which controls tissue specific expression
through a tissue specific promoter. For example, a construct having
a transferrin promoter would express the galectin 11 polypeptide in
the liver of transgenic animals. Therefore, DNA encoding the full
length galectin 11 protein can also be inserted into a vector for
tissue specific expression using the following primers: A 5' primer
containing the underlined SmaI restriction site shown: 5' cgc CCC
GGG GCCT ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:7) and a 3' primer,
containing the underlined Asp 178 restriction site shown: 5' cgc
GGT ACC TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8)
[0744] In addition to expressing the polypeptide of the present
invention in a ubiquitous or tissue specific manner in transgenic
animals, it would also be routine for one skilled in the art to
generate constructs which regulate expression of the polypeptide by
a variety of other means (for example, developmentally or
chemically regulated expression).
[0745] 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.
[0746] 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.
[0747] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of galectin 11 polypeptides, studying
conditions and/or disorders associated with aberrant galectin 11
expression, and in screening for compounds effective in
ameliorating such conditions and/or disorders.
Example 28
Galectin 11 Knock-Out Animals
[0748] Endogenous galectin 11 gene expression can also be reduced
by inactivating or "knocking out" the galectin 11 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.
[0749] 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 galectin 11 polypeptides. 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.
[0750] 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).
[0751] 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.
[0752] 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 galectin 11 polypeptides, studying
conditions and/or disorders associated with aberrant galectin 11
expression, and in screening for compounds effective in
ameliorating such conditions and/or disorders.
Example 29
Assays Detecting Stimulation or Inhibition of B Cell Proliferation
and Differentiation
[0753] 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, IL-5, IL-6, IL-7, IL-10, IL-13, IL-14 and
IL-15. 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.
[0754] One of the best 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.
[0755] In Vitro Assay--Purified galectin 11 protein, or truncated
forms thereof, is assessed for its ability to induce activation,
proliferation, differentiation or inhibition and/or death in B-cell
populations and their precursors. The activity of galectin 11
protein 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).
[0756] 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 2ME, 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 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor
addition. The positive and negative controls are IL2 and medium
respectively.
[0757] In Vivo Assay--BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of Galectin 11 protein, or truncated
forms 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 galectin 11 protein-treated spleens identify the results of the
activity of galectin 11 protein 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.
[0758] Flow cytometric analyses of the spleens from galectin 11
protein-treated mice is used to indicate whether galectin 11
protein specifically increases the proportion of ThB+, CD45R(B220)
dull B cells over that which is observed in control mice.
[0759] 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 galectin 11 protein-treated mice.
[0760] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 30
T Cell Proliferation Assay
[0761] 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 Galectin 11 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 galectin 11 proteins.
[0762] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 31
Effect of Galectin 11 on the Expression of MHC Class II,
Costimulatory and Adhesion Molecules and Cell Differentiation of
Monocytes and Monocyte-Derived Human Dendritic Cells
[0763] 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.
[0764] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of
galectin 11 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).
[0765] 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 galectin 11 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.
[0766] 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.
[0767] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of galectin 11 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).
[0768] 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. galectin 11, agonists,
or antagonists of galectin 11 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 gradient (Sigma). Monocytes are isolated from PBMC by
counterflow centrifugal elutriation.
[0769] 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
incubaed at room temperature for 5 minutes before FACScan analysis.
PI uptake has been demonstrated to correlate with DNA fragmentation
in this experimental paradigm.
[0770] 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
galectin 11 and under the same conditions, but in the absence of
galectin 11. For IL-12 production, the cells are primed overnight
with IFN (100 U/ml) in presence of galectin 11. 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.)) and applying the standard
protocols provided with the kit.
[0771] Oxidative burst. Purified monocytes are plated in 96-w plate
at 2-1.times.10.sup.5 cell/well. Increasing concentrations of
galectin 11 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.
[0772] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 32
Galectin 11 Biological Effects
[0773] Astrocyte and Neuronal Assays.
[0774] Recombinant galectin 11, expressed in Escherichia coli and
purified as described above, can be tested for activity in
promoting the survival, neurite outgrowth, or phenotypic
differentiation of cortical neuronal cells and for inducing the
proliferation of glial fibrillary acidic protein immunopositive
cells, astrocytes. The selection of cortical cells for the bioassay
is based on the prevalent expression of FGF-1 and FGF-2 in cortical
structures and on the previously reported enhancement of cortical
neuronal survival resulting from FGF-2 treatment. A thymidine
incorporation assay, for example, can be used to elucidate galectin
11's activity on these cells.
[0775] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke, P. et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary cortical neuronal
culture paradigm, the ability of galectin 11 to induce neurite
outgrowth can be compared to the response achieved with FGF-2
using, for example, a thymidine incorporation assay.
[0776] Fibroblast and Endothelial Cell Assays.
[0777] Human lung fibroblasts are obtained from Clonetics (San
Diego, Calif.) and maintained in growth media from Clonetics.
Dermal microvascular endothelial cells are obtained from Cell
Applications (San Diego, Calif.). For proliferation assays, the
human lung fibroblasts and dermal microvascular endothelial cells
can be cultured at 5,000 cells/well in a 96-well plate for one day
in growth medium. The cells are then incubated for one day in 0.1%
BSA basal medium. After replacing the medium with fresh 0.1% BSA
medium, the cells are incubated with the test proteins for 3 days.
Alamar Blue (Alamar Biosciences, Sacramento, Calif.) is added to
each well to a final concentration of 10%. The cells are incubated
for 4 hr. Cell viability is measured by reading in a CytoFluor
fluorescence reader. For the PGE.sub.2 assays, the human lung
fibroblasts are cultured at 5,000 cells/well in a 96-well plate for
one day. After a medium change to 0.1% BSA basal medium, the cells
are incubated with FGF-2 or galectin 11 with or without IL-1.alpha.
for 24 hours. The supernatants are collected and assayed for
PGE.sub.2 by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6
assays, the human lung fibroblasts are cultured at 5,000 cells/well
in a 96-well plate for one day. After a medium change to 0.1% BSA
basal medium, the cells are incubated with FGF-2 or galectin 11
with or without IL-1.alpha. for 24 hours. The supernatants are
collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge,
Mass.).
[0778] Human lung fibroblasts are cultured with FGF-2 or galectin
11 for 3 days in basal medium before the addition of Alamar Blue to
assess effects on growth of the fibroblasts. FGF-2 should show a
stimulation at 10-2500 ng/ml which can be used to compare
stimulation with galectin 11.
[0779] Parkinson Models.
[0780] The loss of motor function in Parkinson's disease is
attributed to a deficiency of striatal dopamine resulting from the
degeneration of the nigrostriatal dopaminergic projection neurons.
An animal model for Parkinson's that has been extensively
characterized involves the systemic administration of 1-methyl-4
phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is
taken-up by astrocytes and catabolized by monoamine oxidase B to
1-methyl-4-phenyl pyridine (MPP.sup.+) and released. Subsequently,
MPP.sup.+ is actively accumulated in dopaminergic neurons by the
high-affinity reuptake transporter for dopamine. MPP.sup.+ is then
concentrated in mitochondria by the electrochemical gradient and
selectively inhibits nicotidamide adenine disphosphate: ubiquinone
oxidoreductionase (complex I), thereby interfering with electron
transport and eventually generating oxygen radicals.
[0781] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[0782] Based on the data with FGF-2, galectin 11 can be evaluated
to determine whether it has an action similar to that of FGF-2 in
enhancing dopaminergic neuronal survival in vitro and it can also
be tested in vivo for protection of dopaminergic neurons in the
striatum from the damage associated with MPTP treatment. The
potential effect of galectin 11 is first examined in vitro in a
dopaminergic neuronal cell culture paradigm. The cultures are
prepared by dissecting the midbrain floor plate from gestation day
14 Wistar rat embryos. The tissue is dissociated with trypsin and
seeded at a density of 200,000 cells/cm.sup.2 on
polyorthinine-laminin coated glass coverslips. The cells are
maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N1). The cultures are fixed with
paraformaldehyde after 8 days in vitro and are processed for
tyrosine hydroxylase, a specific marker for dopminergic neurons,
immunohistochemical staining. Dissociated cell cultures are
prepared from embryonic rats. The culture medium is changed every
third day and the factors are also added at that time.
[0783] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if galectin 11 acts to prolong the
survival of dopaminergic neurons, it would suggest that galectin 11
may be involved in Parkinson's Disease.
[0784] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 33
The Effect of Galectin 11 on the Growth of Vascular Endothelial
Cells
[0785] 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. Galectin 11 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.
[0786] An increase in the number of HUVEC cells indicates that
galectin 11 may proliferate vascular endothelial cells.
[0787] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 34
Stimulatory Effect of Galectin 11 on the Proliferation of Vascular
Endothelial Cells
[0788] For evaluation of mitogenic activity of growth factors, the
colorimetric 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).
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 Galectin 11 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).
[0789] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 35
Inhibition of PDGF-Induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
[0790] HAoSMC proliferation can be measured, for example, by BrdUrd
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 BrdUrd.
After 24 h, immunocytochemistry is performed by using BrdUrd
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdUrd antibody at
4.degree. C. for 2 h after being exposed to denaturing solution and
then incubated with the streptavidin-peroxidase and
diaminobenzidine. After counterstaining with hematoxylin, the cells
are mounted for microscopic examination, and the BrdUrd-positive
cells are counted. The BrdUrd index is calculated as a percent of
the BrdUrd-positive cells to the total cell number. In addition,
the simultaneous detection of the BrdUrd 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.
6:271(36):21985-21992 (1996).
[0791] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 36
Stimulation of Endothelial Migration
[0792] This example will be used to explore the possibility that
galectin 11 may stimulate lymphatic endothelial cell migration.
[0793] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D;
Falk, W., et al., J. Immunological Methods 1980; 33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 um (Nucleopore Corp. Cambridge, Mass.) 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.
[0794] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 37
Stimulation of Nitric Oxide Production by Endothelial Cells
[0795] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation. Thus,
galectin 11 activity can be assayed by determining nitric oxide
production by endothelial cells in response to galectin 11.
[0796] 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 galectin 11. 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 galectin 11 on nitric oxide release is
examined on HUVEC.
[0797] 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.) (1049).
Calibration of the NO elements is performed according to the
following equation:
2KNO.sub.2+2KI+2H.sub.2SO.sub.462NO+I.sub.2+2H.sub.2O+2K.sub.2SO.sub.4
[0798] The standard calibration curve is obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
mol/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
(1050). 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).
[0799] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 38
Effect of Galectin 11 on Cord Formation in Angiogenesis
[0800] 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.
[0801] 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 ml/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 mg Cell Applications' Chord Formation Medium
containing control buffer or galectin 11 (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.
[0802] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. b-estradiol (1 ng/ml) is used as a negative control. The
appropriate buffer (without protein) is also utilized as a
control.
[0803] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 39
Angiogenic Effect on Chick Chorioallantoic Membrane
[0804] 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 Galectin 11 to
stimulate angiogenesis in CAM can be examined.
[0805] Fertilized eggs of the White Leghorn chick (Gallus gallus)
and the Japanese qual (Coturnix coturnix) are incubated at
37.8.degree. C. and 80% humidity. Differentiated CAM of 16-day-old
chick and 13-day-old qual embryos is studied with the following
methods.
[0806] On Day 4 of development, a window is made into the egg shell
of chick eggs. The embryos are checked for normal development and
the eggs sealed with cellotape. They are further incubated until
Day 13. Thermanox coverslips (Nunc, Naperville, Ill.) are cut into
disks of about 5 mm in diameter. Sterile and salt-free growth
factors 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.
[0807] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 40
Angiogenesis Assay Using a Matrigel Implant in Mouse
[0808] In vivo angiogenesis assay of galectin 11 measures the
ability of an existing capillary network to form new vessels in an
implanted capsule of murine extracellular matrix material
(Matrigel). The protein is mixed with the liquid Matrigel at 4
degree C. and the mixture is then injected subcutaneously in mice
where it solidifies. After 7 days, the solid "plug" of Matrigel is
removed and examined for the presence of new blood vessels.
Matrigel is purchased from Becton Dickinson Labware/Collaborative
Biomedical Products.
[0809] When thawed at 4 degree C. the Matrigel material is a
liquid. The Matrigel is mixed with galectin 11 at 150 ng/ml at 4
degree C. and drawn into cold 3 ml syringes. Female C57B1/6 mice
approximately 8 weeks old are injected with the mixture of Matrigel
and experimental protein at 2 sites at the midventral aspect of the
abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by
cervical dislocation, the Matrigel plugs are removed and cleaned
(i.e., all clinging membranes and fibrous tissue is removed).
Replicate whole plugs are fixed in neutral buffered 10%
formaldehyde, embedded in paraffin and used to produce sections for
histological examination after staining with Masson's Trichrome.
Cross sections from 3 different regions of each plug are processed.
Selected sections are stained for the presence of vWF. The positive
control for this assay is bovine basic FGF (150 ng/ml). Matrigel
alone is used to determine basal levels of angiogenesis.
[0810] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 41
Rescue of Ischemia in Rabbit Lower Limb Model
[0811] To study the in vivo effects of galectin 11 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 galectin 11
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 galectin 11 is used in the
treatment, a single bolus of 500 mg galectin 11 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.
[0812] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 42
Effect of Galectin 11 on Vasodilation
[0813] Since dilation of vascular endothelium is important in
reducing blood pressure, the ability of galectin 11 to affect the
blood pressure in spontaneously hypertensive rats (SHR) is
examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of
the galectin 11 are administered to 13-14 week old spontaneously
hypertensive rats (SHR). Data are expressed as the mean +/-SEM.
Statistical analysis are performed with a paired t-test and
statistical significance is defined as p<0.05 vs. the response
to buffer alone.
[0814] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 43
Rat Ischemic Skin Flap Model
[0815] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. Galectin 11 expression, during the
skin ischemia, is studied using in situ hybridization.
[0816] The study in this model is divided into three parts as
follows:
[0817] a) Ischemic skin
[0818] b) Ischemic skin wounds
[0819] c) Normal wounds
[0820] The experimental protocol includes:
[0821] a) Raising a 3.times.4 cm, single pedicle full-thickness
random skin flap (myocutaneous flap over the lower back of the
animal).
[0822] b) An excisional wounding (4-6 mm in diameter) in the
ischemic skin (skin-flap).
[0823] c) Topical treatment with galectin 11 of the excisional
wounds (day 0, 1, 2, 3, 4 post-wounding) at the following various
dosage ranges: 1 mg to 100 mg.
[0824] d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and
21 post-wounding for histological, immunohistochemical, and in situ
studies.
[0825] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 44
Peripheral Arterial Disease Model
[0826] Angiogenic therapy using galectin 11 is a novel therapeutic
strategy to obtain restoration of blood flow around the ischemia in
case of peripheral arterial diseases. The experimental protocol
includes:
[0827] 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.
[0828] b) Galectin 11 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.
[0829] c) The ischemic muscle tissue is collected after ligation of
the femoral artery at 1, 2, and 3 weeks for the analysis of
galectin 11 expression and histology. Biopsy is also performed on
the other side of normal muscle of the contralateral hindlimb.
[0830] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 45
Ischemic Myocardial Disease Model
[0831] Galectin 11 is evaluated as a potent mitogen capable of
stimulating the development of collateral vessels, and
restructuring new vessels after coronary artery occlusion.
Alteration of galectin 11 expression is investigated in situ. The
experimental protocol includes:
[0832] 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.
[0833] b) Galectin 11 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.
[0834] c) Thirty days after the surgery, the heart is removed and
cross-sectioned for morphometric and in situ analyzes.
[0835] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 46
Rat Corneal Wound Healing Model
[0836] This animal model shows the effect of galectin 11 on
neovascularization. The experimental protocol includes:
[0837] a) Making a 1-1.5 mm long incision from the center of cornea
into the stromal layer.
[0838] b) Inserting a spatula below the lip of the incision facing
the outer corner of the eye.
[0839] c) Making a pocket (its base is 1-1.5 mm form the edge of
the eye).
[0840] d) Positioning a pellet, containing 50 ng-5 ug of galectin
11, within the pocket.
[0841] e) Galectin 11 treatment can also be applied topically to
the corneal wounds in a dosage range of 20 mg-500 mg (daily
treatment for five days).
[0842] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 47
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[0843] A. Diabetic db+/db+ Mouse Model.
[0844] To demonstrate that galectin 11 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); Greenhalgh, D. G. et al., Am. J.
Pathol. 136:1235 (1990)).
[0845] 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. 0:1375-1377(1978)).
[0846] 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)).
[0847] 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 are 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.
[0848] 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.
[0849] 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.
[0850] Galectin 11 is administered using at a range different doses
of galectin 11, from 4 mg to 500 mg per wound per day for 8 days in
vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[0851] 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.
[0852] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) untreated; and 3) treated group.
[0853] 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 is 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations are made using
the following formula: [Open area on day 8]-[Open area on day
1]/[Open area on day 1]
[0854] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) 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
galectin 11. 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.
[0855] 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.
[0856] 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 can serve as a
positive tissue control and human brain tissue can be used as a
negative tissue control. Each specimen includes 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.
[0857] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[0858] B. Steroid Impaired Rat Model
[0859] 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: Antiinflammatory 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: Antiinflammatory 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)).
[0860] To demonstrate that galectin 11 can accelerate the healing
process, the effects of multiple topical applications of galectin
11 on full thickness excisional skin wounds in rats in which
healing has been impaired by the systemic administration of
methylprednisolone is assessed.
[0861] 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 are 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.
[0862] 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.
[0863] 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 is no longer visible and the wound is covered by
a continuous epithelium.
[0864] Galectin 11 is administered using at a range different doses
of galectin 11, from 4 mg to 500 mg per wound per day for 8 days in
vehicle. Vehicle control groups received 50 mL of vehicle
solution.
[0865] 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.
[0866] Four groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) are evaluated: 1) Untreated group 2)
Vehicle placebo control 3) galectin 11 treated groups.
[0867] 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 is 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations are made using the following
formula: [Open area on day 8]-[Open area on day 1]/[Open area on
day 1]
[0868] 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 is 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 is improved by treatment with galectin 11. A
calibrated lens micrometer is used by a blinded observer to
determine the distance of the wound gap.
[0869] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[0870] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 48
Lymphadema Animal Model
[0871] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of Galectin 11 in lymphangiogenesis and
reestablishment 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.
[0872] 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.
[0873] 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.
[0874] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. 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.
[0875] 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 .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[0876] 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.
[0877] 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.
[0878] 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 (Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[0879] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca2+ comparison.
[0880] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[0881] 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 is observed under fluorescent microscopy for
lymphatics.
[0882] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 49
Suppression of TNF Alpha-Induced Adhesion Molecule Expression by
Galectin 11
[0883] The recruitment of lymphocytes to areas of inflammation and
angiogenesis involves specific receptor-ligand interactions between
cell surface adhesion molecules (CAMs) on lymphocytes and the
vascular endothelium. The adhesion process, in both normal and
pathological settings, follows a multi-step cascade that involves
intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion
molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1
(E-selectin) expression on endothelial cells (EC). The expression
of these molecules and others on the vascular endothelium
determines the efficiency with which leukocytes may adhere to the
local vasculature and extravasate into the local tissue during the
development of an inflammatory response. The local concentration of
cytokines and growth factor participate in the modulation of the
expression of these CAMs.
[0884] Tumor necrosis factor alpha (TNF-a), a potent
proinflammatory cytokine, is a stimulator of all three CAMs on
endothelial cells and may be involved in a wide variety of
inflammatory responses, often resulting in a pathological
outcome.
[0885] The potential of galectin 11 to mediate a suppression of
TNF-a induced CAM expression can be examined. A modified ELISA
assay which uses ECs as a solid phase absorbent is employed to
measure the amount of CAM expression on TNF-a treated ECs when
co-stimulated with a member of the FGF family of proteins.
[0886] To perform the experiment, human umbilical vein endothelial
cell (HUVEC) cultures are obtained from pooled cord harvests and
maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.)
supplemented with 10% FCS and 1% penicillin/streptomycin in a 37
degree C. humidified incubator containing 5% CO2. HUVECs are seeded
in 96-well plates at concentrations of 1.times.104 cells/well in
EGM medium at 37 degree C. for 18-24 hrs or until confluent. The
monolayers are subsequently washed 3 times with a serum-free
solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100
mg/ml streptomycin, and treated with a given cytokine and/or growth
factor(s) for 24 h at 37 degree C. Following incubation, the cells
are then evaluated for CAM expression.
[0887] Human Umbilical Vein Endothelial cells (HUVECs) are grown in
a standard 96 well plate to confluence. Growth medium is removed
from the cells and replaced with 90 ul of 199 Medium (10% FBS).
Samples for testing and positive or negative controls are added to
the plate in triplicate (in 10 ul volumes). Plates are incubated at
37 degree C. for either 5 h (selectin and integrin expression) or
24 h (integrin expression only). Plates are aspirated to remove
medium and 100 .mu.l of 0.1% paraformaldehyde-PBS (with Ca++ and
Mg++) is added to each well. Plates are held at 4.degree. C. for 30
min.
[0888] Fixative is then removed from the wells and wells are washed
1.times. with PBS (+Ca,Mg)+0.5% BSA and drained. Do not allow the
wells to dry. Add 10 .mu.l of diluted primary antibody to the test
and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and
Anti-E-selectin-Biotin are used at a concentration of 10 .mu.g/ml
(1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at
37.degree. C. for 30 min. in a humidified environment. Wells are
washed .times.3 with PBS (+Ca,Mg)+0.5% BSA.
[0889] Then add 20 .mu.l of diluted ExtrAvidin-Alkaline Phosphotase
(1:5,000 dilution) to each well and incubated at 37.degree. C. for
30 min. Wells are washed .times.3 with PBS (+Ca,Mg)+0.5% BSA. 1
tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of
glycine buffer (pH 10.4). 100 .mu.l of pNPP substrate in glycine
buffer is added to each test well. Standard wells in triplicate are
prepared from the working dilution of the ExtrAvidin-Alkaline
Phosphotase in glycine buffer: 1:5,000
(10.sup.0)>10.sup.-0.5>10.sup.-1>10.sup.-1.5. 5 .mu.l of
each dilution is added to triplicate wells and the resulting AP
content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100
.mu.l of pNNP reagent must then be added to each of the standard
wells. The plate must be incubated at 37.degree. C. for 4 h. A
volume of 50 .mu.l of 3M NaOH is added to all wells. The results
are quantified on a plate reader at 405 nm. The background
subtraction option is used on blank wells filled with glycine
buffer only. The template is set up to indicate the concentration
of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng;
0.18 ng]. Results are indicated as amount of bound AP-conjugate in
each sample.
[0890] The studies described in this example tested activity in
galectin 11 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of galectin 11
polynucleotides (e.g., gene therapy), agonists, and/or antagonists
of galectin 11.
Example 50
Galectin 11 Regulates Cell Cycle and Inhibits Cancer Cell
Proliferation
Carbohydrate Binding
[0891] The amino acid sequence of the C-terminal domain of galectin
11 strays significantly from the conenseus sequence for galectins,
and most of the conserved residues in the region that contact
carbohydrate found in other galectins are not present in galectin
11. Thus, the ability of galectin 11 to bind carbohydrate was
tested. Lysate from HeLa cells transfected with galectin 11.alpha.
or .beta. cDNA were mixed with lactosyl-Sepaharose 4B and the bound
proteins were eluted with the SDS sample buffer. Both unbound
fractions and eluted materials were analyzed by immunoblotting
using antibodies specific for an internal peptide of galectin 11.
Galectin 11 did not bind to lacosyl-Sepharose 4B, while in the same
experiment, lactose binding was obtained for galectin-3, as
expected.
Tissue Distribution of Galectin 11 mRNA
[0892] Northern blot analysis showed that galectin 11 mRNA was
nearly undetectable in many tissues tested, in contrast to
galectin-3 mRNA, which was detected in almost all tissues. The
results were not due to the quality of the galectin 11 cDNA probe,
as strong signals were observed in Northern blot of mRNA from cells
transfected with galectin 11 cDNA, using the same probe. However,
using a more sensitive procedure, RT-PCR, galectin 11 mRNA was
detectable in heart, spleen, thymus and peripheral blood
leukocytes. It was also present at lower levels in lung, skeletal
muscle, kidney, pancreas, prostate, testis, ovary and colon but
virtually undetectable in brain and liver. Galectin 11 mRNA was not
detected in many cell lines tested, but its expression was
confirmed by RT-PCR in peripheral blood monocytes and polynucleated
cells as well as myeloid cell lines, U937, HL-60 and KU-812, the
B-cell line Wil-2, and breast cancer cell line HBL-100. Immunoblot
analysis with the anti-peptide antibodies, however, failed to
detect galectin 11 protein in these cell lines, although the
procedures detected the protein in lysates from galectin 11
transfectants.
Induction of Galectin 11 Expression by Stress Signals
[0893] Since a galectin 11 clone was isolated from a Jurkat cell
library arrested at G1, it was of interest to evaluate the
expression of the message under these conditions, and other
conditions that induce cell stasis. HeLa-S3 and Jurkat cells were
treated with thymidine to synchronize cells at the G1-S border or
theophylline and dibutyryl-cAMP for synchronization of cells at G1.
Both treatments induced galectin 11 expression. AU-rich elements in
galectin 11 mRNA and its restricted expression pattern suggest that
galectin 11 gene product is inducible. Indeed, when HL-60 cells
were treated with PMA plus ionomycin, galectin 11 mRNA was rapidly
induced within 0.5 hr, but the message quickly diminished 2 hr
after the treatment and was no longer detectable 4 hr later.
Cell Cycle Arrest by Ectopic Expression of Galectin 11
[0894] The fact that the galectin 11 was isolated from a cDNA
library derived from a Jurkat cell line arrested at the G1 phase
suggested that this protein may function in regulation of cell
cycle. To test this possibility, a human cervical cancer cell line
HeLa were cotransfected with vectors containing the complete cDNA
for galectin 11 (both alpha and beta) and a plasmid containing
green fluorescent protein (GFP) cDNA.
[0895] Compared with control vector-transfected HeLa cells,
significantly higher percentage of cells expressing HA-tagged
galectin 11 was found at the G1 phase of cell cycle, and
compensatory lower percentages at the S and G2/M phases (data not
shown). Similar results were obtained with a breast cancer cell
line, MCF-7 (data not shown). The effects of the alpha and beta
forms of galectin 11 were comparable.
[0896] Another member of the galectin family, galectin-9, which
also contains two CRD, was studied for comparison. A hemagglutinin
(HA)-tagged galectin-9 was used and galectin 11.quadrature. so that
their expression levels in transfected cells could be compared by
immunoblotting using anti-HA antibodies. Galectin-9 levels were
much higher than galectin 11 levels in the respective
transfectants, even though an identical recipient cell line and
procedure were used. Significantly, while galectin-9 expression did
not have any notable effect on the cell cycle, galectin 11 induced
G1 arrest in a dose-dependent manner.
[0897] To confirm the above findings, a transfection system based
on adenovirus was employed, which is known to have a very high
transfection efficiency in a variety of cell types. Thus,
replication-defective adenoviruses containing galectin 11 were used
to infect Arpe-19 cells. Initial experiments confirmed that close
to 100% of the cells were infected, on the basis of expression of
GFP, as measured by flow cytometry. A significantly higher
percentage of cells transfected with galectin 11 was in the G1
phase, compared with control transfectants. To demonstrate that the
accumulation of cells at G1 was a result of G1 arrest rather than
accelerated M to G1 transition, experiments were conducted in which
nocodazole, a drug known to destroy spindle fibers and thus prevent
the cells from exiting mitosis, was added to the cells after
transfection. Forty-eight hr after the treatment, most cells
infected with the control virus have exited G1 and were blocked at
mitosis, while a significant portion of cells infected with viruses
expressing either the .quadrature. or .beta. isoforms of galectin
11 remained at G1.
Inhibition of Cancer Cell Growth by Ectopic Expression of Galectin
11
[0898] Cell cycle arrest activity of galectin 11 indicated that
cells transfected with galectin 11 would fail to proliferate. A
colony formation assay was performed to formally demonstrate the
suppression of cell growth resulting from the ectopic expression of
galectin 1. HeLa cells were co-transfected with galectin 11 cDNA
together with a construct for puromycin-resistance gene. While the
control transfectants formed numerous colonies, galectin 11
transfectants formed only a few colonies, two weeks after selection
in a puromycin-containing medium. The effect of galectin 11
expression on suppression of cell growth was measured more
quantitatively by using a colorimetric assay. Significantly lower
numbers of cells were obtained fromgalectin 11 transfectants as
compared to control transfectants.
[0899] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention. Indeed,
various modifications of the invention, in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and accompanying drawings. Such
modifications are intended to fall within the scope of the appended
claims.
[0900] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[0901] Further, the Sequence Listing submitted herewith in paper
and computer readable form is herein incorporated by reference in
its entirely.
Sequence CWU 1
1
27 1 865 DNA Homo sapiens 1 tttgtggagg gcagcagaga gtacccagct
ggacatcctt tcctgctgat gagccccagg 60 ctggaggtgc cctgctcaca
tgctcttccc cagggtctct cgcctgggca ggtcatcata 120 gtacggggac
tggtcttgca agagccgaag cattttactg tgagcctgag ggaccaggct 180
gcccatgctc ctgtgacact cagggcctcc ttcgcagaca gaactctggc ctggatctcc
240 cgctgggggc agaagaaact gatctcagcc cccttcctct tttaccccca
gagattcttt 300 gaggtgctgc tcctgttcca ggagggaggg ctgaagctgg
cgctcaatgg gcaggggctg 360 ggggccacca gcatgaacca gcaggccctg
gagcagctgc gggagctccg gatcagtgga 420 agtgtccagc tctactgtgt
ccactcctga aggatggttc caggaaatac cgcagaaaac 480 aagagtcagc
cactccccag ggccccactc tcctcccctc attaaaccat ccacctgaac 540
accagcacat cagggcctgg ttcacctctg gggtcacgag actgagtcta caggagcttt
600 gggcctgagg gaaggcacaa gagtgcaaag gttcctcgaa ctctgcacct
tcctccacca 660 ggagcctggg atatggctcc atctgccttc agggcctgga
ctgcactcac agaggcaagt 720 gttgtagact aacaaagata ctccaaaata
caatggctta aagaatgtgg tcatttattc 780 tttattattt atttatttgt
ggtcaaataa ataaataagg ttatttattt aaaaaaaaaa 840 aaaaaaaaaa
aaaaaaaaaa aaaaa 865 2 133 PRT Homo sapiens 2 Met Ser Pro Arg Leu
Glu Val Pro Cys Ser His Ala Leu Pro Gln Gly 1 5 10 15 Leu Ser Pro
Gly Gln Val Ile Ile Val Arg Gly Leu Val Leu Gln Glu 20 25 30 Pro
Lys His Phe Thr Val Ser Leu Arg Asp Gln Ala Ala His Ala Pro 35 40
45 Val Thr Leu Arg Ala Ser Phe Ala Asp Arg Thr Leu Ala Trp Ile Ser
50 55 60 Arg Trp Gly Gln Lys Lys Leu Ile Ser Ala Pro Phe Leu Phe
Tyr Pro 65 70 75 80 Gln Arg Phe Phe Glu Val Leu Leu Leu Phe Gln Glu
Gly Gly Leu Lys 85 90 95 Leu Ala Leu Asn Gly Gln Gly Leu Gly Ala
Thr Ser Met Asn Gln Gln 100 105 110 Ala Leu Glu Gln Leu Arg Glu Leu
Arg Ile Ser Gly Ser Val Gln Leu 115 120 125 Tyr Cys Val His Ser 130
3 145 PRT Homo sapiens 3 Met Ser Ser Phe Ser Thr Gln Thr Pro Tyr
Pro Asn Leu Ala Val Pro 1 5 10 15 Phe Phe Thr Ser Ile Pro Asn Gly
Leu Tyr Pro Ser Lys Ser Ile Val 20 25 30 Ile Ser Gly Val Val Leu
Ser Asp Ala Lys Arg Phe Gln Ile Asn Leu 35 40 45 Arg Cys Gly Gly
Asp Ile Ala Phe His Leu Asn Pro Arg Phe Asp Glu 50 55 60 Asn Ala
Val Val Arg Asn Thr Gln Ile Asn Asn Ser Trp Gly Pro Glu 65 70 75 80
Glu Arg Ser Leu Pro Gly Ser Met Pro Phe Ser Arg Gly Gln Arg Phe 85
90 95 Ser Val Trp Ile Leu Cys Glu Gly His Cys Phe Lys Val Ala Val
Asp 100 105 110 Gly Gln His Ile Cys Glu Tyr Ser His Arg Leu Met Asn
Leu Pro Asp 115 120 125 Ile Asn Thr Leu Glu Val Ala Gly Asp Ile Gln
Leu Thr His Val Glu 130 135 140 Thr 145 4 318 PRT Homo sapiens 4
Met Met Leu Ser Leu Asn Asn Leu Gln Asn Ile Ile Tyr Asn Pro Val 1 5
10 15 Ile Pro Phe Val Gly Thr Ile Pro Asp Gln Leu Asp Pro Gly Thr
Leu 20 25 30 Ile Val Ile Arg Gly His Val Pro Ser Asp Ala Asp Arg
Phe Gln Val 35 40 45 Asp Leu Gln Asn Gly Ser Ser Met Lys Pro Arg
Ala Asp Val Ala Phe 50 55 60 His Phe Asn Pro Arg Phe Lys Arg Ala
Gly Cys Ile Val Cys Asn Thr 65 70 75 80 Leu Ile Asn Glu Lys Trp Gly
Arg Glu Glu Ile Thr Tyr Asp Thr Pro 85 90 95 Phe Gln Lys Glu Lys
Lys Ser Phe Glu Ile Val Ile Met Val Leu Lys 100 105 110 Ala Lys Phe
Gln Val Ala Val Asn Gly Lys His Thr Leu Leu Tyr Gly 115 120 125 His
Arg Ile Gly Pro Glu Lys Ile Asp Thr Leu Gly Ile Tyr Gly Lys 130 135
140 Val Asn Ile His Ser Ile Gly Phe Ser Phe Ser Ser Asp Leu Gln Ser
145 150 155 160 Thr Gln Ala Ser Ser Leu Glu Leu Thr Glu Ile Ser Arg
Glu Asn Val 165 170 175 Pro Lys Ser Gly Thr Pro Gln Leu Arg Leu Pro
Phe Ala Ala Arg Leu 180 185 190 Asn Thr Pro Met Gly Pro Gly Arg Thr
Val Val Val Lys Gly Glu Val 195 200 205 Asn Ala Asn Ala Lys Ser Phe
Asn Val Asp Leu Leu Ala Gly Lys Ser 210 215 220 Lys Asp Ile Ala Leu
His Leu Asn Pro Arg Leu Asn Ile Lys Ala Phe 225 230 235 240 Val Arg
Asn Ser Phe Leu Gln Glu Ser Trp Gly Glu Glu Glu Arg Asn 245 250 255
Ile Thr Ser Phe Pro Phe Ser Pro Gly Met Tyr Phe Glu Met Ile Ile 260
265 270 Tyr Cys Asp Val Arg Glu Phe Lys Val Ala Val Asn Gly Val His
Ser 275 280 285 Leu Glu Tyr Lys His Arg Phe Lys Glu Leu Ser Ser Ile
Asp Thr Leu 290 295 300 Glu Ile Asn Gly Asp Ile His Leu Leu Glu Val
Arg Ser Trp 305 310 315 5 5 PRT Homo sapiens Site (3) Xaa equals
any amino acid 5 Trp Ser Xaa Trp Ser 1 5 6 28 DNA Homo sapiens 6
cgcaagcttt caggagtgga cacagtag 28 7 32 DNA Homo sapiens 7
cgccccgggg cctatgagcc ccaggctgga gg 32 8 28 DNA Homo sapiens 8
cgcggtacct caggagtgga cacagtag 28 9 44 DNA Homo sapiens 9
cgccccgggg ccatcatggc ctatcatgag ccccaggctg gagg 44 10 23 DNA Homo
sapiens 10 cgccgccacc atgagcccca ggc 23 11 22 DNA Homo sapiens 11
ggaatctaga tcaggagtgg ac 22 12 865 DNA Homo sapiens 12 tttttttttt
tttttttttt tttttttttt tttttaaata aataacctta tttatttatt 60
tgaccacaaa taaataaata ataaagaata aatgaccaca ttctttaagc cattgtattt
120 tggagtatct ttgttagtct acaacacttg cctctgtgag tgcagtccag
gccctgaagg 180 cagatggagc catatcccag gctcctggtg gaggaaggtg
cagagttcga ggaacctttg 240 cactcttgtg ccttccctca ggcccaaagc
tcctgtagac tcagtctcgt gaccccagag 300 gtgaaccagg ccctgatgtg
ctggtgttca ggtggatggt ttaatgaggg gaggagagtg 360 gggccctggg
gagtggctga ctcttgtttt ctgcggtatt tcctggaacc atccttcagg 420
agtggacaca gtagagctgg acacttccac tgatccggag ctcccgcagc tgctccaggg
480 cctgctggtt catgctggtg gcccccagcc cctgcccatt gagcgccagc
ttcagccctc 540 cctcctggaa caggagcagc acctcaaaga atctctgggg
gtaaaagagg aagggggctg 600 agatcagttt cttctgcccc cagcgggaga
tccaggccag agttctgtct gcgaaggagg 660 ccctgagtgt cacaggagca
tgggcagcct ggtccctcag gctcacagta aaatgcttcg 720 gctcttgcaa
gaccagtccc cgtactatga tgacctgccc aggcgagaga ccctggggaa 780
gagcatgtga gcagggcacc tccagcctgg ggctcatcag caggaaagga tgtccagctg
840 ggtactctct gctgccctcc acaaa 865 13 733 DNA Homo sapiens 13
gggatccgga gcccaaatct tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg
60 aattcgaggg tgcaccgtca gtcttcctct tccccccaaa acccaaggac
accctcatga 120 tctcccggac tcctgaggtc acatgcgtgg tggtggacgt
aagccacgaa gaccctgagg 180 tcaagttcaa ctggtacgtg gacggcgtgg
aggtgcataa tgccaagaca aagccgcggg 240 aggagcagta caacagcacg
taccgtgtgg tcagcgtcct caccgtcctg caccaggact 300 ggctgaatgg
caaggagtac aagtgcaagg tctccaacaa agccctccca acccccatcg 360
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc
420 catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct 480 atccaagcga catcgccgtg gagtgggaga gcaatgggca
gccggagaac aactacaaga 540 ccacgcctcc cgtgctggac tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg 600 acaagagcag gtggcagcag
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660 acaaccacta
cacgcagaag agcctctccc tgtctccggg taaatgagtg cgacggccgc 720
gactctagag gat 733 14 86 DNA Homo sapiens 14 gcgcctcgag atttccccga
aatctagatt tccccgaaat gatttccccg aaatgatttc 60 cccgaaatat
ctgccatctc aattag 86 15 27 DNA Homo sapiens 15 gcggcaagct
ttttgcaaag cctaggc 27 16 271 DNA Homo sapiens 16 ctcgagattt
ccccgaaatc tagatttccc cgaaatgatt tccccgaaat gatttccccg 60
aaatatctgc catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
120 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
ttttttttat 180 ttatgcagag gccgaggccg cctcggcctc tgagctattc
cagaagtagt gaggaggctt 240 ttttggaggc ctaggctttt gcaaaaagct t 271 17
32 DNA Homo sapiens 17 gcgctcgagg gatgacagcg atagaacccc gg 32 18 31
DNA Homo sapiens 18 gcgaagcttc gcgactcccc ggatccgcct c 31 19 12 DNA
Homo sapiens 19 ggggactttc cc 12 20 73 DNA Homo sapiens 20
gcggcctcga ggggactttc ccggggactt tccggggact ttccgggact ttccatcctg
60 ccatctcaat tag 73 21 27 DNA Homo sapiens 21 gcggcaagct
ttttgcaaag cctaggc 27 22 256 DNA Homo sapiens 22 ctcgagggga
ctttcccggg gactttccgg ggactttccg ggactttcca tctgccatct 60
caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
120 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
cagaggccga 180 ggccgcctcg gcctctgagc tattccagaa gtagtgagga
ggcttttttg gaggcctagg 240 cttttgcaaa aagctt 256 23 30 DNA Homo
sapiens 23 cgcccatgga tgagccccag gctggaggtg 30 24 1337 DNA Homo
sapiens CDS (95)..(919) 24 agccctttct ccaaacctgc atggatgagt
ttcttttctt gttcaggtgg ttccttatgt 60 cacgacgatt tttggaggcc
tgcatgcagg caag atg gtc atg ctg caa gga gtg 115 Met Val Met Leu Gln
Gly Val 1 5 gtc cct cta gat gca cac agg ttt cag gtg gac ttc cag tgt
ggc tgc 163 Val Pro Leu Asp Ala His Arg Phe Gln Val Asp Phe Gln Cys
Gly Cys 10 15 20 agc ctg tgt ccc cgg cca gat atc gcc ttc cac ttc
aac cct cgc ttc 211 Ser Leu Cys Pro Arg Pro Asp Ile Ala Phe His Phe
Asn Pro Arg Phe 25 30 35 cat acc acc aag ccc cat gtc atc tgc aac
acc ctg cat ggt gga cgc 259 His Thr Thr Lys Pro His Val Ile Cys Asn
Thr Leu His Gly Gly Arg 40 45 50 55 tgg caa agg gag gcc cgg tgg ccc
cac ctg gcc ctg cga aga ggc tcc 307 Trp Gln Arg Glu Ala Arg Trp Pro
His Leu Ala Leu Arg Arg Gly Ser 60 65 70 agc ttc ctc atc ctc ttt
ctc ttc ggg aat gag gaa gtg aag gtg agt 355 Ser Phe Leu Ile Leu Phe
Leu Phe Gly Asn Glu Glu Val Lys Val Ser 75 80 85 gtg aat gga cag
cac ttt ctc cac ttc cgc tac cgg ctc cca ctg tct 403 Val Asn Gly Gln
His Phe Leu His Phe Arg Tyr Arg Leu Pro Leu Ser 90 95 100 cat gtg
gac acg ctg ggt ata ttt ggt gac atc ctg gta gag gct gtt 451 His Val
Asp Thr Leu Gly Ile Phe Gly Asp Ile Leu Val Glu Ala Val 105 110 115
gga ttc ctg aac atc aat cca ttt gtg gag ggc agc aga gag tac cca 499
Gly Phe Leu Asn Ile Asn Pro Phe Val Glu Gly Ser Arg Glu Tyr Pro 120
125 130 135 gct gga cat cct ttc ctg ctg atg agc ccc agg ctg gag gtg
ccc tgc 547 Ala Gly His Pro Phe Leu Leu Met Ser Pro Arg Leu Glu Val
Pro Cys 140 145 150 tca cat gct ctt ccc cag ggt ctc tcg cct ggg cag
gtc atc ata gta 595 Ser His Ala Leu Pro Gln Gly Leu Ser Pro Gly Gln
Val Ile Ile Val 155 160 165 cgg gga ctg gtc ttg caa gag ccg aag cat
ttt act gtg agc ctg agg 643 Arg Gly Leu Val Leu Gln Glu Pro Lys His
Phe Thr Val Ser Leu Arg 170 175 180 gac cag gct gcc cat gct cct gtg
aca ctc agg gcc tcc ttc gca gac 691 Asp Gln Ala Ala His Ala Pro Val
Thr Leu Arg Ala Ser Phe Ala Asp 185 190 195 aga act ctg gcc tgg atc
tcc cgc tgg ggg cag aag aaa ctg atc tca 739 Arg Thr Leu Ala Trp Ile
Ser Arg Trp Gly Gln Lys Lys Leu Ile Ser 200 205 210 215 gcc ccc ttc
ctc ttt tac ccc cag aga ttc ttt gag gtg ctg ctc ctg 787 Ala Pro Phe
Leu Phe Tyr Pro Gln Arg Phe Phe Glu Val Leu Leu Leu 220 225 230 ttc
cag gag gga ggg ctg aag ctg gcg ctc aat ggg cag ggg ctg ggg 835 Phe
Gln Glu Gly Gly Leu Lys Leu Ala Leu Asn Gly Gln Gly Leu Gly 235 240
245 gcc acc agc atg aac cag cag gcc ctg gag cag ctg cgg gag ctc cgg
883 Ala Thr Ser Met Asn Gln Gln Ala Leu Glu Gln Leu Arg Glu Leu Arg
250 255 260 atc agt gga agt gtc cag ctc tac tgt gtc cac tcc
tgaaggatgg 929 Ile Ser Gly Ser Val Gln Leu Tyr Cys Val His Ser 265
270 275 ttccaggaaa taccgcagaa aacaagagtc agccactccc cagggcccca
ctctcctccc 989 ctcattaaac catccacctg aacaccagca catcagggcc
tggttcacct ctggggtcac 1049 gagactgagt ctacaggagc tttgggcctg
agggaaggca caagagtgca aaggttcctc 1109 gaactctgca ccttcctcca
ccaggagcct gggatatggc tccatctgcc ttcagggcct 1169 ggactgcact
cacagaggca agtgttgtag actaacaaag atactccaaa atacaatggc 1229
ttaaagaatg tggtcattta ttctttatta tttatttatt tgtggtcaaa taaataaata
1289 aggttattta tttaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1337 25
275 PRT Homo sapiens 25 Met Val Met Leu Gln Gly Val Val Pro Leu Asp
Ala His Arg Phe Gln 1 5 10 15 Val Asp Phe Gln Cys Gly Cys Ser Leu
Cys Pro Arg Pro Asp Ile Ala 20 25 30 Phe His Phe Asn Pro Arg Phe
His Thr Thr Lys Pro His Val Ile Cys 35 40 45 Asn Thr Leu His Gly
Gly Arg Trp Gln Arg Glu Ala Arg Trp Pro His 50 55 60 Leu Ala Leu
Arg Arg Gly Ser Ser Phe Leu Ile Leu Phe Leu Phe Gly 65 70 75 80 Asn
Glu Glu Val Lys Val Ser Val Asn Gly Gln His Phe Leu His Phe 85 90
95 Arg Tyr Arg Leu Pro Leu Ser His Val Asp Thr Leu Gly Ile Phe Gly
100 105 110 Asp Ile Leu Val Glu Ala Val Gly Phe Leu Asn Ile Asn Pro
Phe Val 115 120 125 Glu Gly Ser Arg Glu Tyr Pro Ala Gly His Pro Phe
Leu Leu Met Ser 130 135 140 Pro Arg Leu Glu Val Pro Cys Ser His Ala
Leu Pro Gln Gly Leu Ser 145 150 155 160 Pro Gly Gln Val Ile Ile Val
Arg Gly Leu Val Leu Gln Glu Pro Lys 165 170 175 His Phe Thr Val Ser
Leu Arg Asp Gln Ala Ala His Ala Pro Val Thr 180 185 190 Leu Arg Ala
Ser Phe Ala Asp Arg Thr Leu Ala Trp Ile Ser Arg Trp 195 200 205 Gly
Gln Lys Lys Leu Ile Ser Ala Pro Phe Leu Phe Tyr Pro Gln Arg 210 215
220 Phe Phe Glu Val Leu Leu Leu Phe Gln Glu Gly Gly Leu Lys Leu Ala
225 230 235 240 Leu Asn Gly Gln Gly Leu Gly Ala Thr Ser Met Asn Gln
Gln Ala Leu 245 250 255 Glu Gln Leu Arg Glu Leu Arg Ile Ser Gly Ser
Val Gln Leu Tyr Cys 260 265 270 Val His Ser 275 26 1330 DNA Homo
sapiens CDS (25)..(912) 26 agccctttct ccaaacctgc atgg atg agt ttc
ttt tct tgt tca ggt ggt 51 Met Ser Phe Phe Ser Cys Ser Gly Gly 1 5
tcc tta tgt cac gac gat ttt tgg agg cct gca tgc agg caa gat ggt 99
Ser Leu Cys His Asp Asp Phe Trp Arg Pro Ala Cys Arg Gln Asp Gly 10
15 20 25 cat gct gca agg agt ggt ccc tct aga tgc aca cag gtg gac
ttc cag 147 His Ala Ala Arg Ser Gly Pro Ser Arg Cys Thr Gln Val Asp
Phe Gln 30 35 40 tgt ggc tgc agc ctg tgt ccc cgg cca gat atc gcc
ttc cac ttc aac 195 Cys Gly Cys Ser Leu Cys Pro Arg Pro Asp Ile Ala
Phe His Phe Asn 45 50 55 cct cgc ttc cat acc acc aag ccc cat gtc
atc tgc aac acc ctg cat 243 Pro Arg Phe His Thr Thr Lys Pro His Val
Ile Cys Asn Thr Leu His 60 65 70 ggt gga cgc tgg caa agg gag gcc
cgg tgg ccc cac ctg gcc ctg cga 291 Gly Gly Arg Trp Gln Arg Glu Ala
Arg Trp Pro His Leu Ala Leu Arg 75 80 85 aga ggc tcc agc ttc ctc
atc ctc ttt ctc ttc ggg aat gag gaa gtg 339 Arg Gly Ser Ser Phe Leu
Ile Leu Phe Leu Phe Gly Asn Glu Glu Val 90 95 100 105 aag gtg agt
gtg aat gga cag cac ttt ctc cac ttc cgc tac cgg ctc 387 Lys Val Ser
Val Asn Gly Gln His Phe Leu His Phe Arg Tyr Arg Leu 110 115 120 cca
ctg tct cat gtg gac acg ctg ggt ata ttt ggt gac atc ctg gta 435 Pro
Leu Ser His Val Asp Thr Leu Gly Ile Phe Gly Asp Ile Leu Val 125 130
135 gag gct gtt gga ttc ctg aac atc aat cca ttt gtg gag ggc agc aga
483 Glu Ala Val Gly Phe Leu Asn Ile Asn Pro Phe Val Glu Gly Ser Arg
140 145 150
gag tac cca gct gga cat cct ttc ctg ctg atg agc ccc agg ctg gag 531
Glu Tyr Pro Ala Gly His Pro Phe Leu Leu Met Ser Pro Arg Leu Glu 155
160 165 gtg ccc tgc tca cat gct ctt ccc cag ggt ctc tcg cct ggg cag
gtc 579 Val Pro Cys Ser His Ala Leu Pro Gln Gly Leu Ser Pro Gly Gln
Val 170 175 180 185 atc ata gta cgg gga ctg gtc ttg caa gag ccg aag
cat ttt act gtg 627 Ile Ile Val Arg Gly Leu Val Leu Gln Glu Pro Lys
His Phe Thr Val 190 195 200 agc ctg agg gac cag gct gcc cat gct cct
gtg aca ctc agg gcc tcc 675 Ser Leu Arg Asp Gln Ala Ala His Ala Pro
Val Thr Leu Arg Ala Ser 205 210 215 ttc gca gac aga act ctg gcc tgg
atc tcc cgc tgg ggg cag aag aaa 723 Phe Ala Asp Arg Thr Leu Ala Trp
Ile Ser Arg Trp Gly Gln Lys Lys 220 225 230 ctg atc tca gcc ccc ttc
ctc ttt tac ccc cag aga ttc ttt gag gtg 771 Leu Ile Ser Ala Pro Phe
Leu Phe Tyr Pro Gln Arg Phe Phe Glu Val 235 240 245 ctg ctc ctg ttc
cag gag gga ggg ctg aag ctg gcg ctc aat ggg cag 819 Leu Leu Leu Phe
Gln Glu Gly Gly Leu Lys Leu Ala Leu Asn Gly Gln 250 255 260 265 ggg
ctg ggg gcc acc agc atg aac cag cag gcc ctg gag cag ctg cgg 867 Gly
Leu Gly Ala Thr Ser Met Asn Gln Gln Ala Leu Glu Gln Leu Arg 270 275
280 gag ctc cgg atc agt gga agt gtc cag ctc tac tgt gtc cac tcc 912
Glu Leu Arg Ile Ser Gly Ser Val Gln Leu Tyr Cys Val His Ser 285 290
295 tgaaggatgg ttccaggaaa taccgcagaa aacaagagtc agccactccc
cagggcccca 972 ctctcctccc ctcattaaac catccacctg aacaccagca
catcagggcc tggttcacct 1032 ctggggtcac gagactgagt ctacaggagc
tttgggcctg agggaaggca caagagtgca 1092 aaggttcctc gaactctgca
ccttcctcca ccaggagcct gggatatggc tccatctgcc 1152 ttcagggcct
ggactgcact cacagaggca agtgttgtag actaacaaag atactccaaa 1212
atacaatggc ttaaagaatg tggtcattta ttctttatta tttatttatt tgtggtcaaa
1272 taaataaata aggttattta tttaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaa 1330 27 296 PRT Homo sapiens 27 Met Ser Phe Phe Ser Cys
Ser Gly Gly Ser Leu Cys His Asp Asp Phe 1 5 10 15 Trp Arg Pro Ala
Cys Arg Gln Asp Gly His Ala Ala Arg Ser Gly Pro 20 25 30 Ser Arg
Cys Thr Gln Val Asp Phe Gln Cys Gly Cys Ser Leu Cys Pro 35 40 45
Arg Pro Asp Ile Ala Phe His Phe Asn Pro Arg Phe His Thr Thr Lys 50
55 60 Pro His Val Ile Cys Asn Thr Leu His Gly Gly Arg Trp Gln Arg
Glu 65 70 75 80 Ala Arg Trp Pro His Leu Ala Leu Arg Arg Gly Ser Ser
Phe Leu Ile 85 90 95 Leu Phe Leu Phe Gly Asn Glu Glu Val Lys Val
Ser Val Asn Gly Gln 100 105 110 His Phe Leu His Phe Arg Tyr Arg Leu
Pro Leu Ser His Val Asp Thr 115 120 125 Leu Gly Ile Phe Gly Asp Ile
Leu Val Glu Ala Val Gly Phe Leu Asn 130 135 140 Ile Asn Pro Phe Val
Glu Gly Ser Arg Glu Tyr Pro Ala Gly His Pro 145 150 155 160 Phe Leu
Leu Met Ser Pro Arg Leu Glu Val Pro Cys Ser His Ala Leu 165 170 175
Pro Gln Gly Leu Ser Pro Gly Gln Val Ile Ile Val Arg Gly Leu Val 180
185 190 Leu Gln Glu Pro Lys His Phe Thr Val Ser Leu Arg Asp Gln Ala
Ala 195 200 205 His Ala Pro Val Thr Leu Arg Ala Ser Phe Ala Asp Arg
Thr Leu Ala 210 215 220 Trp Ile Ser Arg Trp Gly Gln Lys Lys Leu Ile
Ser Ala Pro Phe Leu 225 230 235 240 Phe Tyr Pro Gln Arg Phe Phe Glu
Val Leu Leu Leu Phe Gln Glu Gly 245 250 255 Gly Leu Lys Leu Ala Leu
Asn Gly Gln Gly Leu Gly Ala Thr Ser Met 260 265 270 Asn Gln Gln Ala
Leu Glu Gln Leu Arg Glu Leu Arg Ile Ser Gly Ser 275 280 285 Val Gln
Leu Tyr Cys Val His Ser 290 295
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