U.S. patent application number 10/601036 was filed with the patent office on 2004-10-28 for methods of screening for modulators of cell proliferation and methods of diagnosing cell proliferation states.
Invention is credited to Beraud, Christophe, Finer, Jeffrey T., Mak, John, Sakowicz, Roman, Wood, Kenneth W..
Application Number | 20040214249 10/601036 |
Document ID | / |
Family ID | 23697770 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214249 |
Kind Code |
A1 |
Wood, Kenneth W. ; et
al. |
October 28, 2004 |
Methods of screening for modulators of cell proliferation and
methods of diagnosing cell proliferation states
Abstract
Described herein are methods that can be used for diagnosis and
prognosis of cellular proliferation. Also described herein are
methods that can be used to screen candidate bioactive agents for
the ability to modulate cellular proliferation. Additionally,
methods and molecular targets (genes and their products) for
therapeutic intervention in cancers are described.
Inventors: |
Wood, Kenneth W.; (Foster
City, CA) ; Finer, Jeffrey T.; (Foster City, CA)
; Beraud, Christophe; (Palm Springs, CA) ; Mak,
John; (San Bruno, CA) ; Sakowicz, Roman;
(Foster City, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
23697770 |
Appl. No.: |
10/601036 |
Filed: |
June 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10601036 |
Jun 19, 2003 |
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09428156 |
Oct 27, 1999 |
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6617115 |
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Current U.S.
Class: |
435/7.23 |
Current CPC
Class: |
G01N 33/57484 20130101;
G01N 33/502 20130101; G01N 2500/04 20130101; G01N 33/5011 20130101;
G01N 33/57496 20130101 |
Class at
Publication: |
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
1. A method of screening drug candidates comprising: (a) providing
a cell that expresses recombinant human KSP or a fragment thereof;
(b) adding a drug candidate to said cell under conditions where the
drug candidate is taken up by the cell; and (c) determining the
effect of said drug candidate on the bioactivity of said
recombinant human KSP.
2-59. (Canceled)
60. A method of assessing an individual's risk for a
hyper-proliferative disorder, comprising: (a) determining the
expression level of KSP in a sample obtained from the individual;
and (b) comparing the KSP expression level in the sample with the
expression level of KSP in a control of known proliferation state;
and (c) assessing the individual's risk for the hyper-proliferative
disorder on the basis of the comparison of step (b).
61. The method of claim 60, wherein comparing comprises comparing
the KSP expression level in the sample with a control that is
representative of normal cells not in a hyper-proliferative state;
and assessing comprises identifying the individual as at risk for
the hyper-proliferative disorder if there is a difference in KSP
expression levels between the sample and the control.
62. The method of claim 60, wherein determining comprises
determining the expression level of a plurality of target molecules
correlated with cell proliferation, wherein one of the target
molecules is KSP; comparing comprises comparing the expression
levels of each of the plurality of target molecules with the
expression level of the same target molecules in the control.
63. The method of claim 62, wherein the plurality of target
molecules include a plurality of kinesins.
64. The method of claim 60, wherein determining comprises
determining the amount of nucleic acid encoding KSP.
65. The method of claim 64, wherein the amount of nucleic acid
encoding KSP is determined by the extent of binding to probes of a
nucleic acid probe array that specifically hybridize to nucleic
acids encoding KSP.
66. The method of claim 64, wherein the amount of nucleic acid
encoding KSP is determined by in situ hybridization.
67. The method of claim 64, wherein the nucleic acid is DNA.
68. The method of claim 64, wherein the nucleic acid is RNA.
69. The method of claim 60, wherein determining comprises
determining the amount of KSP protein in the sample.
70. The method of claim 69, wherein the amount of KSP protein is
determined by mass spectroscopy.
71. The method of claim 69, wherein the amount of KSP protein is
determined by an immunological method.
72. The method of claim 70, wherein the immunological method is an
enzyme-linked immunoassay assay (ELISA).
73. The method of claim 69, wherein the KSP protein level is
determined by two-dimensional gel electrophoresis.
74. The method of claim 60, wherein the hyper-proliferative
disorder is a cancer.
75. The method of claim 60, further comprising determining whether
a variant form of a cell proliferation gene is present in the
sample, the presence of a variant cell proliferation gene being an
indication that the individual is at risk for the
hyper-proliferative disorder.
76. The method of claim 75, wherein the cell proliferation gene is
KSP.
77. The method of claim 61, wherein the hyper-proliferative
disorder is a cancer; and determining comprises determining the
amount of nucleic acid encoding KSP from the extent of binding to
probes of a nucleic acid probe array that specifically hybridize to
nucleic acids encoding KSP or by in situ hybridization.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the use of nucleic acids encoding
the kinesin KSP and their gene products to identify modulators of
cell proliferation and their use in diagnosis, prognosis and
treatment of cell proliferation states and disorders, for example
cancer.
BACKGROUND OF THE INVENTION
[0002] Cancer is the second-leading cause of death in
industrialized nations. Effective therapeutics include the taxanes
and vinca alkyloids, agents which act on microtubules. Microtubules
are the primary structural element of the mitotic spindle. The
mitotic spindle is responsible for distribution of replicate copies
of the genome to each of the two daughter cells that result from
cell division. It is presumed that it is the disruption of the
mitotic spindle by these drugs that results in inhibition of cancer
cell division, and also induction of cancer cell death. However,
microtubules also form other types of cellular structures,
including tracks for intracellular transport in nerve processes.
Therefore, the taxanes have side effects that limit their
usefulness. Furthermore, taxanes and vinca alkaloids specifically
target microtubule polymerization dynamics. There are additional
dynamics of the mitotic spindle that these compounds do not
target.
[0003] Therefore, it is desirable to identify agents and
compositions which are specific and therapeutically effective
against cancer. It is further desirable to identify agents and
compositions which have a novel mechanism of action. It is further
desirable to provide methods of diagnosis of hyper or hypo
proliferation disorders. Additionally, it is desirable to identify
agents and compositions which modulate cell proliferation. Cell
proliferation modulation is desirable in a number of cases as
discussed below, for example, for treatment of any hyper or hypo
proliferation disorder, wound healing, transplantation procedures
and for use in the agricultural arena. It is thus desirable to
provide such methods of treatment. Moreover, it is desirable to
provide assays to quickly identify such agents and
compositions.
SUMMARY OF THE INVENTION
[0004] Provided herein are assays for screening for bioactive
agents which affect cell proliferation. Also provided herein are
methods of diagnosing proliferation states in a cell which are
useful for identifying cell proliferation disorders such as cancer.
Also provided are methods of prognosis and methods of treatment
including treatment for cancer. As is further described below, a
number of compositions and methods are provided.
[0005] In one aspect, a method of screening drug candidates is
provided. In one embodiment, said method comprises providing a cell
that expresses recombinant human KSP or a fragment thereof and
adding a drug candidate to said cell under conditions where the
drug candidate is taken up by the cell. The method further includes
determining the effect of said drug candidate on the bioactivity of
said recombinant human KSP. The bioactivity of recombinant human
KSP, or particularly the changes in the presence of a drug
candidate, can be determined by assays such as those for
determining cellular proliferation, cellular viability, and
cellular morphology. In a further aspect of the invention, any
changes in bioactivity of recombinant human KSP can be determined
by assays for determining changes in the mitotic spindle,
particularly inhibition of mitosis, and ATP hydrolysis. The methods
herein may also determine the bioactivity of recombinant human KSP
in the presence and absence of candidate agents by performing
assays determining the effect on apoptosis and necrosis.
[0006] The methods provided herein can be performed on single
individual cells or a population of cells. The cell can be any kind
of cell including but not limited to a lymphocyte, cancer cell or
an endothelial cell. In one aspect, wherein cancer cells are
utilized, cancer growth or inhibition can be determined, and
wherein endothelial cells are utilized, angiogenesis or inhibition
thereof can be determined.
[0007] In another aspect of the invention, a method of screening
for a bioactive agent capable of binding to a cellular
proliferation protein is provided. Preferably, the cellular
proliferation protein is human KSP or a fragment thereof. In one
embodiment, said method comprises combining said cellular
proliferation protein and a candidate bioactive agent, wherein said
candidate bioactive agent is an exogenous agent, and determining
the binding of said candidate agent to said cellular proliferation
protein.
[0008] In a further aspect herein, a method of screening for a
candidate protein capable of binding to a cellular proliferation
protein, wherein said cellular proliferation protein is KSP or a
fragment thereof, is provided. In a preferred method, said method
comprises combining a nucleic acid encoding said cellular
proliferation protein and a nucleic acid encoding a candidate
protein, wherein an identifiable marker is expressed wherein said
candidate protein binds to said cellular proliferation protein.
[0009] Also provided herein is a method for screening for a
bioactive agent capable of interfering with the binding of a
cellular proliferation protein, wherein said cellular proliferation
protein is KSP or a fragment thereof, and an antibody which binds
to said cellular proliferation protein. In one embodiment, the
method comprises combining a cellular proliferation protein,
wherein said cellular proliferation protein is KSP or fragment
thereof, a candidate bioactive agent and an antibody which binds to
said cellular proliferation protein and determining the binding of
said cellular proliferation protein and said antibody.
[0010] In a further aspect of the invention herein, a method or
screening for a bioactive agent capable of modulating the activity
of a cellular proliferation protein, wherein said cellular
proliferation protein is human KSP or a fragment thereof, is
provided. In one aspect, said method comprises combining said
cellular proliferation protein and a candidate bioactive agent,
wherein said candidate bioactive agent is an exogenous agent, and
determining the effect of said candidate agent on the activity of
said cellular proliferation protein.
[0011] Also provided herein is a method of screening drug
candidates comprising providing a cell that expresses KSP, adding a
drug candidate to said cell, and determining the effect of said
drug candidate on the expression of KSP. In a further aspect the
method includes comparing the level of expression in the absence of
said drug candidate to the level of expression in the presence of
said drug candidate, wherein the concentration of said drug
candidate can vary when present, and wherein said comparison can
occur after addition or removal of the drug candidate. In a
preferred embodiment, the expression of said KSP is decreased as a
result of the introduction of the drug candidate. Preferably, the
cell utilized is a tumor cell.
[0012] In a further aspect, a method of evaluating the effect of a
candidate drug on cellular proliferation (a candidate cellular
proliferation drug) is provided which comprises administering said
drug to a patient, removing a cell sample from said patient, and
determining the expression profile of said cell, wherein said
expression profile includes a KSP gene. In another aspect, the
method includes comparing said expression profile to an expression
profile of a healthy individual.
[0013] In another aspect herein, a method of diagnosing a
hyper-proliferative disorder in an individual is provided herein
comprising determining the level of expression a KSP gene in an
individual and comparing said level to a standard or control level
of expression, wherein an increase indicates that the individual
has a hyper-proliferative disorder, such as, but not limited to,
cancer.
[0014] Also provided herein is a method of evaluating the effect of
a candidate cellular proliferation drug comprising administering
said drug to a patient wherein said patient has cancer and has been
identified as expressing KSP at a level higher than an individual
not having cancer, removing a cell sample from said patient, and
determining the effect on KSP activity, wherein said KSP activity
is mitosis.
[0015] In the methods provided herein, the cells can come from a
variety of sources. For example, samples can be from, but are not
limited to, a blood sample, a urine sample, a buccal sample, a PAP
smear, cerebral spinal fluid, and any tissue including, breast
tissue, lung tissue and colon tissue. In one embodiment, the
patient has cancer.
[0016] Also provided herein is a method for inhibiting cellular
proliferation, said method comprising administering to a cell a
composition comprising an antibody to KSP, wherein said antibody is
conjugated to a ligand. In one aspect, the ligand of the antibody
is tumor cell specific. In another aspect, the ligand facilitates
said antibody entry to said cell. Moreover, the antibody can be a
humanized antibody. The methods of inhibition can be performed in
vitro on cells or in vivo on an individual. In one embodiment, the
cells are cancerous. In a further embodiment, the individual has
cancer. Another method of inhibiting cellular proliferation in a
cell or individual is provided herein which comprises administering
to a cell or individual a composition comprising antisense
molecules to KSP.
[0017] In yet another embodiment herein, a method for inhibiting
cellular proliferation is provided which comprises administering to
a cell a composition comprising an inhibitor of KSP. In one
embodiment, the inhibitor is of human KSP or a fragment thereof. In
one embodiment, the inhibitor is specific to human KSP. In one
embodiment, KSP inhibitors are any agent which disrupts or inhibits
KSP activity as further described herein. In one aspect of the
invention, the inhibitor of KSP is a small molecule as further
defined herein. Generally, small molecules have a molecular weight
of between 50 kD and 2000 kD, and in some cases, less than 1500 kD,
or less than 1000 kD or less than 500 kD. Examples of KSP
inhibitors include but are not limited to small molecules,
ribozymes, antisense molecules and antibodies. KSP inhibitors are
further described herein and in the application filed Oct. 27,
1999, entitled Methods and Compositions Utilizing Quinazolinones
(serial number not yet received, named inventor Jeffrey T. Finer),
incorporated by reference in its entirety. The composition which is
administered to a cell further comprises an acceptable
pharmaceutical carrier in one embodiment. The composition can have
a variety of formulations, including, but not limited to those for
parental, oral or topical administration.
[0018] The methods of inhibiting cellular proliferation can be
performed in vitro or in vivo. More particularly, the compositions
can be administered to cells in vitro or in an individual. The
individual may have a disease or be at risk for disease. Disease
states which can be treated by the methods herein are further
described below. In one case, the individual has cancer or is at
risk for restenosis. The cell can be any cell, preferably a cancer
cell. Other preferred cell types include but are not limited to
endothelial cells and metastatic cancer cells. In one embodiment,
the method of inhibiting by the KSP inhibitor is by disruption of
mitosis or induction of apoptosis.
[0019] In a further aspect of the invention, a biochip comprising a
nucleic acid segment from KSP, wherein said biochip comprises fewer
than 1000 nucleic acid probes, is provided. Methods of screening
and diagnosing conditions with said biochip are also provided
herein.
[0020] Other aspects of the invention will become apparent to the
skilled artisan by the following description of the invention.
DETAILED DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows a cDNA sequence for human KSP, GenBank
accession number X85137, wherein the start and stop codons are
shown underlined and in bold, beginning at positions 11 and 3182,
respectively.
[0022] FIG. 2 shows an amino acid sequence encoding human KSP.
[0023] FIG. 3 shows a nucleic acid sequence encoding a fragment of
KSP, termed KSPL360 herein. Portions differing from the sequences
of FIGS. 1 and 2 are indicated in bold typeface and are underlined.
Residues at the C-terminus include a myc epitope and a 6-histidine
tag.
[0024] FIG. 4 shows an amino acid sequence encoding KSPL360.
[0025] FIG. 5 shows a nucleic acid sequence encoding a fragment of
KSP, termed KSP-K491 herein. Portions differing from the sequences
of FIGS. 1 and 2 are indicated in bold typeface and are underlined.
Residues at the C-terminus include a myc epitope and a 6-histidine
tag.
[0026] FIG. 6 shows an amino acid sequence encoding KSP-K491.
[0027] FIG. 7 shows a nucleic acid sequence encoding a fragment of
KSP, termed KSP-S553 herein. Portions differing from the sequences
of FIGS. 1 and 2 are indicated in bold typeface and are underlined.
Residues at the C-terminus include a myc epitope and a 6-histidine
tag.
[0028] FIG. 8 shows an amino acid sequence encoding KSP-S553.
[0029] FIG. 9 shows a nucleic acid sequence encoding a fragment of
KSP, termed KSP-K368 herein. Portions differing from the sequences
of FIGS. 1 and 2 are indicated in bold typeface and are
underlined.
[0030] FIG. 10 shows an amino acid sequence encoding KSP-K368.
[0031] FIG. 11 is a graph showing KSP mRNA levels in matched normal
and tumor tissue from breast, lung and colon. mRNA levels were
measured by quantitative PCR relative to a standard. The relative
magnitudes of overexpression in each tumor sample relative to the
matched normal tissue are displayed above each pair. All values are
normalized to the level of KSP mRNA expression observed in cultured
HeLa cells.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Provided herein are assays for screening for bioactive
agents which affect cell proliferation. Also provided herein are
methods of diagnosing proliferation states in a cell which are
useful for identifying cell proliferation disorders such as cancer.
Also provided are methods of prognosis and methods of treatment
including treatment for cancer. As is further described below, a
number of compositions and methods are provided.
[0033] In one aspect, the assays or methods of diagnosis provided
herein include the use of a cellular proliferation protein or
nucleic acid. The terms "cell proliferation" and "cellular
proliferation" are used herein interchangeably. Additionally, the
cellular proliferation protein and nucleic acid can be referred to
herein as "cellular proliferation sequences" wherein the context
will indicate whether the sequence is an amino acid sequence,
nucleic acid sequence, or either.
[0034] In a preferred embodiment, the cellular proliferation
sequence is KSP. KSP belongs to an evolutionarily conserved kinesin
subfamily of plus end-directed microtubule motors that assemble
into bipolar homotetramers consisting of antiparallel homodimers.
During mitosis KSP associates with microtubules of the mitotic
spindle. Microinjection of antibody directed against KSP into cells
prevents spindle pole separation during prometaphase, giving rise
to monopolar spindles and causing mitotic arrest. KSP and related
kinesins bundle antiparallel microtubules and slide them relative
to one another, thus forcing the two spindle poles apart. KSP may
also mediate in anaphase B spindle elongation and focussing of
microtubules at the spindle pole.
[0035] Human KSP has been reported on (also termed HsEg5). Galgio,
et al., J. Cell Biol., 135(2):399-414 (1996); Kaiser, et al., JBC,
274(27):18925-31 (1999); Blangy, et al., Cell, 83:1159-69 (1995);
Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy, et al.,
Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead, et al.,
Arthritis Rheum., 39:1635-42 (1996); GenBank accession numbers:
X85137, NM.sub.--004523 and U37426. Moreover, a fragment of the KSP
gene (TRIP5) has been reported on. Lee, et al., Mol Endocrinol.,
9:243-54 (1995); GenBank accession number L40372. Also see,
Whitehead and Rattner, J. Cell Sci., 111:2551-61 (1998).
[0036] Xenopus KSP homologs (Eg5) have also been reported on.
Walczak, et al., Curr Biol., 8(16):903-13 (1998); Le Guellec, et
al., Mol. Cell Biol., 11 (6):3395-8 (1991); Sawin, et al., Nature,
359:540-3 (1992); Sawin and Mitchison, Mol Biol Cell, 5:217-26
(1994); Sawin and Mitchison, PNAS, 92:4289-93 (1995); Kapoor and
Mitchison, PNAS, 96:9106-11 (1999); Lockhart and Cross,
Biochemistry, 35(7):2365-73 (1996); Crevel, et al, J. Mol. Biol.,
273:160-170 (1997). Additionally, Drosophila KLP61F/KRP130 has been
reported on. Heck, et al., J Cell Biol, 123:665-79 (1993); Cole, et
al., J. Biol. Chem., 269(37):22913-6 (1994); Barton, et al., Mol.
Biol. Cell, 6:1563-74 (1995).
[0037] In the preferred embodiment herein, a sequence as shown in
the figures is utilized. As indicated herein, in some embodiments a
fragment of KSP is utilized. Preferred protein fragments are shown
in FIGS. 2, 4, 6, and 8. In one embodiment, the cellular
proliferation fragment shown in FIG. 4 is preferred. Preferred
fragments of KSP have kinesin activity as further described below.
Moreover, in one embodiment, KSP peptides or fragments have at
least one, and preferably at least two epitope tags. In a preferred
embodiment, a KSP fragment comprises a myc epitope and a histidine
tag.
[0038] In another preferred embodiment herein, the cellular
proliferation protein is non-glycosylated. For example, in one
embodiment the protein is, for example, human, expressed in
bacteria, for example, E. Coli. Moreover, phosphorylation and/or
methylation of KSP as used herein may differ from KSP as found in
its native form within a cell.
[0039] Thus, while it is preferred that the cellular proliferation
sequences are from humans, sequences from other organisms may be
useful in animal models of disease and drug evaluation; thus, in
alternative embodiments, other sequences are provided such as from
vertebrates, including mammals, including rodents (rats, mice,
hamsters, guinea pigs, etc.), primates, farm animals (including
sheep, goats, pigs, cows, horses, etc), Xenopus, and
Drosophila.
[0040] In another embodiment, the sequences are naturally-occurring
allelic variants of the sequences set forth in the figures. In
another embodiment, the sequences are sequence variants as further
described herein.
[0041] In one embodiment, a cellular proliferation sequence can be
initially identified by substantial nucleic acid and/or amino acid
sequence homology to the cellular proliferation sequences outlined
herein. Such homology can be based upon the overall nucleic acid or
amino acid sequence, and is generally determined as outlined below,
using either homology programs or hybridization conditions.
[0042] Thus, in one embodiment, a nucleic acid is a "cellular
proliferation nucleic acid" if the overall homology of the nucleic
acid sequence to the nucleic acid sequences of FIG. 1, 3, 5, 7 or 9
(the nucleic acid figures) is preferably greater than about 75%,
more preferably greater than about 80%, even more preferably
greater than about 85% and most preferably greater than 90%. In
some embodiments the homology will be as high as about 93 to 95 or
98%. Homology as used herein is in reference to sequence similarity
or identity, with identity being preferred. This homology will be
determined using standard techniques known in the art, including,
but not limited to, the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biool. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, PNAS
USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Drive, Madison, Wis.), the Best Fit sequence program described by
Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably
using the default settings, or by inspection.
[0043] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments. It can also plot a tree
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method
is similar to that described by Higgins & Sharp CABIOS
5:151-153 (1989). Useful PILEUP parameters including a default gap
weight of 3.00, a default gap length weight of 0.10, and weighted
end gaps.
[0044] Another example of a useful algorithm is the BLAST
algorithm, described in Altschul et al., J. Mol. Biol. 215,
403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A
particularly useful BLAST program is the WU-BLAST-2 program which
was obtained from Altschul et al., Methods in Enzymology, 266:
460-480 (1996); http://blast.wustl/edu/b- last/REACRCE.html].
WU-BLAST-2 uses several search parameters, most of which are set to
the default values. The adjustable parameters are set with the
following values: overlap span=1, overlap fraction=0.125, word
threshold (T)=11. The HSP S and HSP S2 parameters are dynamic
values and are established by the program itself depending upon the
composition of the particular sequence and composition of the
particular database against which the sequence of interest is being
searched; however, the values may be adjusted to increase
sensitivity. A % amino acid sequence identity value is determined
by the number of matching identical residues divided by the total
number of residues of the "longer" sequence in the aligned region.
The "longer" sequence is the one having the most actual residues in
the aligned region (gaps introduced by WU-Blast-2 to maximize the
alignment score are ignored).
[0045] Thus, "percent (%) nucleic acid sequence identity" is
defined as the percentage of nucleotide residues in a candidate
sequence that are identical with the nucleotide residues of the
sequence shown in the nucleic acid figures. A preferred method
utilizes the BLASTN module of WU-BLAST-2 set to the default
parameters, with overlap span and overlap fraction set to 1 and
0.125, respectively.
[0046] The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer nucleosides than those of the nucleic acid
figures, it is understood that the percentage of homology will be
determined based on the number of homologous nucleosides in
relation to the total number of nucleosides. Thus, for example,
homology of sequences shorter than those of the sequences
identified herein and as discussed below, will be determined using
the number of nucleosides in the shorter sequence.
[0047] In one embodiment, the cellular proliferation nucleic acid
is determined through hybridization studies. Thus, for example,
nucleic acids which hybridize under high stringency to the nucleic
acid sequences identified in the figures, or a complement, are
considered a cellular proliferation sequence in one embodiment
herein. High stringency conditions are known in the art; see for
example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d
Edition, 1989, and Short Protocols in Molecular Biology, ed.
Ausubel, et al., both of which are hereby incorporated by
reference. Stringent conditions are sequence-dependent and will be
different in different circumstances. Longer sequences hybridize
specifically at higher temperatures. An extensive guide to the
hybridization of nucleic acids is found in Tijssen, Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes, "Overview of principles of hybridization and the strategy
of nucleic acid assays" (1993). Generally, stringent conditions are
selected to be about 5-10.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
pH. The Tm is the temperature (under defined ionic strength, pH and
nucleic acid concentration) at which 50% of the probes
complementary to the target hybridize to the target sequence at
equilibrium (as the target sequences are present in excess, at Tm,
50% of the probes are occupied at equilibrium). Stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g. greater than 50 nucleotides). Stringent conditions may
also be achieved with the addition of destabilizing agents such as
formamide.
[0048] In another embodiment, less stringent hybridization
conditions are used; for example, moderate or low stringency
conditions may be used, as are known in the art; see Maniatis and
Ausubel, supra, and Tijssen, supra.
[0049] In addition, in one embodiment the cellular proliferation
nucleic acid sequences of the invention are fragments of larger
genes, i.e. they are nucleic acid segments. "Genes" in this context
includes coding regions, non-coding regions, and mixtures of coding
and non-coding regions. Accordingly, as will be appreciated by
those in the art, using the sequences provided herein, additional
sequences of the cellular proliferation genes can be obtained,
using techniques well known in the art for cloning either longer
sequences or the full length sequences; see Maniatis et al., and
Ausubel, et al., supra, hereby expressly incorporated by
reference.
[0050] Once the cellular proliferation nucleic acid is identified,
it can be cloned and, if necessary, its constituent parts
recombined to form the entire cellular proliferation nucleic acid.
Once isolated from its natural source, e.g., contained within a
plasmid or other vector or excised therefrom as a linear nucleic
acid segment, the recombinant cellular proliferation nucleic acid
can be further-used as a probe to identify and isolate other
cellular proliferation nucleic acids, for example additional coding
regions. It can also be used as a "precursor" nucleic acid to make
modified or variant cellular proliferation nucleic acids and
proteins. "Recombinant" as used herein refers to a nucleic acid or
protein which is not in its native state. For example, the nucleic
acid can be genetically engineered, isolated, inserted into a
man-made vector or be in a cell wherein it is not natively
expressed in order to be considered recombinant.
[0051] In another aspect, the cellular proliferation nucleic acid
and protein sequences are differentially expressed in cells having
varying states of cellular proliferation, including cancer cells
which over proliferate compared to non cancerous cells. As outlined
below, cellular proliferation sequences include those that are
up-regulated (i.e. expressed at a higher level) during cellular
proliferation, as well as those that are down-regulated (i.e.
expressed at a lower level) in cellular proliferation. In a
preferred embodiment, the cellular proliferation sequences are
upregulated during cellular proliferation in their native state,
ie., without the administration of modulators or therapeutics.
[0052] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences and as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes
8:91-98 (1994)). The term nucleic acid is used interchangeably with
gene, cDNA, and mRNA encoded by a gene.
[0053] The cellular proliferation nucleic acids of the present
invention are used in several ways. In a preferred embodiment,
cellular proliferation nucleic acids encoding cellular
proliferation proteins are used to make a variety of expression
vectors to express cellular proliferation proteins which can then
be used in screening assays, as described below. The expression
vectors may be either self-replicating extrachromosomal vectors or
vectors which integrate into a host genome. Generally, these
expression vectors include transcriptional and translational
regulatory nucleic acid operably linked to the nucleic acid
encoding the cellular proliferation protein. The term "control
sequences" refers to DNA sequences necessary for the expression of
an operably linked coding sequence in a particular host organism.
The control sequences that are suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells are known to utilize
promoters, polyadenylation signals, and enhancers.
[0054] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express the cellular proliferation
protein; for example, transcriptional and translational regulatory
nucleic acid sequences from Bacillus are preferably used to express
the cellular proliferation protein in Bacillus. Numerous types of
appropriate expression vectors, and suitable regulatory sequences
are known in the art for a variety of host cells.
[0055] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0056] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0057] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a procaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0058] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0059] The cellular proliferation proteins of the present invention
can be produced by culturing a host cell transformed with an
expression vector containing nucleic acid encoding a cellular
proliferation protein, under the appropriate conditions to induce
or cause expression of the cellular proliferation protein. The
conditions appropriate for cellular proliferation protein
expression will vary with the choice of the expression vector and
the host cell, and will be easily ascertained by one skilled in the
art through routine experimentation. For example, the use of
constitutive promoters in the expression vector will require
optimizing the growth and proliferation of the host cell, while the
use of an inducible promoter requires the appropriate growth
conditions for induction. In addition, in some embodiments, the
timing of the harvest is important. For example, the baculoviral
systems used in insect cell expression are lytic viruses, and thus
harvest time selection can be crucial for product yield.
[0060] Appropriate host cells include yeast, bacteria,
archaebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melangaster
cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus
subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO,
COS, HeLa cells, THP1 cell line (a macrophage cell line) and human
cells and cell lines.
[0061] In one embodiment, the cellular proliferation proteins are
expressed in mammalian cells. Mammalian expression systems are also
known in the art, and include retroviral systems. A preferred
expression vector system is a retroviral vector system such as is
generally described in PCT/US97/01019 and PCT/US97/01048, both of
which are hereby expressly incorporated by reference. Of particular
use as mammalian promoters are the promoters from mammalian viral
genes, since the viral genes are often highly expressed and have a
broad host range. Examples include the SV40 early promoter, mouse
mammary tumor virus LTR promoter, adenovirus major late promoter,
herpes simplex virus promoter, and the CMV promoter. Typically,
transcription termination and polyadenylation sequences recognized
by mammalian cells are regulatory regions located 3' to the
translation stop codon and thus, together with the promoter
elements, flank the coding sequence. Examples of transcription
terminator and polyadenlytion signals include those derived form
SV40.
[0062] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0063] In a preferred embodiment, cellular proliferation proteins
are expressed in bacterial systems. Bacterial expression systems
are well known in the art. Promoters from bacteriophage may also be
used and are known in the art. In addition, synthetic promoters and
hybrid promoters are also useful; for example, the tac promoter is
a hybrid of the trp and lac promoter sequences. Furthermore, a
bacterial promoter can include naturally occurring promoters of
non-bacterial origin that have the ability to bind bacterial RNA
polymerase and initiate transcription. In addition to a functioning
promoter sequence, an efficient ribosome binding site is desirable.
The expression vector may also include a signal peptide sequence
that provides for secretion of the cellular proliferation protein
in bacteria. The protein is either secreted into the growth media
(gram-positive bacteria) or into the periplasmic space, located
between the inner and outer membrane of the cell (gram-negative
bacteria). The expession vector may also include an epitope tag
providing for affinity purification of the cellular proliferation
protein. The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways. These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others. The bacterial
expression vectors are transformed into bacterial host cells using
techniques well known in the art, such as calcium chloride
treatment, electroporation, and others.
[0064] In one embodiment, cellular proliferation proteins are
produced in insect cells. Expression vectors for the transformation
of insect cells, and in particular, baculovirus-based expression
vectors, are well known in the art.
[0065] In another embodiment, cellular proliferation protein is
produced in yeast cells. Yeast expression systems are well known in
the art, and include expression vectors for Saccharomyces
cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha,
Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
[0066] The cellular proliferation protein may also be made as a
fusion protein, using techniques well known in the art. Thus, for
example, for the creation of monoclonal antibodies, if the desired
epitope is small, the cellular proliferation protein may be fused
to a carrier protein to form an immunogen. Alternatively, the
cellular proliferation protein may be made as a fusion protein to
increase expression, or for other reasons. For example, when the
cellular proliferation protein is a cellular proliferation peptide,
the nucleic acid encoding the peptide may be linked to other
nucleic acid for expression purposes.
[0067] In one embodiment, the cellular proliferation nucleic acids,
proteins and antibodies of the invention are labeled. By "labeled"
herein is meant that a compound has at least one element, isotope
or chemical compound attached to enable the detection of the
compound. In general, labels fall into three classes: a) isotopic
labels, which may be radioactive or heavy isotopes; b) immune
labels, which may be antibodies or antigens; and c) colored or
fluorescent dyes. The labels may be incorporated into the cellular
proliferation nucleic acids, proteins and antibodies at any
position. For example, the label should be capable of producing,
either directly or indirectly, a detectable signal. The detectable
moiety may be a radioisotope, such as .sup.3H, .sup.14C, .sup.32P,
.sup.35S, or .sup.125I, a fluorescent or chemiluminescent compound,
such as fluorescein isothiocyanate, rhodamine, or luciferin, or an
enzyme, such as alkaline phosphatase, beta-galactosidase or
horseradish peroxidase. Any method known in the art for conjugating
the antibody to the label may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982).
[0068] Accordingly, the present invention also provides cellular
proliferation protein sequences. A cellular proliferation protein
of the present invention may be identified in several ways.
"Protein" in this sense includes proteins, polypeptides, and
peptides. As will be appreciated by those in the art, the nucleic
acid sequences of the invention can be used to generate protein
sequences.
[0069] Also included within one embodiment of cellular
proliferation proteins are amino acid variants of the naturally
occurring sequences, as determined herein. Preferably, the variants
are preferably greater than about 75% homologous to the wild-type
sequence, more preferably greater than about 80%, even more
preferably greater than about 85% and most preferably greater than
90%. In some embodiments the homology will be as high as about 93
to 95 or 98%. As for nucleic acids, homology in this context means
sequence similarity or identity, with identity being preferred.
This homology will be determined using standard techniques known in
the art as are outlined above for the nucleic acid homologies. The
proteins of the present invention may be shorter or longer than the
wild type amino acid sequences. Thus, in a preferred embodiment,
included within the definition of cellular proliferation proteins
are portions or fragments of the wild type sequences. Preferred
fragments have a binding domain to a modulating agent or antibody
as discussed below. In addition, as outlined above, the cellular
proliferation nucleic acids of the invention may be used to obtain
additional coding regions, and thus additional protein sequence,
using techniques known in the art.
[0070] In one embodiment, the cellular proliferation proteins are
derivative or variant cellular proliferation proteins as compared
to the wild-type sequence. That is, as outlined more fully below,
the derivative cellular proliferation peptide will contain at least
one amino acid substitution, deletion or insertion, with amino acid
substitutions being particularly preferred. The amino acid
substitution, insertion or deletion or combination thereof may
occur at any residue within the cellular proliferation peptide.
These variants ordinarily are prepared by site specific mutagenesis
of nucleotides in the DNA encoding the cellular proliferation
protein, using cassette or PCR mutagenesis or other techniques well
known in the art, to produce DNA encoding the variant, and
thereafter expressing the DNA in recombinant cell culture as
outlined above. However, variant cellular proliferation protein
fragments having up to about 100-150 residues may be prepared by in
vitro synthesis using established techniques. Amino acid sequence
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the cellular proliferation
protein amino acid sequence. The variants typically exhibit the
same qualitative biological activity as the naturally occurring
analogue, although variants can also be selected which have
modified characteristics as will be more fully outlined below.
[0071] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed cellular
proliferation variants screened for the optimal combination of
desired activity. Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are well known,
for example, M13 primer mutagenesis and PCR mutagenesis. Screening
of the mutants is done using assays of cellular proliferation
protein activities.
[0072] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0073] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the cellular proliferation protein are desired,
substitutions are generally made in accordance with the following
chart:
1 CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys
Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln
Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met,
Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0074] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in Chart I. For example, substitutions may be made
which more significantly affect: the structure of the polypeptide
backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl is substituted
for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0075] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
naturally-occurring analogue, although variants also are selected
to modify the characteristics of the cellular proliferation
proteins as needed. Alternatively, the variant may be designed such
that the biological activity of the cellular proliferation protein
is altered.
[0076] Covalent modifications of cellular proliferation
polypeptides are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of a cellular proliferation polypeptide with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N-or C-terminal residues of a cellular proliferation
polypeptide. Derivatization with bifunctional agents is useful, for
instance, for crosslinking cellular proliferation protein to a
water-insoluble support matrix or surface for use in the method for
purifying anti-KSP antibodies or screening assays, as is more fully
described below. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl- )dithio]propioimidate.
[0077] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains [T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine,
and amidation of any C-terminal carboxyl group.
[0078] Another type of covalent modification of the cellular
proliferation polypeptide included within the scope of this
invention comprises altering the native glycosylation pattern of
the polypeptide. "Altering the native glycosylation pattern" is
intended for purposes herein to mean deleting one or more
carbohydrate moieties found in native sequence cellular
proliferation polypeptide, and/or adding one or more glycosylation
sites that are not present in the native sequence cellular
proliferation polypeptide.
[0079] Addition of glycosylation sites to cellular proliferation
polypeptides may be accomplished by altering the amino acid
sequence thereof. The alteration may be made, for example, by the
addition of, or substitution by, one or more serine or threonine
residues to the native sequence cellular proliferation polypeptide
(for O-linked glycosylation sites). The cellular proliferation
amino acid sequence may optionally be altered through changes at
the DNA level, particularly by mutating the DNA encoding the
cellular proliferation polypeptide at preselected bases such that
codons are generated that will translate into the desired amino
acids.
[0080] Another means of increasing the number of carbohydrate
moieties on the cellular proliferation polypeptide is by chemical
or enzymatic coupling of glycosides to the polypeptide. Such
methods are described in the art, e.g., in WO 87/05330 published 11
Sep. 1987, and in Aplin and Wriston, cellular proliferation Crit.
Rev. Biochem., pp. 259-306 (1981).
[0081] Removal of carbohydrate moieties present on the cellular
proliferation polypeptide may be accomplished chemically or
enzymatically or by mutational substitution of codons encoding for
amino acid residues that serve as targets for glycosylation.
Chemical deglycosylation techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem.
Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., Meth. Enzymol.,
138:350 (1987).
[0082] Another type of covalent modification of cellular
proliferation comprises linking the cellular proliferation
polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in
the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
[0083] The cellular proliferation polypeptides of the present
invention may also be modified in one embodiment in a way to form
chimeric molecules comprising a cellular proliferation polypeptide
fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of a
cellular proliferation polypeptide with a tag polypeptide which
provides an epitope to which an anti-tag antibody can selectively
bind. Preferred tags include the myc epitope and 6-histidine. The
epitope tag is generally placed at the amino- or carboxyl-terminus
of the cellular proliferation polypeptide. The presence of such
epitope-tagged forms of a cellular proliferation polypeptide can be
detected using an antibody against the tag polypeptide as further
discussed below. Also, provision of the epitope tag enables the
cellular proliferation polypeptide to be readily purified by
affinity purification using an anti-tag antibody or another type of
affinity matrix that binds to the epitope tag. In an alternative
embodiment, the chimeric molecule may comprise a fusion of a
cellular proliferation polypeptide with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule, such a fusion could be to the Fc region of an
IgG molecule.
[0084] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194
(1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.,
266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag
[Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397
(1990)].
[0085] Also included with the definition of cellular proliferation
protein in one embodiment are other cellular proliferation proteins
of the cellular proliferation family, and cellular proliferation
proteins from other organisms, which are cloned and expressed as
outlined below. Thus, probe or degenerate polymerase chain reaction
(PCR) primer sequences may be used to find other related cellular
proliferation proteins from humans or other organisms. As will be
appreciated by those in the art, particularly useful probe and/or
PCR primer sequences include the unique areas of the cellular
proliferation nucleic acid sequence. As is generally known in the
art, preferred PCR primers are from about 15 to about 35
nucleotides in length, with from about 20 to about 30 being
preferred, and may contain inosine as needed. The conditions for
the PCR reaction are well known in the art.
[0086] In addition, as is outlined herein, cellular proliferation
proteins can be made that are longer than those depicted in the
figures, for example, by the elucidation of additional sequences,
the addition of epitope or purification tags, the addition of other
fusion sequences, etc.
[0087] Cellular proliferation proteins may also be identified as
being encoded by cellular proliferation nucleic acids. Thus, in one
embodiment, cellular proliferation proteins are encoded by nucleic
acids that will hybridize to the sequences of the nucleic acid
figures, or their complements, as outlined herein.
[0088] In a preferred embodiment, the cellular proliferation
protein is purified or isolated after expression. Cellular
proliferation proteins may be isolated or purified in a variety of
ways known to those skilled in the art depending on what other
components are present in the sample. Standard purification methods
include electrophoretic, molecular, immunological and
chromatographic techniques, including ion exchange, hydrophobic,
affinity, and reverse-phase HPLC chromatography, and
chromatofocusing. For example, the cellular proliferation protein
may be purified using a standard anti-KSP antibody column.
Ultrafiltration and diafiltration techniques, in conjunction with
protein concentration, are also useful. For general guidance in
suitable purification techniques, see Scopes, R., Protein
Purification, Springer-Verlag, NY (1982). The degree of
purification necessary will vary depending on the use of the
cellular proliferation protein. In some instances no purification
will be necessary.
[0089] The terms "isolated" "purified" or "biologically pure" refer
to material that is substantially or essentially free from
components which normally accompany it as found in its native
state. Purity and homogeneity are typically determined using
analytical chemistry techniques such as polyacrylamide gel
electrophoresis or high performance liquid chromatography. A
protein that is the predominant species present in a preparation is
substantially purified. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure. In a preferred embodiment, a
protein is considered pure wherein it is determined that there is
no contaminating activity.
[0090] Once expressed and purified if necessary, the cellular
proliferation proteins and nucleic acids are useful in a number of
applications. In a number of methods provided herein, wherein
either the nucleic acid or a protein is used, a candidate bioactive
agent is used to determine the effect on the cellular proliferation
sequence, cellular proliferation, cancer, etc., as further
discussed below.
[0091] In preferred embodiments, the bioactive agents modulate the
cellular proliferation sequences or expression profiles provided
herein. In a particularly preferred embodiment, the candidate agent
suppresses a cellular proliferation phenotype, for example to
inhibit proliferation, inhibit tumor growth, or to a normal tissue
fingerprint as further discussed below. Similarly, the candidate
agent preferably suppresses a severe cellular proliferation
phenotype. Suppression might take the form of cell or tumor growth
arrest, with continued viability. Alternatively, suppression may
take the form of inducing cell death of cells, thereby eliminating
proliferation. As further discussed below, preferred bioactive
agents are identified which cause cell death selectively of tumor
cells or proliferating cells. Generally a plurality of assay
mixtures are run in parallel with different agent concentrations to
obtain a differential response to the various concentrations.
Typically, one of these concentrations serves as a negative
control, i.e., at zero concentration or below the level of
detection.
[0092] The term "candidate bioactive agent" or "drug candidate" or
grammatical equivalents as used herein describes any molecule,
e.g., protein, oligopeptide, small organic molecule,
polysaccharide, polynucleotide, purine analog, etc., to be tested
for bioactive agents that are capable of directly or indirectly
altering either the cellular proliferation phenotype or the
expression of a cellular proliferation sequence, including both
nucleic acid sequences and protein sequences. In other cases,
alteration of cellular proliferation protein binding and/or
activity is screened. In the case where protein binding or activity
is screened, preferred embodiments exclude molecules already known
to bind to that particular protein, for example, polymer structures
such as microtubules, and energy sources such as ATP. Preferred
embodiments of assays herein include candidate agents which do not
bind the cellular proliferation protein in its endogenous native
state termed herein as "exogenous" agents. In another preferred
embodiment, exogenous agents further exclude antibodies to KSP.
[0093] Candidate agents can encompass numerous chemical classes,
though typically they are organic molecules, preferably small
organic compounds having a molecular weight of more than 100 and
less than about 2,500 daltons. Small molecules are further defined
herein as having a molecular weight of between 50 kD and 2000 kD.
In another embodiment, small molecules have a molecular weight of
less than 1500, or less than 1200, or less than 1000, or less than
750, or less than 500 kD. In one embodiment, a small molecule as
used herein has a molecular weight of about 100 to 200 kD.
Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Particularly preferred are peptides.
[0094] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and
animal extracts are available or readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means. Known pharmacological agents may be subjected to
directed or random chemical modifications, such as acylation,
alkylation, esterification, amidification to produce structural
analogs.
[0095] In a preferred embodiment, the candidate bioactive agents
are proteins. By "protein" herein is meant at least two covalently
attached amino acids, which includes proteins, polypeptides,
oligopeptides and peptides. The protein may be made up of naturally
occurring amino acids and peptide bonds, or synthetic
peptidomimetic structures. Thus "amino acid", or "peptide residue",
as used herein means both naturally occurring and synthetic amino
acids. For example, homo-phenylalanine, citrulline and noreleucine
are considered amino acids for the purposes of the invention.
"Amino acid" also includes imino acid residues such as proline and
hydroxyproline. The side chains may be in either the (R) or the (S)
configuration. In the preferred embodiment, the amino acids are in
the (S) or L-configuration. If non-naturally occurring side chains
are used, non-amino acid substituents may be used, for example to
prevent or retard in vivo degradations.
[0096] In a preferred embodiment, the candidate bioactive agents
are naturally occurring proteins or fragments of naturally
occurring proteins. Thus, for example, cellular extracts containing
proteins, or random or directed digests of proteinaceous cellular
extracts, may be used. In this way libraries of procaryotic and
eucaryotic proteins may be made for screening in the methods of the
invention. Particularly preferred in this embodiment are libraries
of bacterial, fungal, viral, and mammalian proteins, with the
latter being preferred, and human proteins being especially
preferred.
[0097] In a preferred embodiment, the candidate bioactive agents
are peptides of from about 5 to about 30 amino acids, with from
about 5 to about 20 amino acids being preferred, and from about 7
to about 15 being particularly preferred. The peptides may be
digests of naturally occurring proteins as is outlined above,
random peptides, or "biased" random peptides. By "randomized" or
grammatical equivalents herein is meant that each nucleic acid and
peptide consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0098] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of nucleic acid binding domains, the
creation of cysteines, for cross-linking, prolines for SH-3
domains, serines, threonines, tyrosines or histidines for
phosphorylation sites, etc., or to purines, etc.
[0099] In a preferred embodiment, the candidate bioactive agents
are nucleic acids. By "nucleic acid" or "oligonucleotide" or
grammatical equivalents herein means at least two nucleotides
covalently linked together. A nucleic acid of the present invention
will generally contain phosphodiester bonds, although in some
cases, as outlined below, nucleic acid analogs are included that
may have alternate backbones, comprising, for example,
phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and
references therein; Letsinger, J. Org. Chem. 35:3800 (1970);
Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al.,
Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805
(1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate
(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);
Carlsson et al., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy et al., Proc. Natl. Acad.
Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)
pp169-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments. In addition, mixtures of naturally occurring nucleic
acids and analogs can be made. Alternatively, mixtures of different
nucleic acid analogs, and mixtures of naturally occurring nucleic
acids and analogs may be made. The nucleic acids may be single
stranded or double stranded, as specified, or contain portions of
both double stranded or single stranded sequence. The nucleic acid
may be DNA, both genomic and cDNA, RNA or a hybrid, where the
nucleic acid contains any combination of deoxyribo- and
ribo-nucleotides, and any combination of bases, including uracil,
adenine, thymine, cytosine, guanine, inosine, xathanine
hypoxathanine, isocytosine, isoguanine, etc.
[0100] As described above generally for proteins, nucleic acid
candidate bioactive agents may be naturally occurring nucleic
acids, random nucleic acids, or "biased" random nucleic acids. For
example, digests of procaryotic or eucaryotic genomes may be used
as is outlined above for proteins.
[0101] In a preferred embodiment, the candidate bioactive agents
are organic chemical moieties, a wide variety of which are
available in the literature.
[0102] In a preferred embodiment, as outlined above, screens may be
done on individual genes and gene products (proteins). In a
preferred embodiment, the gene or protein has been identified as
described below as a differentially expressed gene important in a
particular state. Thus, in one embodiment, screens are designed to
first find candidate agents that can bind to differentially
expressed proteins, and then these agents may be used in assays
that evaluate the ability of the candidate agent to modulate
differentially expressed activity. Thus, as will be appreciated by
those in the art, there are a number of different assays which may
be run; binding assays and activity assays.
[0103] In a preferred embodiment, binding assays are provided. In
one embodiment, the methods comprise combining a cellular
proliferation protein and a candidate bioactive agent in the
presence or absence of microtubules, and determining the binding of
the candidate agent to the cellular proliferation protein.
Preferred embodiments utilize the human cellular proliferation
protein, although other mammalian proteins may also be used as
discussed above, for example for the development of animal models
of human disease. In some embodiments, as outlined herein, variant
or derivative cellular proliferation proteins may be used.
[0104] Generally, in a preferred embodiment of the methods herein,
the cellular proliferation protein or the candidate agent is
non-diffusably bound to an insoluble support having isolated sample
receiving areas (e.g. a microtiter plate, an array, etc.). The
insoluble supports may be made of any composition to which the
compositions can be bound, is readily separated from soluble
material, and is otherwise compatible with the overall method of
screening. The surface of such supports may be solid or porous and
of any convenient shape. Examples of suitable insoluble supports
include microtiter plates, arrays, membranes and beads. These are
typically made of glass, plastic (e.g., polystyrene),
polysaccharides, nylon or nitrocellulose, teflon.TM., etc.
Microtiter plates and arrays are especially convenient because a
large number of assays can be carried out simultaneously, using
small amounts of reagents and samples. The particular manner of
binding of the composition is not crucial so long as it is
compatible with the reagents and overall methods of the invention,
maintains the activity of the composition and is nondiffusable.
Preferred methods of binding include the use of antibodies (which
do not sterically block either the ligand binding site or
activation sequence when the protein is bound to the support),
direct binding to "sticky" or ionic supports, chemical
crosslinking, the synthesis of the protein or agent on the surface,
etc. Following binding of the protein or agent, excess unbound
material is removed by washing. The sample receiving areas may then
be blocked through incubation with bovine serum albumin (BSA),
casein or other innocuous protein or other moiety.
[0105] In a preferred embodiment, the cellular proliferation
protein is bound to the support, and, in the presence or absence of
microtubules, a candidate bioactive agent is added to the assay.
Alternatively, the candidate agent is bound to the support and the
cellular proliferation protein is added. Novel binding agents
include specific antibodies, non-natural binding agents identified
in screens of chemical libraries, peptide analogs, etc. A wide
variety of assays may be used for this purpose, including labeled
in vitro protein-protein binding assays, electrophoretic mobility
shift assays, immunoassays for protein binding, functional assays
(phosphorylation assays, etc.) and the like.
[0106] Moreover, in another aspect, screening assays are performed
herein where neither the drug candidate nor cellular proliferation
protein are bound to a solid support. Soluble assays are known in
the art. In one embodiment, binding of a cellular proliferation
protein, or fragment thereof, to a drug candidate can be determined
by changes in fluorescence of either the cellular proliferation
protein or the drug candidate, or both. Fluorescence may be
intrinsic or conferred by labeling either component with a
fluorophor. As an example that is not meant to be limiting, binding
could be detected by fluorescence polarization.
[0107] The determination of the binding of the candidate bioactive
agent to the cellular proliferation protein may be done in a number
of ways. In a preferred embodiment, the candidate bioactive agent
is labelled, and binding determined directly. For example, this may
be done by attaching all or a portion of the cellular proliferation
protein to a solid support, adding a labelled candidate agent (for
example a fluorescent label), washing off excess reagent, and
determining whether the label is present on the solid support.
Various blocking and washing steps may be utilized as is known in
the art.
[0108] By "labeled" herein is meant that the compound is either
directly or indirectly labeled with a label which provides a
detectable signal, e.g. radioisotope, fluorofers including
organo-metallic fluorescent compounds, enzyme, antibodies,
particles such as magnetic particles, chemiluminescers, or specific
binding molecules, etc. Specific binding molecules include pairs,
such as biotin and streptavidin, digoxin and antidigoxin etc. For
the specific binding members, the complementary member would
normally be labeled with a molecule which provides for detection,
in accordance with known procedures, as outlined above. The label
can directly or indirectly provide a detectable signal.
[0109] In some embodiments, only one of the components is labeled.
For example, the proteins (or proteinaceous candidate agents) may
be labeled at tyrosine positions using .sup.125I, or with
fluorophores. Alternatively, more than one component may be labeled
with different labels; using .sup.125I for the proteins, for
example, and a fluorophor for the candidate agents.
[0110] In a preferred embodiment, the binding of the candidate
bioactive agent is determined through the use of competitive
binding assays. In this embodiment, the competitor is a binding
moiety known to bind to the target molecule (i.e. cellular
proliferation protein), such as ATP, microtubules, an antibody,
peptide; binding partner, ligand, etc. Under certain circumstances,
there may be competitive binding as between the bioactive agent and
the binding moiety, with the binding moiety displacing the
bioactive agent.
[0111] In one embodiment, the candidate bioactive agent is labeled.
Either the candidate bioactive agent, or the competitor, or both,
is added first to the protein for a time sufficient to allow
binding, if present. Incubations may be performed at any
temperature which facilitates optimal activity, typically between 4
and 40.degree. C. Incubation periods are selected for optimum
activity, but may also be optimized to facilitate rapid high
through put screening. Typically between 0.1 and 1 hour will be
sufficient. Excess reagent is generally removed or washed away. The
second component is then added, and the presence or absence of the
labeled component is followed, to indicate binding.
[0112] In a preferred embodiment, the competitor is added first,
followed by the candidate bioactive agent. Displacement of the
competitor is an indication that the candidate bioactive agent is
binding to the cellular proliferation protein and thus is capable
of binding to, and potentially modulating, the activity of the
cellular proliferation protein. In this embodiment, either
component can be labeled. Thus, for example, if the competitor is
labeled, the presence of label in the wash solution indicates
displacement by the agent. Alternatively, if the candidate
bioactive agent is labeled, the presence of the label on the
support indicates displacement.
[0113] In an alternative embodiment, the candidate bioactive agent
is added first, with incubation and washing, followed by the
competitor. The absence of binding by the competitor may indicate
that the bioactive agent is bound to the cellular proliferation
protein with a higher affinity. Thus, if the candidate bioactive
agent is labeled, the presence of the label on the support, coupled
with a lack of competitor binding, may indicate that the candidate
agent is capable of binding to the cellular proliferation
protein.
[0114] In another aspect herein, proteins which bind to KSP or a
fragment thereof are identified. Genetic systems have been
described to detect protein-protein interactions. The first work
was done in yeast systems, namely the "yeast two-hybrid" system.
The basic system requires a protein-protein interaction in order to
turn on transcription of a reporter gene. Subsequent work was done
in mammalian cells. See Fields et al., Nature 340:245 (1989);
Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA
89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 (1991); Chien
et al., PNAS USA 88:9578 (1991); and U.S. Pat. Nos. 5,283,173,
5,667,973, 5,468,614, 5,525,490, and 5,637,463.
[0115] In a preferred embodiment, the binding site of the cellular
proliferation protein is identified and provided herein. This can
be done in a variety of ways. For example, once the cellular
proliferation protein has been identified as binding to a bioactive
agent, the protein is fragmented or modified and the assays
repeated to identify the necessary components for binding.
[0116] In a preferred embodiment, the methods comprise differential
screening to identify bioactive agents that are capable of
modulating the activity of the cellular proliferation proteins. In
this embodiment, the methods comprise combining a cellular
proliferation protein and a competitor in a first sample. A second
sample comprises a candidate bioactive agent, a cellular
proliferation protein and a competitor. The binding of the
competitor is determined for both samples, and a change, or
difference in binding between the two samples indicates the
presence of an agent capable of binding to the cellular
proliferation protein and, in one embodiment, modulating its
activity. Methods of determining modulation of activity are further
described below. That is, if the binding of the competitor is
different in the second sample relative to the first sample, the
agent is capable of binding to the cellular proliferation
protein.
[0117] Alternatively, a preferred embodiment utilizes differential
screening to identify drug candidates that, in the presence or
absence of microtubules, bind to the native cellular proliferation
protein, but cannot bind to modified cellular proliferation
proteins. The structure of the cellular proliferation protein may
be modeled, and used in rational drug design to synthesize agents
that interact with that site. Drug candidates that affect cellular
proliferation bioactivity are also identified by screening drugs
for the ability to either enhance or reduce the activity of the
protein in the presence or absence of microtubules.
[0118] Positive controls and negative controls may be used in the
assays. Preferably all control and test samples are performed in at
least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding
of the agent to the protein. Following incubation, all samples are
washed free of non-specifically bound material and the amount of
bound, generally labeled agent determined. For example, where a
radiolabel is employed, the samples may be counted in a
scintillation counter to determine the amount of bound
compound.
[0119] A variety of other reagents may be included in the screening
assays. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc which may be used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Also reagents that otherwise improve the efficiency
of the assay, such as protease inhibitors, nuclease inhibitors,
anti-microbial agents, etc., may be used. The mixture of components
may be added in any order that provides for the requisite
binding.
[0120] Screening for agents that modulate the activity of cellular
proliferation proteins may also be done. In a preferred embodiment,
methods for screening for a bioactive agent capable of modulating
the activity of cellular proliferation proteins comprise the steps
of adding a candidate bioactive agent to a sample of cellular
proliferation proteins in the presence or absence of microtubules,
as above, and determining an alteration in the biological activity
of cellular proliferation proteins. "Modulating the activity of
cellular proliferation" includes an increase in activity, a
decrease in activity, or a change in the type or kind of activity
present. Thus, in this embodiment, the candidate agent should both
bind to cellular proliferation proteins (although this may not be
necessary), and alter its biological or biochemical activity as
defined herein. The methods include both in vitro screening
methods, as are generally outlined above, and in vivo screening of
cells for alterations in the presence, distribution, activity or
amount of cellular proliferation proteins.
[0121] Thus, in this embodiment, the methods comprise combining a
cellular proliferation sample and a candidate bioactive agent, and
evaluating the effect on cellular proliferation activity. By
"cellular proliferation protein activity" or grammatical
equivalents herein is meant at least one of the cellular
proliferation protein's biological activities, including, but not
limited to, kinesin activity, regulation of spindle pole
separation, mitosis, mitotic spindle assembly, satisfaction of the
mitotic cell cycle checkpoint, cell cycle progression, apoptosis,
cell proliferation, mitotic and involvement in tumor growth. An
inhibitor of cellular proliferation activity is the inhibition of
any one or more cellular proliferation protein activities.
[0122] Kinesin activity is known in the art and includes one or
more kinesin activities. Kinesin activities include the ability to
affect ATP hydrolysis, microtubule binding, gliding and
polymerization/depolymerizat- ion (effects on microtubule
dynamics), binding to other proteins of the spindle, binding to
proteins involved in cell-cycle control, or serving as a substrate
to other enzymes, such as kinases or proteases and specific kinesin
cellular activities such as spindle separation.
[0123] Methods of performing motility assays are well known to
those of skill in the art (see, e.g., Hall, et al. (1996), Biophys.
J., 71:3467-3476, Turner et al., 1996, Anal. Biochem. 242(1):20-5;
Gittes et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al.,
1995, J. Exp. Biol. 198: 1809-15; Winkelmann et al., 1995, Biophys.
J. 68: 2444-53; Winkelmann et al., 1995, Biophys. J. 68: 72S, and
the like).
[0124] In addition to the assays described above, methods known in
the art for determining ATPase activity can be used. Preferably,
solution based assays are utilized. Alternatively, conventional
methods are used. For example, P.sub.i release from kinesin can be
quantified. In one preferred embodiment, the ATPase activity assay
utilizes 0.3 M PCA (perchloric acid) and malachite green reagent
(8.27 mM sodium molybdate II, 0.33 mM malachite green oxalate, and
0.8 mM Triton X-100). To perform the assay, 10 .mu.L of reaction is
quenched in 90 .mu.L of cold 0.3 M PCA. Phosphate standards are
used so data can be converted to mM inorganic phosphate released.
When all reactions and standards have been quenched in PCA, 100
.mu.L of malachite green reagent is added to the to relevant wells
in e.g., a microtiter plate. The mixture is developed for 10-15
minutes and the plate is read at an absorbance of 650 nm. If
phosphate standards were used, absorbance readings can be converted
to mM P.sub.i and plotted over time. Additionally, ATPase assays
known in the art include the luciferase assay.
[0125] In another preferred method, kinesin activity is measured by
the methods disclosed in Ser. No. 09/314,464, filed May 18, 1999,
entitled, Compositions and Assay Utilizing ADP or Phosphate for
Detecting Protein Modulators.
[0126] In a preferred embodiment, the activity of the cellular
proliferation protein is increased; in another preferred
embodiment, the activity of the cellular proliferation protein is
decreased. Thus, bioactive agents that are antagonists are
preferred in some embodiments, and bioactive agents that are
agonists may be preferred in other embodiments.
[0127] In one aspect of the invention, cells containing cellular
proliferation sequences are used in drug screening assays by
evaluating the effect of drug candidates on cellular proliferation.
Cell type include normal cells, and more preferably cells with
abnormal proliferative rates including tumor cells, most preferably
human tumor cells. Methods of assessing cellular proliferation are
known in the art and include growth and viability assays using
cultured cells. In such assays, cell populations are monitored for
growth and or viability, often over time and comparing samples
incubated with various concentrations of the bioactive agent or
without the bioactive agent. Cell number can be quantified using
agents that such as 3-(4,5-dimethylthiazol-2-yl)-2,5-dip-
henyltetrazolim bromide (MTT),
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-
thoxyphenyl)-2-(4-sufophenyl)-2H-tetrazolium (MTS) [U.S. Pat. No.
5,185,450] and Alamar Blue which are converted to colored or
fluorescent compounds in the presence of metabolically active
cells. Alternatively, dyes that bind to cellular protein such as
sulforhodamine B (SRB) or crystal violet can be used to quantify
cell number. Alternatively, cells can be directly counted using a
particle counter, such as a Coulter Counter.RTM. manufactured by
Beckman Coulter, or counted using a microscope to observe cells on
a hemocytometer. Preferably, cells counted using the hemocytometer
are observed in a solution of trypan blue to distinguish viable
from dead cells. Other methods of quantifying cell number are known
to those skilled in the art. These assays can be performed on any
of the cells, including those in a state of necrosis.
[0128] Moreover, apoptosis can be determined by methods known in
the art. For example, markers for apoptosis are known, and TUNEL
(TdT-mediated dUTP-fluorescein nick end labeling) kits can be
bought commercially, for example, Boehringer Mannheim kit, catalog
no.168795.
[0129] In a preferred embodiment, the methods comprise adding a
candidate bioactive agent, as defined above, to a cell comprising
cellular proliferation proteins. Preferred cell types include
almost any cell. The cells contain a nucleic acid, preferably
recombinant, that encodes a cellular proliferation protein. In a
preferred embodiment, a library of candidate agents are tested on a
plurality of cells.
[0130] In one aspect, the assays are evaluated in the presence or
absence or previous or subsequent exposure to physiological
signals, for example hormones, antibodies, peptides, antigens,
cytokines, growth factors, action potentials, pharmacological
agents including chemotherapeutics, radiation, carcinogenics, or
other cells (i.e. cell-cell contacts). In another example, the
determinations are determined at different stages of the cell cycle
process.
[0131] In one aspect of the invention, the cellular proliferation
sequences and cells containing cellular proliferation sequences are
used in drug screening assays by evaluating the effect of drug
candidates on a "gene expression profile" or expression profile
genes. In a preferred embodiment, the expression profiles are used,
preferably in conjunction with high throughput screening techniques
to allow monitoring for expression profile genes after treatment
with a candidate agent. See, Zlokarnik, et al., Science 279, 84-8
(1998).
[0132] In one aspect, the expression levels of genes are determined
for different cellular states in the cellular proliferation
phenotype; that is, the expression levels of genes in normal tissue
in proliferating and non-proliferating states, and in abnormal
cellular proliferation tissue (and in some cases, for varying
severities of cellular proliferation that relate to prognosis, as
outlined below) are evaluated to provide expression profiles.
Abnormal states include cancer states and other hyper or hypo
proliferation states as further defined below.
[0133] An expression profile of a particular cell state or point of
development is essentially a "fingerprint" of the state; while two
states may have any particular gene similarly expressed, the
evaluation of a number of genes simultaneously allows the
generation of a gene expression profile that is unique to the state
of the cell. By comparing expression profiles of cells in different
states, information regarding which genes are important (including
both up- and down-regulation of genes) in each of these states is
obtained. Then, diagnosis may be done or confirmed: does tissue
from a particular patient have the gene expression profile of
normal or abnormal cellular proliferation tissue.
[0134] "Differential expression," or grammatical equivalents as
used herein, refers to both qualitative as well as quantitative
differences in the genes' temporal and/or cellular expression
patterns within and among the cells. Thus, a differentially
expressed gene can qualitatively have its expression altered,
including an activation or inactivation, in, for example, normal
versus abnormal cellular proliferation tissue. That is, genes may
be turned on or turned off in a particular state, relative to
another state or have a different timing pattern, for example,
cancerous cells may have genes which stay on. As is apparent to the
skilled artisan, any comparison of two or more states can be made
and repeated at various time points. Such a qualitatively regulated
gene will exhibit an expression pattern within a state or cell type
which is detectable by standard techniques in one such state or
cell type, but is not detectable in both. Alternatively, the
determination is quantitative in that expression is increased or
decreased; that is, the expression of the gene is either
upregulated, resulting in an increased amount of transcript, or
downregulated, resulting in a decreased amount of transcript. The
degree to which expression differs need only be large enough to
quantify via standard characterization techniques as outlined
below, such as by use of Affymetrix GeneChip.TM. expression arrays,
Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby
expressly incorporated by reference. Other techniques include, but
are not limited to, quantitative reverse transcriptase PCR,
Northern analysis and RNase protection. As outlined above,
preferably the change in expression (i.e. upregulation or
downregulation) is at least about 50%, more preferably at least
about 100%, more preferably at least about 150%, more preferably,
at least about 200%, with from 300 to at least 1000% being
especially preferred.
[0135] As will be appreciated by those in the art, this may be done
by evaluation at either the gene transcript, or the protein level;
that is, the amount of gene expression may be monitored using
nucleic acid probes to the DNA or RNA equivalent of the gene
transcript, and the quantification of gene expression levels, or,
alternatively, the final gene product itself (protein) can be
monitored, for example through the use of antibodies to the
cellular proliferation protein and standard immunoassays (ELISAs,
etc.) or other techniques, including mass spectroscopy assays, 2D
gel electrophoresis assays, etc. Thus, the proteins corresponding
to cellular proliferation genes, i.e. those identified as being
important in a cellular proliferation phenotype, can be evaluated
in a cellular proliferation diagnostic test.
[0136] In a preferred embodiment nucleic acids encoding the
cellular proliferation protein are detected. Although DNA or RNA
encoding the cellular proliferation protein may be detected, of
particular interest are methods wherein the mRNA encoding a
cellular proliferation protein is detected. The presence of mRNA in
a sample is an indication that the cellular proliferation gene has
been transcribed to form the mRNA, and suggests that the protein is
expressed. Probes to detect the mRNA can be any
nucleotide/deoxynucleotide probe that is complementary to and base
pairs with the mRNA and includes but is not limited to
oligonucleotides, cDNA or RNA. Probes also should contain a
detectable label, as defined herein. In one method the mRNA is
detected after immobilizing the nucleic acid to be examined on a
solid support such as nylon membranes and hybridizing the probe
with the sample. Following washing to remove the non-specifically
bound probe, the label is detected. In another method detection of
the mRNA is performed in situ. In this method permeabilized cells
or tissue samples are contacted with a detectably labeled nucleic
acid probe for sufficient time to allow the probe to hybridize with
the target mRNA. Following washing to remove the non-specifically
bound probe, the label is detected. For example a digoxygenin
labeled riboprobe (RNA probe) that is complementary to the mRNA
encoding a cellular proliferation protein is detected by binding
the digoxygenin with an anti-digoxygenin secondary antibody and
developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl
phosphate.
[0137] In one case, having identified a particular gene as up
regulated in cellular proliferation, candidate bioactive agents may
be screened to modulate this gene's response; preferably to down
regulate the gene, although in some circumstances to up regulate
the gene. "Modulation" thus includes both an increase and a
decrease in gene expression or a change in temporal pattern. The
preferred amount of modulation will depend on the original change
of the gene expression in normal versus tumor tissue, with changes
of at least 10%, preferably 50%, more preferably 100-300%, and in
some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4
fold increase in tumor compared to normal tissue, a decrease of
about four fold is desired; a 10 fold decrease in tumor compared to
normal tissue gives a 10 fold increase in expression for a
candidate agent is desired.
[0138] In a preferred embodiment, gene expression monitoring is
done and a number of genes, i.e. an expression profile, is
monitored simultaneously, although multiple protein expression
monitoring can be done as well.
[0139] In one embodiment, the cellular proliferation nucleic acid
probes are attached to biochips as outlined below for the detection
and quantification of cellular proliferation sequences in a
particular cell.
[0140] Generally, in a preferred embodiment, a candidate bioactive
agent is added to the cells prior to analysis. Any cell can be
used, including normal and abnormal cells, including tumor and
non-tumor mammalian, preferably human cells. In some cases, plant
cells are used. After the candidate agent has been added and the
cells allowed to incubate for some period of time, the sample
containing the target sequences to be analyzed is added to the
biochip. If required, the target sequence is prepared using known
techniques. For example, the sample may be treated to lyse the
cells, using known lysis buffers, electroporation, etc., with
purification and/or amplification such as PCR occurring as needed,
as will be appreciated by those in the art. For example, an in
vitro transcription with labels covalently attached to the
nucleosides is done. Generally, the nucleic acids are labeled with
biotin-FITC or PE, or with cy3 or cy5.
[0141] In a preferred embodiment, the target sequence is labeled
with, for example, a fluorescent, a chemiluminescent, a chemical,
or a radioactive signal, to provide a means of detecting the target
sequence's specific binding to a probe. The label also can be an
enzyme, such as, alkaline phosphatase or horseradish peroxidase,
which when provided with an appropriate substrate produces a
product that can be detected. Alternatively, the label can be a
labeled compound or small molecule, such as an enzyme inhibitor,
that binds but is not catalyzed or altered by the enzyme. The label
also can be a moiety or compound, such as, an epitope tag or biotin
which specifically binds to streptavidin. For the example of
biotin, the streptavidin is labeled as described above, thereby,
providing a detectable signal for the bound target sequence. As
known in the art, unbound labeled streptavidin is removed prior to
analysis.
[0142] As will be appreciated by those in the art, these assays can
be direct hybridization assays or can comprise "sandwich assays",
which include the use of multiple probes, as is generally outlined
in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117,
5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802,
5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of
which are hereby incorporated by reference. In this embodiment, in
general, the target nucleic acid is prepared as outlined above, and
then added to the biochip comprising a plurality of nucleic acid
probes, under conditions that allow the formation of a
hybridization complex.
[0143] A variety of hybridization conditions may be used in the
present invention, including high, moderate and low stringency
conditions as outlined above. The assays are generally run under
stringency conditions which allows formation of the label probe
hybridization complex only in the presence of target. Stringency
can be controlled by altering a step parameter that is a
thermodynamic variable, including, but not limited to, temperature,
formamide concentration, salt concentration, chaotropic salt
concentration pH, organic solvent concentration, etc.
[0144] These parameters may also be used to control non-specific
binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus
it may be desirable to perform certain steps at higher stringency
conditions to reduce non-specific binding.
[0145] The reactions outlined herein may be accomplished in a
variety of ways, as will be appreciated by those in the art.
Components of the reaction may be added simultaneously, or
sequentially, in any order, with preferred embodiments outlined
below. In addition, the reaction may include a variety of other
reagents may be included in the assays. These include reagents like
salts, buffers, neutral proteins, e.g. albumin, detergents, etc
which may be used to facilitate optimal hybridization and
detection, and/or reduce non-specific or background interactions.
Also reagents that otherwise improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc., may be used, depending on the sample preparation
methods and purity of the target.
[0146] Once the assay is run, the data is analyzed to determine the
expression levels, and changes in expression levels as between
states, of individual genes, forming a gene expression profile.
[0147] In one aspect, the screens are done to identify drugs or
bioactive agents that modulate the cellular proliferation
phenotype. Specifically, there are several types of screens that
can be run. A preferred embodiment is in the screening of candidate
agents that can induce or suppress a particular expression profile,
thus preferably generating the associated phenotype. That is,
candidate agents that can mimic or produce an expression profile in
cellular proliferation similar to the expression profile of normal
non-cancerous tissue is expected to result in a suppression of the
cellular proliferation phenotype. Thus, in this embodiment,
mimicking an expression profile, or changing one profile to
another, is the goal.
[0148] In a preferred embodiment, as for the diagnosis and
prognosis applications discussed below, having identified the
differentially expressed genes important in any one state as
further described below, screens can be run to alter the expression
of the genes individually. That is, screening for modulation of
regulation of expression of a single gene can be done; that is,
rather than try to mimic all or part of an expression profile,
screening for regulation of individual genes can be done. Thus, for
example, particularly in the case of target genes whose presence,
absence or temporal pattern is unique between two states, screening
is done for modulators of the target gene expression. In a
preferred embodiment, the target gene encodes the cellular
proliferation protein described herein. Thus, screening of
candidate agents that modulate the cellular proliferation phenotype
either at the gene expression level or the protein level can be
done.
[0149] In addition screens can be done for novel genes that are
induced in response to a candidate agent. After identifying a
candidate agent based upon its ability to suppress a cellular
proliferation expression pattern leading to a normal expression
pattern, or modulate a single cellular proliferation gene
expression profile so as to mimic the expression of the gene from
normal tissue, a screen as described above can be performed to
identify genes that are specifically modulated in response to the
agent. Comparing expression profiles between normal tissue and
agent treated cellular proliferation tissue reveals genes that are
not expressed in normal tissue or cellular proliferation tissue,
but are expressed in agent treated tissue. These agent specific
sequences can be identified and used by any of the methods
described herein for cellular proliferation genes or proteins. In
particular these sequences and the proteins they encode find use in
marking or identifying agent treated cells. In addition, antibodies
can be raised against the agent induced proteins and used to target
novel therapeutics to the treated cellular proliferation tissue
sample.
[0150] In one embodiment, a candidate agent is administered to a
population of cellular proliferation cells, that thus has an
associated cellular proliferation expression profile. By
"administration" or "contacting" herein is meant that the candidate
agent is added to the cells in such a manner as to allow the agent
to act upon the cell, whether by uptake and intracellular action,
or by action at the cell surface. In some embodiments, nucleic acid
encoding a proteinaceous candidate agent (i.e. a peptide) may be
put into a viral construct such as a retroviral construct and added
to the cell, such that expression of the peptide agent is
accomplished; see PCT US97/01019, hereby expressly incorporated by
reference. The phrase "under conditions which allow the cell to
uptake the candidate agent" means that the cell is biologically
involved in the uptake and intracellular action, or by action at
the cell surface in that the agent is not injected into the cell.
It is understood that targeting ligands and biochemically agents
can be used to facilitate the uptake, however, this differs from
mechanical injection. Mechanical injection is explicitly excluded
from the definition of "taken up by the cell" as used herein, and
is excluded from conditions inducive to high throughput assays as
used herein.
[0151] Once the candidate agent has been administered to the cells,
the cells can be washed if desired and are allowed to incubate
under preferably physiological conditions for some period of time.
The cells are then harvested and a new gene expression profile is
generated, as outlined herein.
[0152] Thus, for example, cellular proliferation tissue may be
screened for agents that reduce or suppress the cellular
proliferation phenotype. A change in at least one gene of the
expression profile indicates that the agent has an effect on
cellular proliferation activity. By defining such a signature for
the cellular proliferation phenotype, screens for new drugs that
alter the phenotype can be devised. With this approach, the drug
target need not be known and need not be represented in the
original expression screening platform, nor does the level of
transcript for the target protein need to change.
[0153] In all the methods provided herein, bioactive agents are
identified. Similarly, compounds which interfere with binding or
interaction between the cellular proliferation protein and an
identified binding or modulating agent can be identified. Moreover,
transgenic models as discussed below may be used to identify
bioactive agents. Compounds with pharmacological activity are able
to enhance or interfere with the activity of the cellular
proliferation protein. The compounds can be used in further assays
so as to confirm activity wherein necessary or optimize conditions
including varying the identified molecules. In a preferred
embodiment, the agents are used as therapeutics as discussed
below.
[0154] In a further aspect of the present invention, methods of
modulating cellular proliferation in cells or organisms are
provided. In one embodiment, the methods comprise administering to
a cell an anti-cellular proliferation antibody as further discussed
below that reduces or eliminates the biological activity of an
endogeneous cellular proliferation protein. In a preferred
embodiment, a nucleic acid encoding said antibody is administered.
Agents identified to modulate cellular proliferation can also be
used. Alternatively, the methods comprise administering to a cell
or organism a composition comprising a cellular proliferation
sequence.
[0155] In a preferred embodiment, for example when the cellular
proliferation sequence is down-regulated in cellular proliferation,
the activity of the cellular proliferation gene is increased by
increasing the amount of cellular proliferation in the cell, for
example by overexpressing the endogeneous cellular proliferation or
by administering a gene encoding the cellular proliferation
sequence, using known gene-therapy techniques, for example. In a
preferred embodiment, the gene therapy techniques include the
incorporation of the exogeneous gene using enhanced homologous
recombination (EHR), for example as described in PCT/US93/03868,
hereby incorporated by reference in its entirety. Alternatively,
for example when the cellular proliferation sequence is
up-regulated in cellular proliferation, the activity of the
endogeneous cellular proliferation gene is decreased, for example
by the administration of a cellular proliferation antisense nucleic
acid. Preferably, as discussed below, cellular proliferation is
inhibited.
[0156] Thus, in one embodiment, a method of inhibiting cell
division is provided. In a preferred embodiment, a method of
inhibiting tumor growth is provided. In a further embodiment,
methods of treating cells or individuals with cancer are provided.
The method comprises administration of a cellular proliferation
inhibitor.
[0157] In one embodiment, a cellular proliferation inhibitor is an
antibody as discussed above and further described below. In another
embodiment, the cellular proliferation inhibitor is an antisense
molecule as discussed above. Antisense molecules as used herein
include antisense or sense oligonucleotides comprising a
singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target mRNA (sense) or DNA (antisense) sequences for
cellular proliferation molecules. A preferred antisense molecule is
for KSP or for a ligand or activator thereof. Antisense or sense
oligonucleotides, according to the present invention, comprise a
fragment generally at least about 14 nucleotides, preferably from
about 14 to 30 nucleotides. The ability to derive an antisense or a
sense oligonucleotide, based upon a cDNA sequence encoding a given
protein is described in, for example, Stein and Cohen (Cancer Res.
48:2659, 1988) and van der Krol et al. (BioTechniques 6:958,
1988).
[0158] Antisense molecules may be introduced into a cell containing
the target nucleotide sequence by formation of a conjugate with a
ligand binding molecule, as described in WO 91/04753. Suitable
ligand binding molecules include, but are not limited to, cell
surface receptors, growth factors, other cytokines, or other
ligands that bind to cell surface receptors. Preferably,
conjugation of the ligand binding molecule does not substantially
interfere with the ability of the ligand binding molecule to bind
to its corresponding molecule or receptor, or block entry of the
sense or antisense oligonucleotide or its conjugated version into
the cell. Alternatively, a sense or an antisense oligonucleotide
may be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. It is understood that the use of
antisense molecules or knock out and knock in models may also be
used in screening assays as discussed above, in addition to methods
of treatment. Moreover, knock out models can include knocking out
expression, rather than the genome, such as by the use ribozymes.
In one case, ribozymes are a preferred KSP inhibitor.
[0159] As discussed above, the methods and compositions herein are
not limited to cancer. Disease states which can be treated by the
methods and compositions provided herein include, but are not
limited to, cancer (further discussed below), restenosis,
autoimmune disease, arthritis, graft rejection, inflammatory bowel
disease, proliferation induced after medical procedures, including,
but not limited to, surgery, angioplasty, and the like. It is
appreciated that in some cases the cells may not be in a hyper or
hypo proliferation state (abnormal state) and still require
treatment. For example, during wound healing, the cells may be
proliferating "normally", but proliferation enhancement may be
desired. Similarly, as discussed above, in the agriculture arena,
cells may be in a "normal" state, but proliferation modulation may
be desired to enhance a crop by directly enhancing growth of a
crop, or by inhibiting the growth of a plant or organism which
adversely affects the crop. Thus, in one embodiment, the invention
herein includes application to cells or individuals afflicted or
impending affliction with any one of these disorders or states.
[0160] The compositions and methods provided herein are
particularly deemed useful for the treatment of cancer including
solid tumors such as skin, breast, brain, cervical carcinomas,
testicular carcinomas, etc. More particularly, cancers that may be
treated by the compositions and methods of the invention include,
but are not limited to: Cardiac: sarcoma (angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,
fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma
(squamous cell, undifferentiated small cell, undifferentiated large
cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial
adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;
Gastrointestinal: esophagus (squamous cell carcinoma,
adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma,
lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,
insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma),
small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's
sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma),
large bowel (adenocarcinoma, tubular adenoma, villous adenoma,
hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma,
Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and
urethra (squamous cell carcinoma, transitional cell carcinoma,
adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis
(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,
choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,
fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma
(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom,
angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic
sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous
histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma
(reticulum cell sarcoma), multiple myeloma, malignant giant cell
tumor chordoma, osteochronfroma (osteocartilaginous exostoses),
benign chondroma, chondroblastoma, chondromyxofibroma, osteoid
osteoma and giant cell tumors; Nervous system: skull (osteoma,
hemangioma, granuloma, xanthoma, osteitis deformans), meninges
(meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma,
medulloblastoma, glioma, ependymoma, germinoma [pinealoma],
glioblastoma multiform, oligodendroglioma, schwannoma,
retinoblastoma, congenital tumors), spinal cord neurofibroma,
meningioma, glioma, sarcoma); Gynecological: uterus (endometrial
carcinoma), cervix (cervical carcinoma, pre-tumor cervical
dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,
mucinous cystadenocarcinoma, unclassified carcinoma],
granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,
dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,
intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),
vagina (clear cell carcinoma, squamous cell carcinoma, botryoid
sarcoma [embryonal rhabdomyosarcoma], fallopian tubes (carcinoma);
Hematologic: blood (myeloid leukemia [acute and chronic], acute
lymphoblastic leukemia, chronic lymphocytic leukemia,
myeloproliferative diseases, multiple myeloma, myelodysplastic
syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant
lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous
cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma,
angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands:
neuroblastoma. The cancer can be solid tumors or metastatic. Thus,
the term "cancerous cell" as provided herein, includes a cell
afflicted by any one of the above identified conditions.
[0161] In another aspect herein, diagnostic assays are provided
herein. In one embodiment, the cellular proliferation sequences are
used in the diagnostic assays. This can be done on an individual
gene or corresponding polypeptide level. In a preferred embodiment,
the expression profiles are used, preferably in conjunction with
high throughput screening techniques to allow monitoring for
expression profile genes and/or corresponding polypeptides. In a
preferred embodiment, in situ hybridization of labeled cellular
proliferation nucleic acid probes to tissue arrays is done. For
example, arrays of tissue samples, including cellular proliferation
tissue in various states and or time points and/or normal tissue,
are made. In situ hybridization as is known in the art can then be
done. It is understood that conventional antibody and protein
localization methods can also be used in diagnostic assays
herein.
[0162] It is understood that when comparing the fingerprints
between an individual and a standard, the skilled artisan can make
a diagnosis as well as a prognosis. It is further understood that
the genes which indicate the diagnosis may differ from those which
indicate the prognosis.
[0163] In a preferred embodiment, the cellular proliferation
sequences are used in prognosis assays. As above, gene expression
profiles can be generated that correlate to cellular proliferation
severity, in terms of long term prognosis. Again, this may be done
on either a protein or gene level, with the use of genes being
preferred. In both the diagnostic and prognostic assays, the
cellular proliferation probes can be attached to biochips as
described below for the detection and quantification of cellular
proliferation sequences in a tissue or patient.
[0164] Accordingly, disorders based on mutant or variant cellular
proliferation genes may also be determined. In one embodiment, the
invention provides methods for identifying cells containing variant
cellular proliferation genes comprising determining all or part of
the sequence of at least one endogenous cellular proliferation
genes in a cell. As will be appreciated by those in the art, this
may be done using any number of sequencing techniques. In a
preferred embodiment, the invention provides methods of identifying
the cellular proliferation genotype of an individual comprising
determining all or part of the sequence of at least one cellular
proliferation gene of the individual. This is generally done in at
least one tissue of the individual, and may include the evaluation
of a number of tissues or different samples of the same tissue. The
method may include comparing the sequence of the sequenced cellular
proliferation gene to a known cellular proliferation gene, i.e. a
wild-type gene.
[0165] The sequence of all or part of the cellular proliferation
gene can then be compared to the sequence of a known cellular
proliferation gene to determine if any differences exist. This can
be done using any number of known homology programs, such as
Bestfit, etc. In a preferred embodiment, the presence of a
difference in the sequence between the cellular proliferation gene
of the patient and the known cellular proliferation gene is
indicative of a disease state or a propensity for a disease state,
as outlined herein.
[0166] In a preferred embodiment, the cellular proliferation genes
are used as probes to determine the number of copies of the
cellular proliferation gene in the genome.
[0167] In another preferred embodiment cellular proliferation genes
are used as probes to determine the chromosomal localization of the
cellular proliferation genes. Information such as chromosomal
localization finds use in providing a diagnosis or prognosis in
particular when chromosomal abnormalities such as translocations,
and the like are identified in cellular proliferation gene
loci.
[0168] Once a determination has been made regarding the
proliferation state of a cell, if desired, the compositions or
agents described herein can be administered. The compounds having
the desired pharmacological activity may be administered in a
physiologically acceptable carrier (also called a pharmaceutically
acceptable carrier) to a host. Depending upon the manner of
introduction, the compounds may be formulated in a variety of ways
as discussed below. The concentration of therapeutically active
compound in the formulation may vary from about 0.1-100 wt. %. The
agents may be administered alone or in combination with other
treatments, e.g., radiation.
[0169] Thus, in a preferred embodiment, cellular proliferation
proteins and modulators are administered as is therapeutic agents.
Similarly, cellular proliferation genes (including both the
full-length sequence, partial sequences, or regulatory sequences of
the cellular proliferation coding regions) can be administered in
gene therapy applications, as is known in the art. These cellular
proliferation genes can include antisense applications, either as
gene therapy (i.e. for incorporation into the genome) or as
antisense compositions, as will be appreciated by those in the
art.
[0170] In the preferred embodiment, the pharmaceutical compositions
are in a water soluble form, such as being present as
pharmaceutically acceptable salts, which is meant to include both
acid and base addition salts. "Pharmaceutically acceptable acid
addition salt" refers to those salts that retain the biological
effectiveness of the free bases and that are not biologically or
otherwise undesirable, formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. "Pharmaceutically acceptable base
addition salts" include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Particularly
preferred are the ammonium, potassium, sodium, calcium, and
magnesium salts. Salts derived from pharmaceutically acceptable
organic non-toxic bases include salts of primary, secondary, and
tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic amines and basic ion exchange resins,
such as isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, and ethanolamine.
[0171] The pharmaceutical compositions can be prepared in various
forms, such as granules, tablets, pills, suppositories, capsules,
suspensions, salves, lotions and the like. Pharmaceutical grade
organic or inorganic carriers and/or diluents suitable for oral and
topical use can be used to make up compositions containing the
therapeutically-active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents. The
pharmaceutical compositions may also include one or more of the
following: carrier proteins such as serum albumin; buffers; fillers
such as microcrystalline cellulose, lactose, corn and other
starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known
in the art, and are used in a variety of formulations.
[0172] The administration of the cellular proliferation proteins
and modulators of the present invention can be done in a variety of
ways as discussed above, including, but not limited to, orally,
subcutaneously, intravenously, intranasally, transdermally,
intraperitoneally, intramuscularly, intrapulmonary, vaginally,
rectally, or intraocularly. In some instances, for example, in the
treatment of wounds and inflammation, the cellular proliferation
proteins and modulators may be directly applied as a solution or
spray.
[0173] In one embodiment, a therapeutically effective dose of a
cellular proliferation protein or modulator thereof is administered
to a patient. By "therapeutically effective dose" herein is meant a
dose that produces the effects for which it is administered. The
exact dose will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques. As
is known in the art, adjustments for cellular proliferation
degradation, systemic versus localized delivery, and rate of new
protease synthesis, as well as the age, body weight, general
health, sex, diet, time of administration, drug interaction and the
severity of the condition may be necessary, and will be
ascertainable with routine experimentation by those skilled in the
art.
[0174] a "patient" for the purposes of the present invention
includes both humans and other animals, particularly mammals, and
organisms. Thus the methods are applicable to both human therapy
and veterinary applications. In the preferred embodiment the
patient is a mammal, and in the most preferred embodiment the
patient is human.
[0175] In a preferred embodiment, cellular proliferation genes are
administered as DNA vaccines, either single genes or combinations
of cellular proliferation genes. Naked DNA vaccines are generally
known in the art. Brower, Nature Biotechnology, 16:1304-1305
(1998).
[0176] In one embodiment, cellular proliferation genes of the
present invention are used as DNA vaccines. Methods for the use of
genes as DNA vaccines are well known to one of ordinary skill in
the art, and include placing a cellular proliferation gene or
portion of a cellular proliferation gene under the control of a
promoter for expression in a cellular proliferation patient. The
cellular proliferation gene used for DNA vaccines can encode
full-length cellular proliferation proteins, but more preferably
encodes portions of the cellular proliferation proteins including
peptides derived from the cellular proliferation protein. In a
preferred embodiment a patient is immunized with a DNA vaccine
comprising a plurality of nucleotide sequences derived from a
cellular proliferation gene. Similarly, it is possible to immunize
a patient with a plurality of cellular proliferation genes or
portions thereof as defined herein. Without being bound by theory,
expression of the polypeptide encoded by the DNA vaccine, cytotoxic
T-cells, helper T-cells and antibodies are induced which recognize
and destroy or eliminate cells expressing cellular proliferation
proteins.
[0177] In a preferred embodiment, the DNA vaccines include a gene
encoding an adjuvant molecule with the DNA vaccine. Such adjuvant
molecules include cytokines that increase the immunogenic response
to the cellular proliferation polypeptide encoded by the DNA
vaccine. Additional or alternative adjuvants are known to those of
ordinary skill in the art and find use in the invention.
[0178] In another preferred embodiment cellular proliferation genes
find use in generating animal models of cellular proliferation. As
is appreciated by one of ordinary skill in the art, when the
cellular proliferation gene identified is repressed or diminished
in cellular proliferation tissue, gene therapy technology wherein
antisense RNA directed to the cellular proliferation gene will also
diminish or repress expression of the gene. An animal generated as
such serves as an animal model of cellular proliferation that finds
use in screening bioactive drug candidates. Similarly, gene
knockout technology, for example as a result of homologous
recombination with an appropriate gene targeting vector, will
result in the absence of the cellular proliferation protein. When
desired, tissue-specific expression or knockout of the cellular
proliferation protein may be necessary.
[0179] It is also possible that the cellular proliferation protein
is overexpressed in cellular proliferation. As such, transgenic
animals can be generated that overexpress the cellular
proliferation protein. Depending on the desired expression level,
promoters of various strengths can be employed to express the
transgene. Also, the number of copies of the integrated transgene
can be determined and compared for a determination of the
expression level of the transgene. Animals generated by such
methods find use as animal models of cellular proliferation and are
additionally useful in screening for bioactive molecules to treat
cellular proliferation.
[0180] In a preferred embodiment, biochips are provided herein.
Nucleic acid probes to cellular proliferation nucleic acids (both
the nucleic acid sequences outlined in the figures and/or the
complements thereof are made. The nucleic acid probes attached to
the biochip are designed to be substantially complementary to the
cellular proliferation nucleic acids, i.e. the target sequence
(either the target sequence of the sample or to other probe
sequences, for example in sandwich assays), such that hybridization
of the target sequence and the probes of the present invention
occurs. As outlined below, this complementarity need not be
perfect; there may be any number of base pair mismatches which will
interfere with hybridization between the target sequence and the
single stranded nucleic acids of the present invention. However, if
the number of mutations is so great that no hybridization can occur
under even the least stringent of hybridization conditions, the
sequence is not a complementary target sequence. Thus, by
"substantially complementary" herein is meant that the probes are
sufficiently complementary to the target sequences to hybridize
under normal reaction conditions, particularly high stringency
conditions, as outlined herein.
[0181] A nucleic acid probe is generally single stranded but can be
partially single and partially double stranded. The strandedness of
the probe is dictated by the structure, composition, and properties
of the target sequence. In general, the nucleic acid probes range
from about 8 to about 100 bases long, with from about 10 to about
80 bases being preferred, and from about 30 to about 50 bases being
particularly preferred. That is, generally whole genes are not
used. In some embodiments, much longer nucleic acids can be used,
up to hundreds of bases.
[0182] In a preferred embodiment, more than one probe per sequence
is used, with either overlapping probes or probes to different
sections of the target being used. That is, two, three, four or
more probes, with three being preferred, are used to build in a
redundancy for a particular target. The probes can be overlapping
(i.e. have some sequence in common), or separate.
[0183] As will be appreciated by those in the art, nucleic acids
can be attached or immobilized to a solid support in a wide variety
of ways. By "immobilized" and grammatical equivalents herein is
meant the association or binding between the nucleic acid probe and
the solid support is sufficient to be stable under the conditions
of binding, washing, analysis, and removal as outlined below. The
binding can be covalent or non-covalent. By "non-covalent binding"
and grammatical equivalents herein is meant one or more of either
electrostatic, hydrophilic, and hydrophobic interactions. Included
in non-covalent binding is the covalent attachment of a molecule,
such as, streptavidin to the support and the non-covalent binding
of the biotinylated probe to the streptavidin. By "covalent
binding" and grammatical equivalents herein is meant that the two
moieties, the solid support and the probe, are attached by at least
one bond, including sigma bonds, pi bonds and coordination bonds.
Covalent bonds can be formed directly between the probe and the
solid support or can be formed by a cross linker or by inclusion of
a specific reactive group on either the solid support or the probe
or both molecules. Immobilization may also involve a combination of
covalent and non-covalent interactions.
[0184] In general, the probes are attached to the biochip in a wide
variety of ways, as will be appreciated by those in the art. As
described herein, the nucleic acids can either be synthesized
first, with subsequent attachment to the biochip, or can be
directly synthesized on the biochip.
[0185] The biochip comprises a suitable solid substrate. By
"substrate" or "solid support" or other grammatical equivalents
herein is meant any material that can be modified to contain
discrete individual sites appropriate for the attachment or
association of the nucleic acid probes and is amenable to at least
one detection method. As will be appreciated by those in the art,
the number of possible substrates are very large, and include, but
are not limited to, glass and modified or functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, etc. In general, the substrates allow optical detection
and do not appreciably fluorescese. a preferred substrate is
described in copending application entitled Reusable Low
Fluorescent Plastic Biochip filed Mar. 15, 1999, herein
incorporated by reference in its entirety.
[0186] Generally the substrate is planar, although as will be
appreciated by those in the art, other configurations of substrates
may be used as well. For example, the probes may be placed on the
inside surface of a tube, for flow-through sample analysis to
minimize sample volume. Similarly, the substrate may be flexible,
such as a flexible foam, including closed cell foams made of
particular plastics.
[0187] In a preferred embodiment, the surface of the biochip and
the probe may be derivatized with chemical functional groups for
subsequent attachment of the two. Thus, for example, the biochip is
derivatized with a chemical functional group including, but not
limited to, amino groups, carboxy groups, oxo groups and thiol
groups, with amino groups being particularly preferred. Using these
functional groups, the probes can be attached using functional
groups on the probes. For example, nucleic acids containing amino
groups can be attached to surfaces comprising amino groups, for
example using linkers as are known in the art; for example, homo-
or hetero-bifunctional linkers as are well known (see 1994 Pierce
Chemical Company catalog, technical section on cross-linkers, pages
155-200, incorporated herein by reference). In addition, in some
cases, additional linkers, such as alkyl groups (including
substituted and heteroalkyl groups) may be used.
[0188] In this embodiment, the oligonucleotides are synthesized as
is known in the art, and then attached to the surface of the solid
support. As will be appreciated by those skilled in the art, either
the 5' or 3' terminus may be attached to the solid support, or
attachment may be via an internal nucleoside.
[0189] In an additional embodiment, the immobilization to the solid
support may be very strong, yet non-covalent.
[0190] For example, biotinylated oligonucleotides can be made,
which bind to surfaces covalently coated with streptavidin,
resulting in attachment.
[0191] Alternatively, the oligonucleotides may be synthesized on
the surface, as is known in the art. For example, photoactivation
techniques utilizing photopolymerization compounds and techniques
are used. In a preferred embodiment, the nucleic acids can be
synthesized in situ, using well known photolithographic techniques,
such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos.
5,700,637 and 5,445,934; and references cited within, all of which
are expressly incorporated by reference; these methods of
attachment form the basis of the Affimetrix GeneChip.TM.
technology.
[0192] In another preferred embodiment anti-cellular proliferation
antibodies are provided. In one case, the cellular proliferation
protein is to be used to generate antibodies, for example for
immunotherapy. Wherein a fragment of the cellular proliferation
protein is used, the cellular proliferation protein should share at
least one epitope or determinant with the full length protein. By
"epitope" or "determinant" herein is meant a portion of a protein
which will generate and/or bind an antibody or T-cell receptor in
the context of MHC. Thus, in most instances, antibodies made to a
smaller cellular proliferation protein will be able to bind to the
full length protein. In a preferred embodiment, the epitope is
unique; that is, antibodies generated to a unique epitope show
little or no cross-reactivity.
[0193] In one embodiment, the term "antibody" includes antibody
fragments, as are known in the art, including Fab, Fab.sub.2,
single chain antibodies (Fv for example), chimeric antibodies,
etc., either produced by the modification of whole antibodies or
those synthesized de novo using recombinant DNA technologies.
[0194] Methods of preparing polyclonal antibodies are known to the
skilled artisan. Polyclonal antibodies can be raised in a mammal,
for example, by one or more injections of an immunizing agent and,
if desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the
KSP or fragment thereof or a fusion protein thereof. It may be
useful to conjugate the immunizing agent to a protein known to be
immunogenic in the mammal being immunized. Examples of such
immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine thyroglobulin, and soybean
trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid a, synthetic trehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art
without undue experimentation.
[0195] The antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such
as those described by Kohler and Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host
animal, is typically immunized with an immunizing agent to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the immunizing agent. Alternatively,
the lymphocytes may be immunized in vitro. The immunizing agent
will typically include the KSP polypeptide or fragment thereof or a
fusion protein thereof. Generally, either peripheral blood
lymphocytes ("PBLs") are used if cells of human origin are desired,
or spleen cells or lymph node cells are used if non-human mammalian
sources are desired. The lymphocytes are then fused with an
immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986) pp.
59-103]. Immortalized cell lines are usually transformed mammalian
cells, particularly myeloma cells of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells may be cultured in a suitable culture medium that
preferably contains one or more substances that inhibit the growth
or survival of the unfused, immortalized cells. For example, if the
parental cells lack the enzyme hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas
typically will include hypoxanthine, aminopterin, and thymidine
("HAT medium"), which substances prevent the growth of
HGPRT-deficient cells.
[0196] In one embodiment, the antibodies are bispecific antibodies.
Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the KSP or a fragment thereof, the other one
is for any other antigen, and preferably for a cell-surface protein
or receptor or receptor subunit, preferably one that is tumor
specific.
[0197] In a preferred embodiment, the antibodies to cellular
proliferation are capable of reducing or eliminating the biological
function of cellular proliferation, as is described below. That is,
the addition of anti-KSP antibodies (either polyclonal or
preferably monoclonal) to cellular proliferation (or cells
containing cellular proliferation) may reduce or eliminate the
cellular proliferation activity. Generally, at least a 25% decrease
in activity is preferred, with at least about 50% being
particularly preferred and about a 95-100% decrease being
especially preferred.
[0198] In a preferred embodiment the antibodies to the cellular
proliferation proteins are humanized antibodies. Humanized forms of
non-human (e.g., murine) antibodies are chimeric molecules of
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues form a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0199] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0200] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boemer et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783 (1992);
Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0201] By immunotherapy is meant treatment of cellular
proliferation with an antibody raised against cellular
proliferation proteins. As used herein, immunotherapy can be
passive or active. Passive immunotherapy as defined herein is the
passive transfer of antibody to a recipient (patient). Active
immunization is the induction of antibody and/or T-cell responses
in a recipient (patient). Induction of an immune response is the
result of providing the recipient with an antigen to which
antibodies are raised. As appreciated by one of ordinary skill in
the art, the antigen may be provided by injecting a polypeptide
against which antibodies are desired to be raised into a recipient,
or contacting the recipient with a nucleic acid capable of
expressing the antigen and under conditions for expression of the
antigen.
[0202] As will be appreciated by one of ordinary skill in the art,
the antibody may be a competitive, non-competitive or uncompetitive
inhibitor of protein binding to the cellular proliferation protein.
Preferably, the antibody is also an antagonist of the cellular
proliferation protein. In one aspect, when the antibody prevents
the binding of other molecules to the cellular proliferation
protein, the antibody prevents growth of the cell. The antibody
also sensitizes the cell to cytotoxic agents, including, but not
limited to TNF-.alpha., TNF-.beta., IL-1, INF-.gamma. and IL-2, or
chemotherapeutic agents including 5FU, vinblastine, actinomycin D,
cisplatin, methotrexate, and the like.
[0203] In another preferred embodiment, the antibody is conjugated
to a therapeutic moiety. In one aspect the therapeutic moiety is a
small molecule that modulates the activity of the cellular
proliferation protein. In another aspect the therapeutic moiety
modulates the activity of molecules associated with or in close
proximity to the cellular proliferation protein.
[0204] In a preferred embodiment, the therapeutic moiety may also
be a cytotoxic agent. In this method, targeting the cytotoxic agent
to tumor tissue or cells, results in a reduction in the number of
afflicted cells, thereby reducing symptoms associated with cellular
proliferation. Cytotoxic agents are numerous and varied and
include, but are not limited to, cytotoxic drugs or toxins or
active fragments of such toxins. Suitable toxins and their
corresponding fragments include diptheria a chain, exotoxin a
chain, ricin a chain, abrin a chain, curcin, crotin, phenomycin,
enomycin and the like. Cytotoxic agents also include radiochemicals
made by conjugating radioisotopes to antibodies raised against
cellular proliferation proteins, or binding of a radionuclide to a
chelating agent that has been covalently attached to the antibody.
Targeting the therapeutic moiety to cellular proliferation proteins
not only serves to increase the local concentration of therapeutic
moiety in the cellular proliferation afflicted area, but also
serves to reduce deleterious side effects that may be associated
with the therapeutic moiety.
[0205] Preferably, the antibody is conjugated to a protein which
facilitates entry into the cell. In one case, the antibody enters
the cell by endocytosis. In another embodiment, a nucleic acid
encoding the antibody is administered to the individual or cell.
The nucleic acid is identified based on the sequence of the
antibody, determined by standard recombinant techniques. Moreover,
wherein the cellular proliferation protein can be targeted within a
cell, i.e., the nucleus, an antibody thereto contains a signal for
that target localization, i.e., a nuclear localization signal.
[0206] In a preferred embodiment, the cellular proliferation
antibodies of the invention specifically bind to cellular
proliferation proteins. By "specifically bind" herein is meant that
the antibodies bind to the protein with a binding constant in the
range of at least 10.sup.-4-10.sup.-6 M.sup.-1, with a preferred
range being 10.sup.-7-10.sup.-9 M.sup.-1.
[0207] It is understood that the examples described above in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are incorporated by reference in their entirety as well as the
sequences cited therein or having a GenBank accession number.
Sequence CWU 1
1
10 1 3762 DNA Human 1 gaattccgtc atggcgtcgc atggcgtcgc atggcgtcgc
vagccaaatt cgtctgcgaa 60 gaagaaagag gagaagggga agaacatcca
ggtggtggtg agatgcagac catttaattt 120 ggcagagcgg aaagctagcg
cccattcaat agtagaatgt gatcctgtac gaaaagaagt 180 tagtgtacga
actggaggat tggctgacaa gagctcaagg aaaacataca cttttgatat 240
ggtgtttgga gcatctacta aacagattga tgtttaccga agtgttgttt gtccaattct
300 ggatgaagtt attatgggct ataattgcac tatctttgcg tatggccaaa
ctggcactgg 360 aaaaactttt acaatggaag gtgaaaggtc acctaatgaa
gagtatacct gggaagagga 420 tcccttggct ggtataattc cacgtaccct
tcatcaaatt tttgagaaac ttactgataa 480 tggtactgaa ttttcagtca
aagtgtctct gttggagatc tataatgaag agctttttga 540 tcttcttaat
ccatcatctg atgtttctga gagactacag atgtttgatg atccccgtaa 600
caagagagga gtgataatta aaggtttaga agaaattaca gtacaagaac aggatgaagt
660 ctatcaaatt ttagaaaagg gggcagcaaa aaggacaact gcagctactc
tgatgaatgc 720 atactctagt cgttcccact cagttttctc tgttacaata
catatgaaag aaactacgat 780 tgatggagaa gagcttgtta aaatcggaaa
gttgaacttg gttgatcttg caggaagtga 840 aaacattggc cgttctggag
ctgttgataa gagagctcgg gaagctggaa atataaatca 900 atccctgttg
actttgggaa gggtcattac tgcccttgta gaaagaacac ctcatgttcc 960
ttatcgagaa tctaaactaa ctagaatcct ccaggattct cttggagggc gtacaagaac
1020 atctataatt gcaacaattt ctcctgcatc tctcaatctt gaggaaactc
tgagtacatt 1080 ggaatatgct catagagcaa agaacatatt gaataagcct
gaagtgaatc agaaactcac 1140 caaaaaagct cttattaagg agtatacgga
ggagatagaa cgtttaaaac gagatcttgc 1200 tgcagcccgt gagaaaaatg
gagtgtatat ttctgaagaa aattttagag tcatgagtgg 1260 aaaattaact
gttcaagaag agcagattgt agaattgatt gaaaaaattg gtgctgttga 1320
ggaggagctg aatagggtta cagagttgtt tatggataat aaaaatgaac ttgaccagtg
1380 taaatctgac ctgcaaaata aaacacaaga acttgaaacc actcaaaaac
atttgcaaga 1440 aactaaatta caacttgtta aagaagaata tatcacatca
gctttggaaa gtactgagga 1500 gaaacttcat gatgctgcca gcaagatgat
taacacagtt gaagaaacta caaaagatgt 1560 atctggtctc cattccaaac
tggatcgtaa gaaggcagtt gaccaacaca atgcagaagc 1620 tcaggatatt
tttggcaaaa acctgaatag tctgtttaat aatatggaag aattaattaa 1680
ggatggcagc tcaaagcaaa aggccatgct agaagtacat aagaccttat ttggtaatgt
1740 gctgtcttcc agtgtctctg cattagatac cattactaca gtagcacttg
gatctctcac 1800 atctattcca gaaaatgtgt ctactcatgt ttctcagatt
tttaatatga tactaaaaga 1860 acaatcatta gcagcagaaa gtaaaactgt
actacaggaa ttgattaatg tactcaagac 1920 tgatcttcta agttcactgg
aaatgatttt atccccaact gtggtgtcta tactgaaaat 1980 caatagtcaa
ctaaagcata ttttcaagac ttcattgaca gtggccgata agatagaaga 2040
tcaaaaaaaa aggaactcag atggctttct cagtatactg tgtaacaatc tacatgaact
2100 acaagaaaat accatttgtt ccttggttga gtcacaaaag caatgtggaa
acctaactga 2160 agacctgaag acaataaagc agacccattc ccaggaactt
tgcaagttaa tgaatctttg 2220 gacagagaga ttctgtgctt tggaggaaaa
gtgtgaaaat atacagaaac cacttagtag 2280 tgtccaggaa aatatacagc
agaaatctaa ggatatagtc aacaaaatga cttttcacag 2340 tcaaaaattt
tgtgctgatt ctgatggctt ctcacaggaa ctcagaaatt ttaaccaaga 2400
aggtacaaaa ttggttgaag aatctgtgaa acactctgat aaactcaatg gcaacctgga
2460 aaaaatatct caagagactg aacagagatg tgaatctctg aacacaagaa
cagtttattt 2520 ttctgaacag tgggtatctt ccttaaatga aagggaacag
gaacttcaca acttattgga 2580 ggttgtaagc caatgttgtg aggcttcaag
ttcagacatc actgagaaat gagatggacg 2640 taaggcagct catgagaaac
agcataacat ttttcttgat cagatgacta ttgatgaaga 2700 taaattgata
gcacaaaatc tagaacttaa tgaaaccata aaaattggtt tgactaagct 2760
taattgcttt ctggaacagg atctgaaact ggatatccca acaggtacga caccacagag
2820 gaaaagttat ttatacccat caacactggt aagaactgaa ccacgtgaac
atctccttga 2880 tcagctgaaa aggaaacagc ctgagctgtt aatgatgcta
aactgttcag aaaacaacaa 2940 agaagagaca attccggatg tggatgtaga
agaggcagtt ctggggcagt atactgaaga 3000 acctctaagt caagagccat
ctgtagatgc tggtgtggat tgttcatcaa ttggcggggt 3060 tccatttttc
cagcataaaa aatcacatgg aaaagacaaa gaaaacagag gcattaacac 3120
actggagagg tctaaagtgg actaaagtgg agagcacttg gttacaaaga gcagattacc
3180 tctgcgagcc cagatcaacc tttaattcac ttgggggttg gcaattttat
ttttaaagaa 3240 aaacttaaaa ataaaacctg aaaccccaga acttgagcct
tgtgtataga ttttaaaaga 3300 atatatatat cagccgggcg cgtggctcta
gctgtaatcc cagctaactt tggaggctga 3360 ggcgggtgga ttgcttgagc
ccaggagttt gagaccagcc tggccaacgt gcgctaaaac 3420 cttcgtctct
gttaaaaatt agccgggcgt ggtgggcaca ctcctgtaat cccagctact 3480
ggggaggctg aggcacgaga atcacttgaa cccagaagcg gggttgcagt gagccaaagg
3540 tacaccacta cactccagcc tgggcaacag agcaagactc ggtctcaaaa
ataaaattta 3600 aaaaagatat aaggcagtac tgtaaattca gttgaatttt
gatatctacc catttttctg 3660 tcatccctat agttcacttt gtattaaatt
gggttcattt tgggatttgc aatgtaaata 3720 cgtatttcta gttttcatat
aaagtagttc ttttaggaat tc 3762 2 1057 PRT Human 2 Met Ala Ser Gln
Pro Asn Ser Ser Ala Lys Lys Lys Glu Glu Lys Gly 1 5 10 15 Lys Asn
Ile Gln Val Val Val Arg Cys Arg Pro Phe Asn Leu Ala Glu 20 25 30
Arg Lys Ala Ser Ala His Ser Ile Val Glu Cys Asp Pro Val Arg Lys 35
40 45 Glu Val Ser Val Arg Thr Gly Gly Leu Ala Asp Lys Ser Ser Arg
Lys 50 55 60 Thr Tyr Thr Phe Asp Met Val Phe Gly Ala Ser Thr Lys
Gln Ile Asp 65 70 75 80 Val Tyr Arg Ser Val Val Cys Pro Ile Leu Asp
Glu Val Ile Met Gly 85 90 95 Tyr Asn Cys Thr Ile Phe Ala Tyr Gly
Gln Thr Gly Thr Gly Lys Thr 100 105 110 Phe Thr Met Glu Gly Glu Arg
Ser Pro Asn Glu Glu Tyr Thr Trp Glu 115 120 125 Glu Asp Pro Leu Ala
Gly Ile Ile Pro Arg Thr Leu His Gln Ile Phe 130 135 140 Glu Lys Leu
Thr Asp Asn Gly Thr Glu Phe Ser Val Lys Val Ser Leu 145 150 155 160
Leu Glu Ile Tyr Asn Glu Glu Leu Phe Asp Leu Leu Asn Pro Ser Ser 165
170 175 Asp Val Ser Glu Arg Leu Gln Met Phe Asp Asp Pro Arg Asn Lys
Arg 180 185 190 Gly Val Ile Ile Lys Gly Leu Glu Glu Ile Thr Val His
Asn Lys Asp 195 200 205 Glu Val Tyr Gln Ile Leu Glu Lys Gly Ala Ala
Lys Arg Thr Thr Ala 210 215 220 Ala Thr Leu Met Asn Ala Tyr Ser Ser
Arg Ser His Ser Val Phe Ser 225 230 235 240 Val Thr Ile His Met Lys
Glu Thr Thr Ile Asp Gly Glu Glu Leu Val 245 250 255 Lys Ile Gly Lys
Leu Asn Leu Val Asp Leu Ala Gly Ser Glu Asn Ile 260 265 270 Gly Arg
Ser Gly Ala Val Asp Lys Arg Ala Arg Glu Ala Gly Asn Ile 275 280 285
Asn Gln Ser Leu Leu Thr Leu Gly Arg Val Ile Thr Ala Leu Val Glu 290
295 300 Arg Thr Pro His Val Pro Tyr Arg Glu Ser Lys Leu Thr Arg Ile
Leu 305 310 315 320 Gln Asp Ser Leu Gly Gly Arg Thr Arg Thr Ser Ile
Ile Ala Thr Ile 325 330 335 Ser Pro Ala Ser Leu Asn Leu Glu Glu Thr
Leu Ser Thr Leu Glu Tyr 340 345 350 Ala His Arg Ala Lys Asn Ile Leu
Asn Lys Pro Glu Val Asn Gln Lys 355 360 365 Leu Thr Lys Lys Ala Leu
Ile Lys Glu Tyr Thr Glu Glu Ile Glu Arg 370 375 380 Leu Lys Arg Asp
Leu Ala Ala Ala Arg Glu Lys Asn Gly Val Tyr Ile 385 390 395 400 Ser
Glu Glu Asn Phe Arg Val Met Ser Gly Lys Leu Thr Val Gln Glu 405 410
415 Glu Gln Ile Val Glu Leu Ile Glu Lys Ile Gly Ala Val Glu Glu Glu
420 425 430 Leu Asn Arg Val Thr Ala Leu Phe Met Asp Asn Lys Asn Glu
Leu Asp 435 440 445 Gln Cys Lys Ser Asp Leu Gln Asn Lys Thr Gln Glu
Leu Glu Thr Thr 450 455 460 Gln Lys His Leu Gln Glu Thr Lys Leu Gln
Leu Val Lys Glu Glu Tyr 465 470 475 480 Ile Thr Ser Ala Leu Glu Ser
Thr Glu Glu Lys Leu His Asp Ala Ala 485 490 495 Ser Lys Leu Leu Asn
Thr Val Glu Glu Thr Thr Lys Asp Val Ser Gly 500 505 510 Leu His Ser
Lys Leu Asp Arg Ala Lys Lys Ala Val Asp Gln His Asn 515 520 525 Ala
Glu Ala Gln Asp Asp Ile Phe Gly Lys Asn Leu Ser Leu Phe Asn 530 535
540 Asn Met Glu Glu Leu Ile Lys Asp Gly Ser Lys Gln Lys Ala Met Leu
545 550 555 560 Glu Val His Lys Thr Leu Phe Gly Asn Leu Leu Ser Ser
Ser Val Ser 565 570 575 Ala Leu Asp Thr Ile Thr Thr Val Ala Leu Gly
Ser Leu Thr Ser Ile 580 585 590 Pro Glu Asn Val Ser Thr His Val Ser
Gln Ile Phe Asn Met Ile Leu 595 600 605 Lys Glu Gln Ser Leu Ala Ala
Glu Ser Lys Thr Val Leu Gln Glu Leu 610 615 620 Ile Asn Val Leu Lys
Thr Asp Leu Leu Ser Ser Leu Glu Met Ile Leu 625 630 635 640 Ser Pro
Thr Val Val Ser Ile Leu Lys Ile Asn Ser Gln Leu Lys His 645 650 655
Ile Phe Lys Thr Ser Leu Thr Val Ala Asp Lys Ile Glu Asp Gln Lys 660
665 670 Lys Arg Asn Ser Asp Gly Phe Leu Ser Ile Leu Cys Asn Asn Leu
Glu 675 680 685 His Glu Leu Gln Glu Asn Thr Ile Cys Ser Leu Val Glu
Ser Gln Lys 690 695 700 Gln Cys Gly Asn Leu Thr Glu Asp Leu Lys Thr
Ile Lys Gln Thr His 705 710 715 720 Ser Gln Glu Leu Cys Lys Leu Met
Asn Trp Thr Glu Arg Phe Cys Ala 725 730 735 Leu Glu Glu Lys Cys Glu
Asn Ile Gln Lys Pro Leu Ser Ser Val Gln 740 745 750 Glu Asn Ile Gln
Gln Lys Ser Lys Asp Ile Val Asn Lys Met Thr Phe 755 760 765 His Ser
Gln Lys Phe Cys Ala Asp Ser Asp Gly Phe Ser Gln Glu Leu 770 775 780
Arg Asn Phe Asn Gln Glu Gly Thr Lys Leu Val Glu Glu Ser Val Lys 785
790 795 800 His Ser Asp Lys Leu Asn Gly Asn Leu Glu Lys Ile Ser Gln
Ile Thr 805 810 815 Glu Gln Arg Cys Glu Ser Leu Asn Thr Arg Thr Val
Tyr Phe Ser Glu 820 825 830 Gln Trp Val Ser Ser Leu Asn Glu Arg Glu
Gln Glu Leu His Asn Leu 835 840 845 Leu Glu Val Val Ser Gln Cys Cys
Glu Ala Ser Ser Ser Asp Ile Thr 850 855 860 Glu Lys Ser Asp Gly Arg
Lys Ala Ala His Glu Lys Gln His Asn Ile 865 870 875 880 Phe Leu Asp
Gln Met Thr Ile Asp Glu Asp Lys Leu Ile Ala Gln Asn 885 890 895 Leu
Glu Leu Asn Glu Thr Ile Lys Ile Gly Leu Thr Lys Leu Asn Cys 900 905
910 Phe Leu Glu Gln Asp Leu Lys Leu Asp Ile Pro Thr Gly Thr Thr Pro
915 920 925 Gln Arg Lys Ser Tyr Leu Tyr Pro Ser Thr Leu Val Arg Thr
Glu Pro 930 935 940 Arg Glu His Leu Leu Asp Gln Leu Lys Arg Lys Gln
Pro Glu Leu Leu 945 950 955 960 Met Met Leu Asn Cys Ser Glu Asn Asn
Lys Glu Glu Thr Ile Pro Asp 965 970 975 Val Asp Val Glu Glu Ala Val
Leu Gly Gln Tyr Thr Glu Glu Pro Leu 980 985 990 Ser Gln Glu Pro Ser
Val Asp Ala Gly Val Asp Cys Ser Ser Ile Gly 995 1000 1005 Gly Val
Pro Phe Phe Gln His Lys Lys Ser His Gly Lys Asp Lys Glu 1010 1015
1020 Asn Arg Gly Ile Asn Thr Leu Glu Arg Ser Lys Val Glu Glu Thr
Thr 1025 1030 1035 1040 Glu His Leu Val Thr Lys Ser Arg Leu Pro Leu
Arg Ala Gln Ile Asn 1045 1050 1055 Leu 3 1149 DNA Human 3
atggcgtgcc agccaaattc gtctgcgaag aagaaagagg agaaggggaa gaacatccag
60 gtggtggtga gatgcagacc atttaatttg gcagagcgga aagctagcgc
ccattcaata 120 gtagaatgtg atcctgtacg aaaagaagtt agtgtacgaa
ctggaggatt ggctgacaag 180 agctcaagga aaacatacac ttttgatatg
gtgtttggag catctactaa acagattgat 240 gtttaccgag gtgttgtttg
tccaattctg gatgaagtta ttatgggcta taattgcact 300 atctttgcgt
atggccaaac tggcactgga aaaactttta caatggaagg tgaaaggtca 360
cctaatgaag agtatacctg ggaagaggat cccttggctg gtataattcc acgtaccctt
420 catcaaattt ttgagaaact tactgataat ggtactgaat tttcagtcaa
agtgtctctg 480 ttggagatct ataatgaaga gctttttgat cttcttaatc
catcatctga tgtttctgag 540 agactacaga tgtttgatga tccccgtaac
aagagaggag tgataattaa aggtttagaa 600 gaaattacag tacacaacaa
ggatgaagtg tatcaaattt tagaaaaggg ggcagcaaaa 660 aggacaactg
cagctactct gatgaatgca tactctagtc gttcccactc agttttctct 720
gttacaatac atatgaaaga aactacgatt gatggagaag agcttgttaa aatcggaaag
780 ttgaacttgg ttgatcttgc aggaagtgaa aacattggcc gttctggagc
tgttgataag 840 agagctcggg aagctggaaa tataaatcaa tccctgttga
ctttgggaag ggtcattact 900 gcccttgtag aaagaacacc tcatgttcct
tatcgagaat ctaaactaac tagaatcctc 960 caggattctc ttggagggcg
tacaagaaca tctataattg caacaatttc tcctgcatct 1020 ctcaatcttg
aggaaactct gagtacattg gaatatgctc atagagcaaa gaacatattg 1080
ctcgagggta ccgagcagaa gctgatcagc gaggaggacc tgatcgagca ccaccaccac
1140 caccactga 1149 4 382 PRT Human 4 Met Ala Cys Gln Pro Asn Ser
Ser Ala Lys Lys Lys Glu Glu Lys Gly 1 5 10 15 Lys Asn Ile Gln Val
Val Val Arg Cys Arg Pro Phe Asn Leu Ala Glu 20 25 30 Arg Lys Ala
Ser Ala His Ser Ile Val Glu Cys Asp Pro Val Arg Lys 35 40 45 Glu
Val Ser Val Arg Thr Gly Gly Leu Ala Asp Lys Ser Ser Arg Lys 50 55
60 Thr Tyr Thr Phe Asp Met Val Phe Gly Ala Ser Thr Lys Gln Ile Asp
65 70 75 80 Val Tyr Arg Ser Val Val Cys Pro Ile Leu Asp Glu Val Ile
Met Gly 85 90 95 Tyr Asn Cys Thr Ile Phe Ala Tyr Gly Gln Thr Gly
Thr Gly Lys Thr 100 105 110 Phe Thr Met Glu Gly Glu Arg Ser Pro Asn
Glu Glu Tyr Thr Trp Glu 115 120 125 Glu Asp Pro Leu Ala Gly Ile Ile
Pro Arg Thr Leu His Gln Ile Phe 130 135 140 Glu Lys Leu Thr Asp Asn
Gly Thr Glu Phe Ser Val Lys Val Ser Leu 145 150 155 160 Leu Glu Ile
Tyr Asn Glu Glu Leu Phe Asp Leu Leu Asn Pro Ser Ser 165 170 175 Asp
Val Ser Glu Arg Leu Gln Met Phe Asp Asp Pro Arg Asn Lys Arg 180 185
190 Gly Val Ile Ile Lys Gly Leu Glu Glu Ile Thr Val His Asn Lys Asp
195 200 205 Glu Val Tyr Gly Ile Leu Glu Lys Gly Ala Ala Lys Arg Thr
Thr Ala 210 215 220 Ala Thr Leu Met Asn Ala Tyr Ser Ser Arg Ser His
Ser Val Phe Ser 225 230 235 240 Val Thr Ile His Met Lys Glu Thr Thr
Ile Asp Gly Glu Glu Leu Val 245 250 255 Lys Ile Gly Lys Leu Asn Leu
Val Asp Leu Ala Gly Ser Glu Asn Ile 260 265 270 Gly Arg Ser Gly Ala
Val Asp Lys Arg Ala Arg Glu Ala Gly Asn Ile 275 280 285 Asn Gln Ser
Leu Leu Thr Leu Gly Arg Val Ile Thr Ala Leu Val Glu 290 295 300 Arg
Thr Pro His Val Pro Tyr Arg Glu Ser Lys Leu Thr Arg Ile Leu 305 310
315 320 Gln Asp Ser Leu Gly Gly Arg Thr Arg Thr Ser Ile Ile Ala Thr
Ile 325 330 335 Ser Pro Ala Ser Leu Asn Leu Glu Glu Thr Leu Ser Thr
Leu Glu Tyr 340 345 350 Ala His Arg Ala Lys Asn Ile Leu Leu Glu Gln
Thr Glu Gly Lys Leu 355 360 365 Ile Ser Glu Glu Asp Leu Ile Glu His
His His His His His 370 375 380 5 1542 DNA Human 5 atggcgtgcc
agccaaattc gtctgcgaag aagaaagagg agaaggggaa gaacatccag 60
gtggtggtga gatgcagacc atttaatttg gcagagcgga aagctagcgc ccattcaata
120 gtagaatgtg atcctgtacg aaaagaagtt agtgtacgaa ctggaggatt
ggctgacaag 180 agctcaagga aaacatacac ttttgatatg gtgtttggag
catctactaa acagattgat 240 gtttaccgag gtgttgtttg tccaattctg
gatgaagtta ttatgggcta taattgcact 300 atctttgcgt atggccaaac
tggcactgga aaaactttta caatggaagg tgaaaggtca 360 cctaatgaag
agtatacctg ggaagaggat cccttggctg gtataattcc acgtaccctt 420
catcaaattt ttgagaaact tactgataat ggtactgaat tttcagtcaa agtgtctctg
480 ttggagatct ataatgaaga gctttttgat cttcttaatc catcatctga
tgtttctgag 540 agactacaga tgtttgatga tccccgtaac aagagaggag
tgataattaa aggtttagaa 600 gaaattacag tacacaacaa ggatgaagtg
tatcaaattt tagaaaaggg ggcagcaaaa 660 aggacaactg cagctactct
gatgaatgca tactctagtc gttcccactc agttttctct 720 gttacaatac
atatgaaaga aactacgatt gatggagaag agcttgttaa aatcggaaag 780
ttgaacttgg ttgatcttgc aggaagtgaa aacattggcc gttctggagc tgttgataag
840 agagctcggg aagctggaaa tataaatcaa tccctgttga ctttgggaag
ggtcattact 900 gcccttgtag aaagaacacc tcatgttcct tatcgagaat
ctaaactaac tagaatcctc 960 caggattctc ttggagggcg tacaagaaca
tctataattg caacaatttc tcctgcatct 1020 ctcaatcttg aggaaactct
gagtacattg gaatatgctc atagagcaaa gaacatattg 1080 aataagcctg
aagtgaatca gaaactcacc aaaaaagctc ttattaagga gtatacggag
1140 gagatagaac gtttaaaacg agatcttgct gcagcccgtg agaaaaatgg
agtgtatatt 1200 tctgaagaaa attttagagt catgagtgga aaattaactg
ttcaagaaga gcagattgta 1260 gaattgattg aaaaaattgg tgctgttgag
gaggagctga atagggttac agagttgttt 1320 atggataata aaaatgaact
tgaccagtgt aaatctgacc tgcaaaataa aacacaagaa 1380 cttgaaacca
ctcaaaaaca tttgcaagaa actaaattac aacttgttaa agaagaatat 1440
atcacatcag ctttggaaag tactgaggag aaactcgagg gtaccgagca gaagctgatc
1500 agcgaggagg acctgatcga gcaccaccac caccaccact ga 1542 6 513 PRT
Human 6 Met Ala Cys Gln Pro Asn Ser Ser Ala Lys Lys Lys Glu Glu Lys
Gly 1 5 10 15 Lys Asn Ile Gln Val Val Val Arg Cys Arg Pro Phe Asn
Leu Ala Glu 20 25 30 Arg Lys Ala Ser Ala His Ser Ile Val Glu Cys
Asp Pro Val Arg Lys 35 40 45 Glu Val Ser Val Arg Thr Gly Gly Leu
Ala Asp Lys Ser Ser Arg Lys 50 55 60 Thr Tyr Thr Phe Asp Met Val
Phe Gly Ala Ser Thr Lys Gln Ile Asp 65 70 75 80 Val Tyr Arg Ser Val
Val Cys Pro Ile Leu Asp Glu Val Ile Met Gly 85 90 95 Tyr Asn Cys
Thr Ile Phe Ala Tyr Gly Gln Thr Gly Thr Gly Lys Thr 100 105 110 Phe
Thr Met Glu Gly Glu Arg Ser Pro Asn Glu Glu Tyr Thr Trp Glu 115 120
125 Glu Asp Pro Leu Ala Gly Ile Ile Pro Arg Thr Leu His Gln Ile Phe
130 135 140 Glu Lys Leu Thr Asp Asn Gly Thr Glu Phe Ser Val Lys Val
Ser Leu 145 150 155 160 Leu Glu Ile Tyr Asn Glu Glu Leu Phe Asp Leu
Leu Asn Pro Ser Ser 165 170 175 Asp Val Ser Glu Arg Leu Gln Met Phe
Asp Asp Pro Arg Asn Lys Arg 180 185 190 Gly Val Ile Ile Lys Gly Leu
Glu Glu Ile Thr Val His Asn Lys Asp 195 200 205 Glu Val Tyr Gly Ile
Leu Glu Lys Gly Ala Ala Lys Arg Thr Thr Ala 210 215 220 Ala Thr Leu
Met Asn Ala Tyr Ser Ser Arg Ser His Ser Val Phe Ser 225 230 235 240
Val Thr Ile His Met Lys Glu Thr Thr Ile Asp Gly Glu Glu Leu Val 245
250 255 Lys Ile Gly Lys Leu Asn Leu Val Asp Leu Ala Gly Ser Glu Asn
Ile 260 265 270 Gly Arg Ser Gly Ala Val Asp Lys Arg Ala Arg Glu Ala
Gly Asn Ile 275 280 285 Asn Gln Ser Leu Leu Thr Leu Gly Arg Val Ile
Thr Ala Leu Val Glu 290 295 300 Arg Thr Pro His Val Pro Tyr Arg Glu
Ser Lys Leu Thr Arg Ile Leu 305 310 315 320 Gln Asp Ser Leu Gly Gly
Arg Thr Arg Thr Ser Ile Ile Ala Thr Ile 325 330 335 Ser Pro Ala Ser
Leu Asn Leu Glu Glu Thr Leu Ser Thr Leu Glu Tyr 340 345 350 Ala His
Arg Ala Lys Asn Ile Leu Asn Lys Pro Glu Val Asn Gln Lys 355 360 365
Leu Thr Lys Lys Ala Leu Ile Lys Glu Tyr Thr Glu Glu Ile Glu Arg 370
375 380 Leu Lys Arg Asp Leu Ala Ala Ala Arg Glu Lys Asn Gly Val Tyr
Ile 385 390 395 400 Ser Glu Glu Asn Phe Arg Val Met Ser Gly Lys Leu
Thr Val Gln Glu 405 410 415 Glu Gln Ile Val Glu Leu Ile Glu Lys Ile
Gly Ala Val Glu Glu Glu 420 425 430 Leu Asn Arg Val Thr Glu Leu Phe
Met Asp Asn Lys Asn Glu Leu Asp 435 440 445 Gln Cys Lys Ser Asp Leu
Gln Asn Lys Thr Gln Glu Leu Glu Thr Thr 450 455 460 Gln Lys His Leu
Gly Glu Thr Lys Leu Gly Leu Val Lys Glu Glu Tyr 465 470 475 480 Ile
Thr Ser Ala Leu Glu Ser Thr Glu Glu Lys Leu Glu Gln Thr Glu 485 490
495 Gly Lys Leu Ile Ser Glu Glu Asp Leu Ile Glu His His His His His
500 505 510 His 7 1728 DNA Human 7 atggcgtgcc agccaaattc gtctgcgaag
aagaaagagg agaaggggaa gaacatccag 60 gtggtggtga gatgcagacc
atttaatttg gcagagcgga aagctagcgc ccattcaata 120 gtagaatgtg
atcctgtacg aaaagaagtt agtgtacgaa ctggaggatt ggctgacaag 180
agctcaagga aaacatacac ttttgatatg gtgtttggag catctactaa acagattgat
240 gtttaccgag gtgttgtttg tccaattctg gatgaagtta ttatgggcta
taattgcact 300 atctttgcgt atggccaaac tggcactgga aaaactttta
caatggaagg tgaaaggtca 360 cctaatgaag agtatacctg ggaagaggat
cccttggctg gtataattcc acgtaccctt 420 catcaaattt ttgagaaact
tactgataat ggtactgaat tttcagtcaa agtgtctctg 480 ttggagatct
ataatgaaga gctttttgat cttcttaatc catcatctga tgtttctgag 540
agactacaga tgtttgatga tccccgtaac aagagaggag tgataattaa aggtttagaa
600 gaaattacag tacacaacaa ggatgaagtg tatcaaattt tagaaaaggg
ggcagcaaaa 660 aggacaactg cagctactct gatgaatgca tactctagtc
gttcccactc agttttctct 720 gttacaatac atatgaaaga aactacgatt
gatggagaag agcttgttaa aatcggaaag 780 ttgaacttgg ttgatcttgc
aggaagtgaa aacattggcc gttctggagc tgttgataag 840 agagctcggg
aagctggaaa tataaatcaa tccctgttga ctttgggaag ggtcattact 900
gcccttgtag aaagaacacc tcatgttcct tatcgagaat ctaaactaac tagaatcctc
960 caggattctc ttggagggcg tacaagaaca tctataattg caacaatttc
tcctgcatct 1020 ctcaatcttg aggaaactct gagtacattg gaatatgctc
atagagcaaa gaacatattg 1080 aataagcctg aagtgaatca gaaactcacc
aaaaaagctc ttattaagga gtatacggag 1140 gagatagaac gtttaaaacg
agatcttgct gcagcccgtg agaaaaatgg agtgtatatt 1200 tctgaagaaa
attttagagt catgagtgga aaattaactg ttcaagaaga gcagattgta 1260
gaattgattg aaaaaattgg tgctgttgag gaggagctga atagggttac agagttgttt
1320 atggataata aaaatgaact tgaccagtgt aaatctgacc tgcaaaataa
aacacaagaa 1380 cttgaaacca ctcaaaaaca tttgcaagaa actaaattac
aacttgttaa agaagaatat 1440 atcacatcag ctttggaaag tactgaggag
aaacttcatg atgctgccag caagctgctt 1500 aacacagttg aagaaactac
aaaagatgta tctggtctcc attccaaact ggatcgtaag 1560 aaggcagttg
accaacacaa tgcagaagct caggatattt ttggcaaaaa cctgaatagt 1620
ctgtttaata atatggaaga attaattaag gatggcagcc tcgagggtac cgagcagaag
1680 ctgatcagcg aggaggacct gatcgagcac caccaccacc accactga 1728 8
575 PRT Human 8 Met Ala Cys Gln Pro Asn Ser Ser Ala Lys Lys Lys Glu
Glu Lys Gly 1 5 10 15 Lys Asn Ile Gln Val Val Val Arg Cys Arg Pro
Phe Asn Leu Ala Glu 20 25 30 Arg Lys Ala Ser Ala His Ser Ile Val
Glu Cys Asp Pro Val Arg Lys 35 40 45 Glu Val Ser Val Arg Thr Gly
Gly Leu Ala Asp Lys Ser Ser Arg Lys 50 55 60 Thr Tyr Thr Phe Asp
Met Val Phe Gly Ala Ser Thr Lys Gln Ile Asp 65 70 75 80 Val Tyr Arg
Ser Val Val Cys Pro Ile Leu Asp Glu Val Ile Met Gly 85 90 95 Tyr
Asn Cys Thr Ile Phe Ala Tyr Gly Gln Thr Gly Thr Gly Lys Thr 100 105
110 Phe Thr Met Glu Gly Glu Arg Ser Pro Asn Glu Glu Tyr Thr Trp Glu
115 120 125 Glu Asp Pro Leu Ala Gly Ile Ile Pro Arg Thr Leu His Gln
Ile Phe 130 135 140 Glu Lys Leu Thr Asp Asn Gly Thr Glu Phe Ser Val
Lys Val Ser Leu 145 150 155 160 Leu Glu Ile Tyr Asn Glu Glu Leu Phe
Asp Leu Leu Asn Pro Ser Ser 165 170 175 Asp Val Ser Glu Arg Leu Gln
Met Phe Asp Asp Pro Arg Asn Lys Arg 180 185 190 Gly Val Ile Ile Lys
Gly Leu Glu Glu Ile Thr Val His Asn Lys Asp 195 200 205 Glu Val Tyr
Gly Ile Leu Glu Lys Gly Ala Ala Lys Arg Thr Thr Ala 210 215 220 Ala
Thr Leu Met Asn Ala Tyr Ser Ser Arg Ser His Ser Val Phe Ser 225 230
235 240 Val Thr Ile His Met Lys Glu Thr Thr Ile Asp Gly Glu Glu Leu
Val 245 250 255 Lys Ile Gly Lys Leu Asn Leu Val Asp Leu Ala Gly Ser
Glu Asn Ile 260 265 270 Gly Arg Ser Gly Ala Val Asp Lys Arg Ala Arg
Glu Ala Gly Asn Ile 275 280 285 Asn Gln Ser Leu Leu Thr Leu Gly Arg
Val Ile Thr Ala Leu Val Glu 290 295 300 Arg Thr Pro His Val Pro Tyr
Arg Glu Ser Lys Leu Thr Arg Ile Leu 305 310 315 320 Gln Asp Ser Leu
Gly Gly Arg Thr Arg Thr Ser Ile Ile Ala Thr Ile 325 330 335 Ser Pro
Ala Ser Leu Asn Leu Glu Glu Thr Leu Ser Thr Leu Glu Tyr 340 345 350
Ala His Arg Ala Lys Asn Ile Leu Asn Lys Pro Glu Val Asn Gln Lys 355
360 365 Leu Thr Lys Lys Ala Leu Ile Lys Glu Tyr Thr Glu Glu Ile Glu
Arg 370 375 380 Leu Lys Arg Asp Leu Ala Ala Ala Arg Glu Lys Asn Gly
Val Tyr Ile 385 390 395 400 Ser Glu Glu Asn Phe Arg Val Met Ser Gly
Lys Leu Thr Val Gln Glu 405 410 415 Glu Gln Ile Val Glu Leu Ile Glu
Lys Ile Gly Ala Val Glu Glu Glu 420 425 430 Leu Asn Arg Val Thr Glu
Leu Phe Met Asp Asn Lys Asn Glu Leu Asp 435 440 445 Gln Cys Lys Ser
Asp Leu Gln Asn Lys Thr Gln Glu Leu Glu Thr Thr 450 455 460 Gln Lys
His Leu Gly Glu Thr Lys Leu Gly Leu Val Lys Glu Glu Tyr 465 470 475
480 Ile Thr Ser Ala Leu Glu Ser Thr Glu Glu Lys Leu His Asp Ala Ala
485 490 495 Ser Lys Leu Leu Asn Thr Val Glu Glu Thr Thr Lys Asp Val
Ser Gly 500 505 510 Leu His Ser Lys Leu Asp Arg Lys Lys Ala Val Asp
Gln His Asn Ala 515 520 525 Glu Ala Gln Asp Ile Phe Gly Lys Asn Leu
Asn Ser Leu Phe Asn Asn 530 535 540 Met Glu Glu Leu Ile Lys Asp Gly
Ser Leu Glu Gln Thr Glu Gly Lys 545 550 555 560 Leu Ile Ser Glu Glu
Asp Leu Ile Glu His His His His His His 565 570 575 9 1107 DNA
Human 9 atggcgtgcc agccaaattc gtctgcgaag aagaaagagg agaaggggaa
gaacatccag 60 gtggtggtga gatgcagacc atttaatttg gcagagcgga
aagctagcgc ccattcaata 120 gtagaatgtg atcctgtacg aaaagaagtt
agtgtacgaa ctggaggatt ggctgacaag 180 agctcaagga aaacatacac
ttttgatatg gtgtttggag catctactaa acagattgat 240 gtttaccgag
gtgttgtttg tccaattctg gatgaagtta ttatgggcta taattgcact 300
atctttgcgt atggccaaac tggcactgga aaaactttta caatggaagg tgaaaggtca
360 cctaatgaag agtatacctg ggaagaggat cccttggctg gtataattcc
acgtaccctt 420 catcaaattt ttgagaaact tactgataat ggtactgaat
tttcagtcaa agtgtctctg 480 ttggagatct ataatgaaga gctttttgat
cttcttaatc catcatctga tgtttctgag 540 agactacaga tgtttgatga
tccccgtaac aagagaggag tgataattaa aggtttagaa 600 gaaattacag
tacacaacaa ggatgaagtg tatcaaattt tagaaaaggg ggcagcaaaa 660
aggacaactg cagctactct gatgaatgca tactctagtc gttcccactc agttttctct
720 gttacaatac atatgaaaga aactacgatt gatggagaag agcttgttaa
aatcggaaag 780 ttgaacttgg ttgatcttgc aggaagtgaa aacattggcc
gttctggagc tgttgataag 840 agagctcggg aagctggaaa tataaatcaa
tccctgttga ctttgggaag ggtcattact 900 gcccttgtag aaagaacacc
tcatgttcct tatcgagaat ctaaactaac tagaatcctc 960 caggattctc
ttggagggcg tacaagaaca tctataattg caacaatttc tcctgcatct 1020
ctcaatcttg aggaaactct gagtacattg gaatatgctc atagagcaaa gaacatattg
1080 aataagcctg aagtgaatca gaaatag 1107 10 368 PRT Human 10 Met Ala
Cys Gln Pro Asn Ser Ser Ala Lys Lys Lys Glu Glu Lys Gly 1 5 10 15
Lys Asn Ile Gln Val Val Val Arg Cys Arg Pro Phe Asn Leu Ala Glu 20
25 30 Arg Lys Ala Ser Ala His Ser Ile Val Glu Cys Asp Pro Val Arg
Lys 35 40 45 Glu Val Ser Val Arg Thr Gly Gly Leu Ala Asp Lys Ser
Ser Arg Lys 50 55 60 Thr Tyr Thr Phe Asp Met Val Phe Gly Ala Ser
Thr Lys Gln Ile Asp 65 70 75 80 Val Tyr Arg Ser Val Val Cys Pro Ile
Leu Asp Glu Val Ile Met Gly 85 90 95 Tyr Asn Cys Thr Ile Phe Ala
Tyr Gly Gln Thr Gly Thr Gly Lys Thr 100 105 110 Phe Thr Met Glu Gly
Glu Arg Ser Pro Asn Glu Glu Tyr Thr Trp Glu 115 120 125 Glu Asp Pro
Leu Ala Gly Ile Ile Pro Arg Thr Leu His Gln Ile Phe 130 135 140 Glu
Lys Leu Thr Asp Asn Gly Thr Glu Phe Ser Val Lys Val Ser Leu 145 150
155 160 Leu Glu Ile Tyr Asn Glu Glu Leu Phe Asp Leu Leu Asn Pro Ser
Ser 165 170 175 Asp Val Ser Glu Arg Leu Gln Met Phe Asp Asp Pro Arg
Asn Lys Arg 180 185 190 Gly Val Ile Ile Lys Gly Leu Glu Glu Ile Thr
Val His Asn Lys Asp 195 200 205 Glu Val Tyr Gly Ile Leu Glu Lys Gly
Ala Ala Lys Arg Thr Thr Ala 210 215 220 Ala Thr Leu Met Asn Ala Tyr
Ser Ser Arg Ser His Ser Val Phe Ser 225 230 235 240 Val Thr Ile His
Met Lys Glu Thr Thr Ile Asp Gly Glu Glu Leu Val 245 250 255 Lys Ile
Gly Lys Leu Asn Leu Val Asp Leu Ala Gly Ser Glu Asn Ile 260 265 270
Gly Arg Ser Gly Ala Val Asp Lys Arg Ala Arg Glu Ala Gly Asn Ile 275
280 285 Asn Gln Ser Leu Leu Thr Leu Gly Arg Val Ile Thr Ala Leu Val
Glu 290 295 300 Arg Thr Pro His Val Pro Tyr Arg Glu Ser Lys Leu Thr
Arg Ile Leu 305 310 315 320 Gln Asp Ser Leu Gly Gly Arg Thr Arg Thr
Ser Ile Ile Ala Thr Ile 325 330 335 Ser Pro Ala Ser Leu Asn Leu Glu
Glu Thr Leu Ser Thr Leu Glu Tyr 340 345 350 Ala His Arg Ala Lys Asn
Ile Leu Asn Lys Pro Glu Val Asn Gln Lys 355 360 365
* * * * *
References