U.S. patent application number 10/153740 was filed with the patent office on 2002-10-10 for cytostatin iii.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Dillon, Patrick J., Gentz, Reiner L., Ni, Jian, Yu, Guo-Liang.
Application Number | 20020147149 10/153740 |
Document ID | / |
Family ID | 26685095 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020147149 |
Kind Code |
A1 |
Ni, Jian ; et al. |
October 10, 2002 |
Cytostatin III
Abstract
The invention relates to Cytostatin III polypeptides,
polynucleotides encoding the polypeptides, methods for producing
the polypeptides, in particular by expressing the polynucleotides,
and agonists and antagonists of the polypeptides. The invention
further relates to methods for utilizing such polynucleotides,
polypeptides, agonists and antagonists for applications, which
relate, in part, to research, diagnostic and clinical arts.
Inventors: |
Ni, Jian; (Germantown,
MD) ; Yu, Guo-Liang; (Berkeley, CA) ; Gentz,
Reiner L.; (Rockville, MD) ; Dillon, Patrick J.;
(Carlsbad, CA) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
9410 KEY WEST AVENUE
ROCKVILLE
MD
20850
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
26685095 |
Appl. No.: |
10/153740 |
Filed: |
May 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10153740 |
May 24, 2002 |
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09734036 |
Dec 12, 2000 |
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6413726 |
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09734036 |
Dec 12, 2000 |
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09307817 |
May 10, 1999 |
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6232291 |
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09307817 |
May 10, 1999 |
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08820825 |
Mar 19, 1997 |
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5945309 |
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60013655 |
Mar 19, 1996 |
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Current U.S.
Class: |
514/19.4 ;
435/320.1; 435/325; 435/69.4; 530/399; 536/23.5 |
Current CPC
Class: |
C07K 14/475 20130101;
C07K 2319/00 20130101 |
Class at
Publication: |
514/12 ;
435/320.1; 435/325; 435/69.4; 530/399; 536/23.5 |
International
Class: |
C07K 014/575; C12P
021/02; C12N 005/06; A61K 038/22; C07H 021/04 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a polynucleotide having at
least a 70% identity to a member selected from the group consisting
of: (a) a polynucleotide encoding a polypeptide comprising an amino
acid sequence as set forth in SEQ ID NO:2; (b) a polynucleotide
which is complementary to the polynucleotide of (a); and (c) a
polynucleotide comprising at least 15 bases of the polynucleotides
of (a) or (b).
2. The polynucleotide of claim 1 wherein the polynucleotide is
DNA.
3. The polynucleotide of claim 1 wherein the polynucleotide is
RNA.
4. The polynucleotide of claim 1 wherein the polynucleotide is
genomic DNA.
5. The polynucleotide of claim 2 which encodes the polypeptide
comprising amino acid 1 to 135 of SEQ ID NO:2.
6. An isolated polynucleotide comprising a polynucleotide having at
least a 70% identity to a member selected from the group consisting
of: (a) a polynucleotide which encodes a mature polypeptide having
the amino acid sequence expressed by the human cDNA contained in
ATCC Deposit No. 97332; (b) a polynucleotide which is complementary
to the polynucleotide of (a); and (c) a polynucleotide comprising
at least 15 bases of the polynucleotide of (a) or (b).
7. The polynucleotide of claim 1 comprising the sequence as set
forth in SEQ ID No. 1 from nucleotide 1 to nucleotide 944.
8. The polynucleotide of claim 1 comprising the sequence as set
forth in SEQ ID No. 1 from nucleotide 94 to nucleotide 498.
9. A vector comprising the DNA of claim 2.
10. A host cell comprising the vector of claim 9.
11. A process for producing a polypeptide comprising: expressing
from the host cell of claim 10 the polypeptide encoded by said
DNA.
12. A process for producing a cell which expresses a polypeptide
comprising genetically engineering the cell with the vector of
claim 9 such that the cell expresses the polypeptide encoded by the
DNA contained in the vector.
13. A polypeptide comprising a member selected from the group
consisting of: (a) a polypeptide comprising an amino acid sequence
set forth in SEQ ID NO:2; and (b) a polypeptide which is at least
70% identical to the polypeptide of (a).
14. The polypeptide of claim 13 wherein the polypeptide comprises
amino acid 1 to amino acid 135 of SEQ ID NO:2.
15. A compound which inhibits activation of the polypeptide of
claim 13.
16. A method for the treatment of a patient having need of
Cytostatin III comprising: administering to the patient a
therapeutically effective amount of the polypeptide of claim
13.
17. The method of claim 16 wherein said therapeutically effective
amount of the polypeptide is administered by providing to the
patient DNA encoding said polypeptide and expressing said
polypeptide in vivo.
18. A method for the treatment of a patient having need to inhibit
a Cytostatin III polypeptide comprising: administering to the
patient a therapeutically effective amount of the compound of claim
15.
19. A process for diagnosing a disease or a susceptibility to a
disease related to an under-expression of the polypeptide of claim
13 comprising: determining a mutation in a nucleic acid sequence
encoding said polypeptide.
20. A diagnostic process comprising: analyzing for the presence of
the polypeptide of claim 13 in a sample derived from a host.
21. A method for identifying compounds which bind to and inhibit
activation of the polypeptide of claim 13 comprising: contacting a
cell expressing on the surface thereof a receptor for the
polypeptide, said receptor being associated with a second component
capable of providing a detectable signal in response to the binding
of a compound to said receptor, with an analytically detectable
Cytostatin III polypeptide and a compound under conditions to
permit binding to the receptor; and determining whether the
compound binds to and inhibits the receptor by detecting the
absence of a signal generated from the interaction of Cytostatin
III with the receptor.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/734,036, filed Dec. 12, 2000, which is a divisional of U.S.
application Ser. No. 09/307,817, filed May 10, 1999, now issued as
U.S. Pat. No. 6,232,291, which is a divisional of U.S. application
Ser. No. 08/820,825, filed Mar. 19, 1997, now issued as U.S. Pat.
No. 5,945,309, which claims benefit of Provisional Application No.
60/013,655, filed Mar. 19, 1996, all of which are hereby
incorporated by reference.
[0002] This invention relates, in part, to newly identified
polynucleotides and polypeptides; variants and derivatives of the
polynucleotides and polypeptides; processes for making the
polynucleotides and the polypeptides, and their variants and
derivatives; agonists and antagonists of the polypeptides; and uses
of the polynucleotides, polypeptides, variants, derivatives,
agonists and antagonists. In particular, in these and in other
regards, the invention relates to polynucleotides and polypeptides
of human Cytostatin III.
BACKGROUND OF THE INVENTION
[0003] The growth and differentiation of cells and the development
of tissues and glands is controlled by autocrine and paracrine
factors, such as systemic hormones and factors that modulate or
mediate the action of hormones, such as growth factors, which
themselves may be hormones.
[0004] For example, peptides that locally signal growth cessation
and stimulate differentiation of cells of the developing epithelium
are very important to mammary gland development. These factors
largely have not been identified or characterized, particularly not
in humans.
[0005] A few factors that play a role in the humoral mediation of
growth and differentiation of cells in tissues and glands, mammary
glands in particular, have been identified in non-human organisms.
One such factor is mammary-derived growth inhibitor ("MDGI").
(Grosse et al., 5. Mammary-Derived Growth Inhibitor (MDGI) in
GENES, ONCOGENES, AND HORMONES: ADVANCES IN CELLULAR AND MOLECULAR
BIOLOGY OF BREAST CANCER, Dickson et al., Eds., Kluwer Academic
Publishers, Boston (1991)). MDGI was first identified in milk and
mammary glands of cows. Subsequently, it was identified in mice. In
mice and cows, at least, MDGI has been shown to inhibit epithelial
cell growth and stimulate epithelial cell differentiation.
[0006] MDGI occurs in at least two forms produced by alternative
routes of post-translational processing. The original form is
referred to as MDGI and the second form is called MDGI-2.
[0007] MDGI is associated primarily with milk fat globule membranes
("MFGM"), as assessed by immunological assays using anti-MDGI
antibodies. Similar time course studies show that MDGI increases
dramatically in mammary glands when lactation begins, following
delivery. MDGI-2 differs from MDGI in this respect. It is found in
mammary glands during pregnancy but not during lactation. (Grosse
et al. cited above and Kurtz et al. J. Cell. Biol. 110: 1779-1789
(1990))
[0008] The roles of the two forms of MDGI and their mechanism(s) of
action are not clearly defined. Mouse and bovine MDGI are
homologous to one another and to a family of low molecular mass
hydrophobic ligand-binding proteins ("low MW HLBP(s)"), which
includes fatty acid-binding proteins ("FABP(s)") from brain, hart,
liver and intestine, myelin P2 protein, the differentiation
associated protein of adipocytes called p422 gastrotropin and
cellular retinoic acid-binding protein ("CRABP"). These proteins
which bind hydrophobic ligands such as long-chain fatty acids,
retinoids and eiconsanoids and they are thought to play roles in
the transport, sequestration, or metabolism of fatty acids and
fatty acid derivatives. However, they are expressed in a
differentiation specific manner, in cells of the mammary gland,
heart, liver, brain and intestine, and they appear not only to play
roles in basal metabolism but also to play important roles in
differentiation and development.
[0009] The homology of MDGI to the low MW HLBPs raises the
possibility that MDGI, at least as part of its function, binds a
hydrophobic ligand, and that binding to this ligand is important to
the mechanism(s) by which MDGI inhibits cell growth and stimulates
differentiation. It should be noted, however, that the other low MW
HLBPs, except gastrotropin, act intracellularly, whereas MDGI acts
extracellularly, at least in vitro. (Yang et al., J. Cell Biol.
127: 1097-1109 (1994).
[0010] Among the low MW HLBPs, MDGI most closely resembles the
fatty acid binding proteins ("FABP"). FABPs have been identified in
brain, heart, liver and intestine. Heart FABP, like MDGI, whether
produced from natural sources or by expression of a cloned gene in
a heterologous host, inhibits growth of normal mammary epithelial
cells ("MEC") of mouse origin. In addition, it stimulates milk
protein synthesis and it stimulates its own expression in these
cells. However, unlike bovine heart FABP, bovine MDGI does not bind
fatty acids, although the two proteins are 95% homologous and it
has been suggested that heart FABP actually may be a form of MDGI.
(Treuner et al., Gene 147: 237-242 (1994)) Thus, even if MDGI is a
low MW HLBP, its substrate affinities are distinct from its close
relatives in the family, and it therefore likely plays a different
physiological role.
[0011] In vivo MDGI is found in capillary endothelial cells and in
the mammary parenchyma, in mice and cows. (See, for instance,
Grosse et al. cited above.) MDGI appears first in the capillary
endothelial cells and later in the secretory epithelial cells. The
location of MDGI in the mammary capillary endothelium is consistent
with a role in regulating endothelial cell proliferation.
[0012] A number of activities of MDGI have been demonstrated in
vitro, as discussed in Grosse et al. cited above, for example. For
instance, it has been shown that MDGI inhibits L(+)-lactate-,
arachidonic acid- and 15-S-hydroxyeicosatetraenoic acid-induced
supersensitivity of neonatal rat heart cells to beta-adrenergic
stimulation. As reported by Burton et al., BBRC 205: 1822-1828
(1994), the induced hypersensitivity is mediated by a small
population of beta 2-adrenergic receptors and, therefore, it has
been suggested that MDGI interferes with the normal function of
these receptors. Interaction with these receptors might also be
part of the mechanism by which MDGI inhibits cells growth. This
activity also raises the possibility that MDGI naturally modulates
the beta-adrenergic sensitivity of cardiac myocytes.
[0013] Furthermore, as reported by Burton et al. cited above,
H-FABP can be a potent inducer of cardiac myotrophy, capable of
stimulating protein synthesis and c-jun expression in myocytes, and
increasing their surface area.
[0014] The effect of MDGI on differentiation of mammary epithelial
cells ("MEC") has been further demonstrated by antisense inhibition
experiments using phosphorothioate oligonucleotides. (Yang at al.
cited above.) These experiments show that MDGI antisense molecules
decrease beta-casein levels and suppress the appearance of alveolar
end buds in organ cultures. Furthermore, MDGI suppresses the
mitogenic effects of epidermal growth factor, and epidermal growth
factor antagonizes the activities of MDGI. MDGI is the first known
growth inhibitor which promotes mammary gland differentiation.
[0015] The regulatory properties of MDGI can be fully mimicked by
an 11-amino acid sequence, which is represented in the carboxyl
terminus of MDGI and a subfamily of the low MW HLBPs.
[0016] Not all mammary epithelial cell lines respond to MDGI in the
same way. MDGF inhibits growth of normal human MEC, passaged for
varying lengths of time. (Yang et al. cited above.) It also
inhibits growth of the mouse mammary malignant epithelial cell
lines mMaCa 20177, the human malignant mammary cell lines MaTu and
T47D and it inhibits the resumption of growth of stationary Ehrlich
ascites carcinoma cells ("EAC") in vitro. In contrast, MDGF
slightly stimulates growth of the human malignant mammary
epithelial cell line MCF7. Finally, MDGI promotes differentiation
of mouse pluripotent embryonic stem cells.
[0017] The mechanism of the effects of MDGI on cells is not known,
as yet. The resumption of growth of stationary Ehrlich ascites
carcinoma cells ("EAC") in vitro is accompanied by a rapid increase
in cellular c-fos, c-myc and c-ras mRNA. The rapid induction of
these genes upon exposure to MDGI underscores the importance of
oncogene expression to growth regulation and evidences a positive
correlation between cell growth and expression of c-fos, c-myc and
c-ras. Furthermore, the effect of MDGI on expression of these genes
indicates that it is a positive effector of cellular protooncogene
expression, either directly or through one or more signaling
pathways, or both.
[0018] It also has been shown that MDGI can function as a potent
tumor suppressor gene. (Huynh et al., Caner Res., 55: 2225 (1995))
Human breast cancer cells transfected with an MDGI expression
construct exhibited differentiated morphology, reduced
proliferation rate, reduced clonogenicity in soft agar, and reduced
tumorgenicity in nude mice. The human homologue of this gene was
mapped to chromosome 1p33-35, a locus previously shown to exhibit
frequent loss of heterozygosity in human breast cancer (about 40%
of tumors). The magnitude of the in vivo and in vitro tumor
suppressor activity of MDGI is comparable to that previously
observed for BRCA1, p53, Rb, and H19.
[0019] The effects of MDGF on cell growth and differentiation, and
on cellular protooncogene expression reiterate the importance of
soluble factors in normal growth and differentiation of cells,
tissues, glands and organs, and their roles in aberrant cell
growth, dysfunction and disease. Clearly, there is a need for
factors that regulate growth and differentiation of normal and
abnormal cells. There is a need, therefore, for identification and
characterization of such factors that modulate growth and
differentiation of cells, both normally and in disease states. In
particular, there is a need to isolate and characterize additional
cytostatins akin to MDGI that modulate growth and differentiation
of cells, such as epithelial cells, particularly mammary epithelial
cells, are essential to the proper development and health of tissue
and organs, such as mammary glands of developing and adult females,
particularly human females, and, among other things, can play a
role in preventing, ameliorating or correcting dysfunctions or
diseases.
SUMMARY OF THE INVENTION
[0020] Toward these ends, and others, it is an object of the
present invention to provide polypeptides, inter alia, that have
been identified as novel cytostatins by homology between the amino
acid sequence set out in FIG. 1 (SEQ ID NO:2) and known amino acid
sequences of other proteins such as MDGI proteins.
[0021] It is a further object of the invention, moreover, to
provide polynucleotides that encode cytostatins, particularly
polynucleotides that encode the polypeptide herein designated
Cytostatin III.
[0022] In a particularly preferred embodiment of this aspect of the
invention the polynucleotide comprises the region encoding human
Cytostatin III in the sequence set out in FIG. 1 (SEQ ID NO:2).
[0023] In accordance with this aspect of the present invention
there is provided an isolated nucleic acid molecule encoding a
mature polypeptide expressed by the human cDNA contained in ATCC
Deposit No. 97332.
[0024] In accordance with this aspect of the invention there are
provided isolated nucleic acid molecules encoding human Cytostatin
III, including mRNAs, cDNAs, genomic DNAs and, in further
embodiments of this aspect of the invention, biologically,
diagnostically, clinically or therapeutically useful variants,
analogs or derivatives thereof, or fragments thereof, including
fragments of the variants, analogs and derivatives.
[0025] Among the particularly preferred embodiments of this aspect
of the invention are naturally occurring allelic variants of human
Cytostatin III.
[0026] It also is an object of the invention to provide Cytostatin
III polypeptides, particularly human Cytostatin III polypeptides,
that modulate growth activity of epithelial cells, stimulate milk
production in both humans and cows and promote involution of the
breast.
[0027] In accordance with this aspect of the invention there are
provided novel polypeptides of human origin referred to herein as
Cytostatin III as well as biologically, diagnostically or
therapeutically useful fragments, variants and derivatives thereof,
variants and derivatives of the fragments, and analogs of the
foregoing.
[0028] Among the particularly preferred embodiments of this aspect
of the invention are variants of human Cytostatin III encoded by
naturally occurring alleles of the human Cytostatin III gene.
[0029] It is another object of the invention to provide a process
for producing the aforementioned polypeptides, polypeptide
fragments, variants and derivatives, fragments of the variants and
derivatives, and analogs of the foregoing.
[0030] In a preferred embodiment of this aspect of the invention
there are provided methods for producing the aforementioned
Cytostatin III polypeptides comprising culturing host cells having
expressibly incorporated therein an exogenously-derived human
Cytostatin III-encoding polynucleotide under conditions for
expression of human Cytostatin III in the host and then recovering
the expressed polypeptide.
[0031] In accordance with another object the invention there are
provided products, compositions, processes and methods that utilize
the aforementioned polypeptides and polynucleotides for research,
biological, clinical and therapeutic purposes, inter alia.
[0032] In accordance with certain preferred embodiments of this
aspect of the invention, there are provided products, compositions
and methods, inter alia, for, among other things: assessing
Cytostatin III expression in cells by determining Cytostatin III
polypeptides or Cytostatin III-encoding mRNA; modulating cell
growth in vitro, ex vivo or in vivo by exposing cells to Cytostatin
III polypeptides or polynucleotides as disclosed herein; assaying
genetic variation and aberrations, such as defects, in Cytostatin
III genes; and administering a Cytostatin III polypeptide or
polynucleotide to an organism to augment Cytostatin III function or
remediate Cytostatin III dysfunction.
[0033] In accordance with certain preferred embodiments of this and
other aspects of the invention there are provided probes that
hybridize to human Cytostatin III sequences.
[0034] In certain additional preferred embodiments of this aspect
of the invention there are provided antibodies against Cytostatin
III polypeptides. In certain particularly preferred embodiments in
this regard, the antibodies are highly selective for human
Cytostatin III.
[0035] In accordance with another aspect of the present invention,
there are provided Cytostatin III agonists. Among preferred
agonists are molecules that mimic Cytostatin III, that bind to
Cytostatin III-binding molecules or receptor molecules, and that
elicit or augment Cytostatin III-induced responses. Also among
preferred agonists are molecules that interact with Cytostatin III
or Cytostatin III polypeptides, or with other modulators of
Cytostatin III activities, and thereby potentiate or augment an
effect of Cytostatin III or more than one effect of Cytostatin
III.
[0036] In accordance with yet another aspect of the present
invention, there are provided Cytostatin III antagonists. Among
preferred antagonists are those which mimic Cytostatin III so as to
bind to Cytostatin III receptor or binding molecules but not elicit
a Cytostatin III-induced response or more than one Cytostatin
III-induced response. Also among preferred antagonists are
molecules that bind to or interact with Cytostatin III so as to
inhibit an effect of Cytostatin III or more than one effect of
Cytostatin III or which prevent expression of Cytostatin III.
[0037] The agonists and antagonists may be used to mimic, augment
or inhibit the action of Cytostatin III polypeptides. They may be
used, for instance, for purposes relating to growth of cells in
vitro or for purposes relating to treatment of disorders associated
with aberrant growth of cells affected by cytostatins, particularly
Cytostatin III.
[0038] In a further aspect of the invention there are provided
compositions comprising a Cytostatin III polynucleotide or a
Cytostatin III polypeptide for administration to cells in vitro, to
cells ex vivo and to cells in vivo, or to a multicellular organism.
In certain particularly preferred embodiments of this aspect of the
invention, the compositions comprise a Cytostatin III
polynucleotide for expression of a Cytostatin III polypeptide in a
host organism for treatment of disease. Particularly preferred in
this regard is expression in a human patient for treatment of a
dysfunction associated with aberrant endogenous activity of
Cytostatin III.
[0039] Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill from the
following description. It should be understood, however, that the
following description and the specific examples, while indicating
preferred embodiments of the invention, are given by way of
illustration only. Various changes and modifications within the
spirit and scope of the disclosed invention will become readily
apparent to those skilled in the art from reading the following
description and from reading the other parts of the present
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] The following drawings depict certain embodiments of the
invention. They are illustrative only and do not limit the
invention otherwise disclosed herein.
[0041] FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino
acid (SEQ ID NO:2) sequence of human Cytostatin III.
[0042] FIG. 2 shows the regions of similarity between amino acid
sequences of cytostatin and MDGI polypeptides (SEQ ID NO:11-15, and
2, respectively).
[0043] FIG. 3 shows structural and functional features of
Cytostatin III deduced by the indicated techniques, as a function
of amino acid sequence.
GLOSSARY
[0044] The following illustrative explanations are provided to
facilitate understanding of certain terms used frequently herein,
particularly in the examples. The explanations are provided as a
convenience and are not limitative of the invention.
[0045] DIGESTION of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes referred to herein are
commercially available and their reaction conditions, cofactors and
other requirements for use are known and routine to the skilled
artisan.
[0046] For analytical purposes, typically, 1 mg of plasmid or DNA
fragment is digested with about 2 units of enzyme in about 20 ml of
reaction buffer. For the purpose of isolating DNA fragments for
plasmid construction, typically 5 to 50 mg of DNA are digested with
20 to 250 units of enzyme in proportionately larger volumes.
[0047] Appropriate buffers and substrate amounts for particular
restriction enzymes are described in standard laboratory manuals,
such as those referenced below, and they are specified by
commercial suppliers.
[0048] Incubation times of about 1 hour at 37.degree. C. are
ordinarily used, but conditions may vary in accordance with
standard procedures, the supplier's instructions and the
particulars of the reaction. After digestion, reactions may be
analyzed, and fragments may be purified by electrophoresis through
an agarose or polyacrylamide gel, using well known methods that are
routine for those skilled in the art.
[0049] GENETIC ELEMENT generally means a polynucleotide comprising
a region that encodes a polypeptide or a region that regulates
transcription or translation or other processes important to
expression of the polypeptide in a host cell, or a polynucleotide
comprising both a region that encodes a polypeptide and a region
operably linked thereto that regulates expression.
[0050] Genetic elements may be comprised within a vector that
replicates as an episomal element; that is, as a molecule
physically independent of the host cell genome. They may be
comprised within mini-chromosomes, such as those that arise during
amplification of transfected DNA by methotrexate selection in
eukaryotic cells. Genetic elements also may be comprised within a
host cell genome; not in their natural state but, rather, following
manipulation such as isolation, cloning and introduction into a
host cell in the form of purified DNA or in a vector, among
others.
[0051] ISOLATED means altered from "by the hand of man" from its
natural state; i.e., that, if it occurs in nature, it has been
changed or removed from its original environment, or both.
[0052] For example, a naturally occurring polynucleotide or a
polypeptide naturally present in a living animal in its natural
state is not "isolated," but the same polynucleotide or polypeptide
separated from the coexisting materials of its natural state is
"isolated", as the term is employed herein. For example, with
respect to polynucleotides, the term isolated means that it is
separated from the chromosome and cell in which it naturally
occurs.
[0053] As part of or following isolation, such polynucleotides can
be joined to other polynucleotides, such as DNAs, for mutagenesis,
to form fusion proteins, and for propagation or expression in a
host, for instance. The isolated polynucleotides, alone or joined
to other polynucleotides such as vectors, can be introduced into
host cells, in culture or in whole organisms. Introduced into host
cells in culture or in whole organisms, such DNAs still would be
isolated, as the term is used herein, because they would not be in
their naturally occurring form or environment. Similarly, the
polynucleotides and polypeptides may occur in a composition, such
as a media formulations, solutions for introduction of
polynucleotides or polypeptides, for example, into cells,
compositions or solutions for chemical or enzymatic reactions, for
instance, which are not naturally occurring compositions, and,
therein remain isolated polynucleotides or polypeptides within the
meaning of that term as it is employed herein.
[0054] LIGATION refers to the process of forming phosphodiester
bonds between two or more polynucleotides, which most often are
double stranded DNAs. Techniques for ligation are well known to the
art and protocols for ligation are described in standard laboratory
manuals and references, such as, for instance, Sambrook et al.,
MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis
et al., pg. 146, as cited below.
[0055] OLIGONUCLEOTIDE(S) refers to relatively short
polynucleotides. Often the term refers to single-stranded
deoxyribonucleotides, but it can refer as well to single-or
double-stranded ribonucleotides, RNA:DNA hybrids and
double-stranded DNAs, among others.
[0056] Oligonucleotides, such as single-stranded DNA probe
oligonucleotides, often are synthesized by chemical methods, such
as those implemented on automated oligonucleotide synthesizers.
However, oligonucleotides can be made by a variety of other
methods, including in vitro recombinant DNA-mediated techniques and
by expression of DNAs in cells and organisms.
[0057] Initially, chemically synthesized DNAs typically are
obtained without a 5' phosphate. The 5' ends of such
oligonucleotides are not substrates for phosphodiester bond
formation by ligation reactions that employ DNA ligases typically
used to form recombinant DNA molecules. Where ligation of such
oligonucleotides is desired, a phosphate can be added by standard
techniques, such as those that employ a kinase and ATP.
[0058] The 3' end of a chemically synthesized oligonucleotide
generally has a free hydroxyl group and, in the presence of a
ligase, such as T4 DNA ligase, readily will form a phosphodiester
bond with a 5' phosphate of another polynucleotide, such as another
oligonucleotide. As is well known, this reaction can be prevented
selectively, where desired, by removing the 5' phosphates of the
other polynucleotide(s) prior to ligation.
[0059] PLASMIDS generally are designated herein by a lower case p
preceded and/or followed by capital letters and/or numbers, in
accordance with standard naming conventions that are familiar to
those of skill in the art.
[0060] Starting plasmids disclosed herein are either commercially
available, publicly available on an unrestricted basis, or can be
constructed from available plasmids by routine application of well
known, published procedures. Many plasmids and other cloning and
expression vectors that can be used in accordance with the present
invention are well known and readily available to those of skill in
the art. Moreover, those of skill readily may construct any number
of other plasmids suitable for use in the invention. The
properties, construction and use of such plasmids, as well as other
vectors, in the present invention will be readily apparent to those
of skill from the present disclosure.
[0061] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. Thus, for instance, polynucleotides as used
herein refers to, among others, single- and double-stranded DNA,
DNA that is a mixture of single-and double-stranded regions,
single- and double-stranded RNA, and RNA that is mixture of single-
and double-stranded regions, hybrid molecules comprising DNA and
RNA that may be single-stranded or, more typically, double-stranded
or a mixture of single- and double-stranded regions.
[0062] In addition, polynucleotide as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0063] As used herein, the term polynucleotide includes DNAs or
RNAs as described above that contain one or more modified bases.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons are "polynucleotides" as that term is intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, are polynucleotides as the term is used herein.
[0064] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term polynucleotide as it is
employed herein embraces such chemically, enzymatically or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple and complex cells, inter alia.
[0065] POLYPEPTIDES, as used herein, includes all polypeptides as
described below. The basic structure of polypeptides is well known
and has been described in innumerable textbooks and other
publications in the art. In this context, the term is used herein
to refer to any peptide or protein comprising two or more amino
acids joined to each other in a linear chain by peptide bonds. As
used herein, the term refers to both short chains, which also
commonly are referred to in the art as peptides, oligopeptides and
oligomers, for example, and to longer chains, which generally are
referred to in the art as proteins, of which there are many
types.
[0066] It will be appreciated that polypeptides, as is well known
and as the term is used herein, generally are formed of the 20
naturally occurring amino acids, and that the amino acids in a
polypeptide generally are joined to one another in a linear chain
by peptide bonds between the alpha carboxyl and the alpha amino
groups of adjacent, succeeding amino acids.
[0067] By convention, the sequence of amino acids in a chain
usually, but not always, is written beginning (on the left and at
the top) with the amino acid having a free alpha amino group. This
amino acid is taken as the amino terminus of the polypeptide, also
referred to as the N-terminus. Each successive amino acid then is
listed in turn, ending with the amino acid having a free carboxyl
group (at bottom and right), which is taken as the carboxyl
terminus of the polypeptide, also called the C-terminus.
[0068] Individual amino acids in a polypeptide commonly are
referred to as amino acid residues, and as residues. Generally, the
amino acids in a polypeptide are numbered beginning with the amino
terminus and proceeding integer by integer and residue by residue
to the carboxyl terminus. However, for polypeptides that first are
synthesized in cells as precursors to a mature form, it also is
common to begin numbering amino acids with the first residue of the
mature form. Then, the upstream residues (i.e., those closer to the
N-terminus) are assigned negative numbers counting back from
residue one (the N-terminus of the mature form) to the N-terminus
of the earliest precursor form. Other numbering schemes also have
been employed, but less commonly.
[0069] Notwithstanding the foregoing general characteristics, it
will be appreciated that polypeptides often contain amino acids
other than the 20 amino acids commonly referred to as the 20
naturally occurring amino acids, and that many amino acids,
including the terminal amino acids, may be modified in a given
polypeptide, either by natural processes, such as processing and
other post-translational modifications, but also by chemical
modification techniques which are well known to the art. Even the
common modifications that occur naturally in polypeptides are too
numerous to list exhaustively here, but they are well described in
basic texts and in more detailed monographs, as well as in a
voluminous research literature, and they are well known to those of
skill in the art.
[0070] Among the known modifications which may be present in
polypeptides of the present are, to name an illustrative few,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cystine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0071] Such modifications are well known to those of skill and have
been described in great detail in the scientific literature.
Several particularly common modifications, glycosylation, lipid
attachment, sulfation, gamma-carboxylation of glutamic acid
residues, hydroxylation and ADP-ribosylation, for instance, are
described in most basic texts, such as, for instance
PROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as, for
example, those provided by Wold, F., Posttranslational Protein
Modifications: Perspectives and Prospects, pgs. 1-12 in
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York (1983); Seifter et al., Analysis for
protein modifications and nonprotein cofactors, Meth. Enzymol. 182:
626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.
663: 48-62 (1992).
[0072] It will be appreciated, as is well known and as noted above,
that polypeptides are not always entirely linear. For instance,
polypeptides may be branched as a result of ubiquitination, and
they may be circular, with or without branching, generally as a
result of posttranslation events, including natural processing
event and events brought about by human manipulation which do not
occur naturally. Circular, branched and branched circular
polypeptides may be synthesized by non-translation natural process
and by entirely synthetic methods, as well.
[0073] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. In fact, blockage of the amino or carboxyl group
in a polypeptide, or both, by a covalent modification, is common in
naturally occurring and synthetic polypeptides and such
modifications may be present in polypeptides of the present
invention, as well. For instance, the amino terminal residue of
polypeptides made in E. coli, prior to proteolytic processing,
almost invariably will be N-formylmethionine.
[0074] The modifications that occur in a polypeptide often will be
a function of how it is made. For polypeptides made by expressing a
cloned gene in a host, for instance, the nature and extent of the
modifications in large part will be determined by the host cell
posttranslational modification capacity and the modification
signals present in the polypeptide amino acid sequence. For
instance, as is well known, glycosylation often does not occur in
bacterial hosts such as E. coli. Accordingly, when glycosylation is
desired, a polypeptide should be expressed in a glycosylating host,
generally a eukaryotic cell. Insect cell often carry out the same
posttranslational glycosylations as mammalian cells and, for this
reason, insect cell expression systems have been developed to
express efficiently mammalian proteins having native patterns of
glycosylation, inter alia. Similar considerations apply to other
modifications.
[0075] It will be appreciated that the same type of modification
may be present in the same or varying degree at several sites in a
given polypeptide. Also, a given polypeptide may contain many types
of modifications.
[0076] In general, as used herein, the term polypeptide encompasses
all such modifications, particularly those that are present in
polypeptides synthesized by expressing a polynucleotide in a host
cell.
[0077] VARIANT(S) of polynucleotides or polypeptides, as the term
is used herein, are polynucleotides or polypeptides that differ
from a reference polynucleotide or polypeptide, respectively.
Variants in this sense are described below and elsewhere in the
present disclosure in greater detail.
[0078] A polynucleotide that differs in nucleotide sequence from
another, reference polynucleotide. Generally, differences are
limited so that the nucleotide sequences of the reference and the
variant are closely similar overall and, in many regions,
identical.
[0079] As noted below, changes in the nucleotide sequence of the
variant may be silent. That is, they may not alter the amino acids
encoded by the polynucleotide. Where alterations are limited to
silent changes of this type a variant will encode a polypeptide
with the same amino acid sequence as the reference. Also as noted
below, changes in the nucleotide sequence of the variant may alter
the amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Such nucleotide changes may result in amino acid
substitutions, additions, deletions, fusions and truncations in the
polypeptide encoded by the reference sequence, as discussed
below.
[0080] A polypeptide that differs in amino acid sequence from
another, reference polypeptide. Generally, differences are limited
so that the sequences of the reference and the variant are closely
similar overall and, in many region, identical.
[0081] A variant and reference polypeptide may differ in amino acid
sequence by one or more substitutions, additions, deletions,
fusions and truncations, which may be present in any
combination.
[0082] Among preferred variants are those that vary from a
reference by conservative amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0083] RECEPTOR MOLECULE, as used herein, refers to molecules which
bind or interact specifically with Cytostatin III polypeptides of
the present invention, including not only classic receptors, which
are preferred, but also other molecules that specifically bind to
or interact with polypeptides of the invention (which also may be
referred to as "binding molecules" and "interaction molecules,"
respectively and as "Cytostatin III binding molecules" and
"Cytostatin III interaction molecules." Binding between
polypeptides of the invention and such molecules, including
receptor or binding or interaction molecules may be exclusive to
polypeptides of the invention, which is very highly preferred, or
it may be highly specific for polypeptides of the invention, which
is highly preferred, or it may be highly specific to a group of
proteins that includes polypeptides of the invention, which is
preferred, or it may be specific to several groups of proteins at
least one of which includes polypeptides of the invention.
[0084] Such molecules generally are proteins, which may be single
or multichain proteins and multisubunit or multiprotein complexes,
such as those of classic cell surface receptors, which are highly
preferred in the invention. Receptor molecules also may be
non-protein molecules that bind to or interact specifically with
polypeptides of the invention.
[0085] Such molecules may occur in membranes, such as classic cell
surface receptors, or they may occur intracellularly, in the
cytosol, inside organelles, or in the surface of organelles, for
instance. Among particularly preferred receptor molecules in this
regard are membrane bound receptors, particularly cell membrane
receptors, especially cell surface receptors. Also among preferred
receptors are those that occur in the membranes of organelles,
particularly nuclear membrane receptors and mitochondrial membrane
receptors.
[0086] Receptors also may be non-naturally occurring, such as
antibodies and antibody-derived reagents that bind specifically to
polypeptides of the invention.
DESCRIPTION OF THE INVENTION
[0087] The present invention relates to novel Cytostatin III
polypeptides and polynucleotides, among other things, as described
in greater detail below. In particular, the invention relates to
polypeptides and polynucleotides of a novel human Cytostatin III,
which is related by amino acid sequence homology to the mammary
derived growth inhibitor ("MDGF") found in cows and mice. The
invention relates especially to Cytostatin III having the
nucleotide and amino acid sequences set out in FIG. 1 (SEQ ID NO: 1
and 2), and to the Cytostatin III nucleotide and amino acid
sequences of the cDNA in ATCC Deposit No. 97332 which is herein
referred to as "the deposited clone" or as the "cDNA of the
deposited clone." It will be appreciated that the nucleotide and
amino acid sequences set out in FIG. 1 (SEQ ID NO:1 and 2) were
obtained by sequencing the human cDNA of the deposited clone.
Hence, the sequence of the deposited clone is controlling as to any
discrepancies between the two and any reference to the sequences of
FIG. 1 (SEQ ID NO:1) include reference to the sequence of the human
cDNA of the deposited clone.
[0088] Polynucleotides
[0089] In accordance with one aspect of the present invention,
there are provided isolated polynucleotides which encode the
Cytostatin III polypeptide having the deduced amino acid sequence
of FIG. 1 (SEQ ID NO:2).
[0090] Using the information provided herein, such as the
polynucleotide sequence set out in FIG. 1 (SEQ ID NO:1), a
polynucleotide of the present invention encoding human Cytostatin
III polypeptide may be obtained using standard cloning and
screening procedures, such as those for cloning cDNAs using mRNA
from cells of a breast lymph node as starting material.
Illustrative of the invention, the polynucleotide set out in FIG. 1
(SEQ ID NO: 1) was discovered in a cDNA library derived from cells
of a human breast lymph node.
[0091] Human Cytostatin III of the invention is structurally
related to other proteins of the cytostatin family of growth
modulating factors, as shown by the results of sequencing the cDNA
encoding human Cytostatin III in the deposited clone. The human
cDNA sequence thus obtained is set out in FIG. 1 (SEQ ID NO:1). It
contains an open reading frame encoding a protein of about 135
amino acid residues with a deduced molecular weight of about 15.9
kDa. The protein exhibits greatest homology to mouse
mammary-derived growth inhibitor ("MDGI"), among known proteins.
The first 133 residues of the Cytostatin III of FIG. 1 (SEQ ID
NO:2) have about 33% identity and about 62% similarity with the
amino acid sequence of mouse MDGI.
[0092] Polynucleotides of the present invention may be in the form
of RNA, such as mRNA, or in the form of DNA, including, for
instance, cDNA and genomic DNA obtained by cloning or produced by
chemical synthetic techniques or by a combination thereof. The DNA
may be double-stranded or single-stranded. Single-stranded DNA may
be the coding strand, also known as the sense strand, or it may be
the non-coding strand, also referred to as the anti-sense
strand.
[0093] A polynucleotide of the present invention may a naturally
occurring sequence, such as that of a naturally occurring allelic
variant, or it may have a sequence that does not occur in nature,
such as a sequence that has been produced, for instance, by in
vitro mutagenesis techniques.
[0094] The coding sequence which encodes the polypeptide may be
identical to the coding sequence of the polynucleotide shown in
FIG. 1 (SEQ ID NO:1). It also may be a polynucleotide with a
different sequence, which, as a result of the redundancy
(degeneracy) of the genetic code, encodes the polypeptide of the
DNA of FIG. 1 (SEQ ID NO:1).
[0095] Polynucleotides of the present invention which encode the
polypeptide of FIG. 1 (SEQ ID NO:2) may include, but are not
limited to the coding sequence for the mature polypeptide, by
itself, the coding sequence for the mature polypeptide and
additional coding sequences, such as those encoding a leader or
secretory sequence, such as a pre-, or pro- or prepro-protein
sequence; the coding sequence of the mature polypeptide, with or
without the aforementioned additional coding sequences, together
with additional, non-coding sequences, including for example, but
not limited to introns and non-coding 5' and 3' sequences, such as
the transcribed, non-translated sequences that play a role in
transcription, mRNA processing--including splicing and
polyadenylation signals, for example--ribosome binding and
stability of mRNA.
[0096] In accordance with the foregoing, the term "polynucleotide
encoding a polypeptide" as used herein encompasses polynucleotides
which include a sequence encoding a polypeptide of the present
invention, particularly the human Cytostatin III having the amino
acid sequence set out in FIG. 1 (SEQ ID NO:2). The term encompasses
polynucleotides that include a single continuous region or
discontinuous regions encoding the polypeptide, together with
additional regions, that also may contain coding and/or non-coding
sequences.
[0097] The present invention further relates to variants of the
herein above described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of FIG. 1 (SEQ ID NO:2). A variant of the
polynucleotide may be a naturally occurring variant such as a
naturally occurring allelic variant, or it may be a variant that is
not known to occur naturally. Such non-naturally occurring variants
of the polynucleotide may be made by mutagenesis techniques,
including those applied to polynucleotides, cells or organisms.
[0098] The present invention includes polynucleotides encoding the
same mature polypeptide as shown in FIG. 1 (SEQ ID NO:2). Further,
the invention includes variants of such polynucleotides that encode
a fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ
ID NO:2). Among variants in this regard are variants that differ
from the aforementioned polynucleotides by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding or non-coding regions or both. Alterations in
the coding regions may produce conservative or non-conservative
amino acid substitutions, deletions or additions.
[0099] Variants of the invention may have a sequence that occurs in
nature or they may have a sequence that does not occur naturally.
As herein above indicated, the polynucleotide may have a coding
sequence which is a naturally occurring allelic variant of the
coding sequence shown in FIG. 1 (SEQ ID NO:1). As known in the art,
an allelic variant is an alternate form of a polynucleotide
sequence which may have a substitution, deletion or addition of one
or more nucleotides.
[0100] Among the particularly preferred embodiments of the
invention in this regard are polynucleotides encoding polypeptides
having the amino acid sequence of Cytostatin III set out in FIG. 1
(SEQ ID NO:2); variants, analogs, derivatives and fragments
thereof, and fragments of the variants, analogs and derivatives
[0101] Further particularly preferred in this regard are
polynucleotides encoding Cytostatin III variants, analogs,
derivatives and fragments, and variants, analogs and derivatives of
the fragments, which have the amino acid sequence of the Cytostatin
III polypeptide of FIG. 1 (SEQ ID NO:2) in which several, a few, 5
to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are
substituted, deleted or added, in any combination. Especially
preferred among these are silent substitutions, additions and
deletions, which do not alter the properties and activities of the
Cytostatin III. Also especially preferred in this regard are
conservative substitutions. Most highly preferred are
polynucleotides encoding polypeptides having the amino acid
sequence of FIG. 1 (SEQ ID NO:2), without substitutions.
[0102] Also particularly preferred in this regard are
polynucleotides encoding a polypeptide having the amino acid
sequence of the Cytostatin III set out in FIG. 1 (SEQ ID NO:2). As
set out elsewhere herein, the polynucleotide may encode the
polypeptide in a continuous region or in a plurality of two or more
discontinuous exons, and it may comprise additional regions as
well, which are unrelated to the coding region or regions.
[0103] Most highly preferred in this regard are polynucleotides
that comprise a region that is more than 85% identical to the
Cytostatin III-encoding portion of the polynucleotide set out in
FIG. 1 (SEQ ID NO:1). Alternatively, most highly preferred are
polynucleotides that comprise a region that is more than 85%
identical to the Cytostatin III-encoding portion of the cDNA the
deposited clone. Among such polynucleotides, those more than 90%
identical to the same are particularly preferred, and, among these
particularly preferred polynucleotides, those with 95% or more
identity are especially preferred. Furthermore, those with 97% or
more identity are highly preferred among those with 95% or more
identity, and among these those with 98% or more and 99% or more
identity are particularly highly preferred, with 99% or more being
the more preferred of these.
[0104] The present invention also includes polynucleotides in which
the sequence encoding the mature polypeptide is fused in the same
reading frame to additional sequences. Such sequences include
signal sequences, which facilitate transport of the nascent protein
into the endoplasmic reticulum, pro-sequences that are associated
with inactive precursor forms of the polypeptide, which may
facilitate trafficking of the protein in a cell or out of a cell or
may improve persistence of the protein in a cell or in an
extracellular compartment. Such sequences also may be added to
facilitate production and purification, or to add additional
functional domains, as discussed elsewhere herein.
[0105] Thus, polynucleotides of the invention may encode, in
addition to a mature cytostatin, particularly Cytostatin III, for
example, a leader sequence, such as a signal peptide which
functions as a secretory sequence for controlling transport of the
polypeptide into the lumen of the endoplasmic reticulum. The leader
sequence may be removed by the host cell, as is generally the case
for signal peptides, yielding another precursor protein or the
mature polypeptide. A precursor protein having a leader sequence
often is called a preprotein.
[0106] A polynucleotide of the present invention may encode a
mature or precursor pre-, pro- or prepropolypeptide as discussed
above, among others, fused to additional amino acids, such as those
which provide additional functionalities. Thus, for instance, the
polypeptide may be fused to a marker sequence, such as a peptide,
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
sequence is a hexa-histidine peptide, such as the tag provided in
the vector pQE-9, among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci.,
USA 86: 821-824 (1989), for instance, hexa-histidine provides for
convenient purification of the fusion protein. Typically, it does
not adversely affect protein structure or function, and it binds
efficiently, selectively and tightly to metal chelate resins,
particularly nickel chelate resins. For instance, as is well known,
hexa-histidine tags often bind especially well to nickel-NTA resin,
which is well known and readily available and can be obtained
commercially from, for instance, Qiagen. Moreover, the
histidine-metal interaction not only is stable to a variety of
conditions useful to remove non-specifically bound material, but
also the fusion polypeptide can be bound and removed under mild,
non-denaturing conditions. The hexa-histidine tag can be fused most
conveniently to the amino or the carboxyl terminus of the
cytostatin polypeptide. A tag of the hexa-histidine type is
particularly useful for bacterial expression.
[0107] Another useful marker sequence in certain other preferred
embodiments is a hemagglutinin ("HA") tag, particularly when a
mammalian cell is used for expression; e.g., COS-7 cells. The HA
tag corresponds to an epitope derived of influenza hemagglutinin
protein, which has been described by Wilson et al., Cell 37: 767
(1984), for instance.
[0108] The present invention further relates to polynucleotides
that hybridize to the herein above-described cytostatin sequences,
particularly Cytostatin III sequences. Preferred in this regard are
polynucleotides that have at least 50% identity to the sequences
described herein above. Particularly preferred are sequences that
have at least 70% identity. In this regard, the present invention
especially relates to polynucleotides which hybridize under
stringent conditions to the herein above-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization
will occur only if there is at least 95% and preferably at least
97% identity between the sequences.
[0109] As discussed additionally herein regarding polynucleotide
assays of the invention, for instance, a probe as discussed above,
derived from the full length Cytostatin III cDNA, including the
entire Cytostatin III cDNA of FIG. 1 (SEQ ID NO:1) or of the
deposited clone, or the coding region of thereof, or any part
thereof useful as a probe, may be used as a hybridization probe for
cDNA and genomic DNA to isolate full-length cDNAs and genomic
clones encoding Cytostatin III and to isolate cDNA and genomic
clones of other genes that have a high sequence similarity to the
human Cytostatin III gene. Such probes generally will comprise at
least 20 bases. Preferably, such probes will have at least 30
bases. Particularly preferred probes will have at least 30 bases
and will have 50 bases or less.
[0110] For example, the coding region of the Cytostatin III gene
may be isolated by screening using the known DNA sequence to
synthesize an oligonucleotide probe. Labeled an oligonucleotide
having a sequence complementary to that of a gene of the present
invention then is used to screen a library of human cDNA, genomic
DNA or mRNA to determine which members of the library the probe
hybridizes to.
[0111] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to human disease, as
further discussed herein relating to polynucleotide assays, inter
alia.
[0112] The polynucleotides also may encode a polypeptide which is
the mature protein plus additional amino or carboxyl-terminal amino
acids, or amino acids interior to the mature polypeptide (when the
mature form has more than one polypeptide chain, for instance).
Such sequences may play a role in processing of a protein from
precursor to a mature form, may facilitate protein trafficking, may
prolong or shorten protein half-life or may facilitate manipulation
of a protein for assay or production, among other things. As
generally is the case in situ, the additional amino acids may be
processed away from the mature protein by cellular enzymes.
[0113] A precursor protein, having the mature form of the
polypeptide fused to one or more prosequences may be an inactive
form of the polypeptide. When prosequences are removed such
inactive precursors generally are activated. Some or all of the
prosequences may be removed before activation. Generally, such
precursors are called proproteins.
[0114] In sum, a polynucleotide of the present invention may encode
a mature protein, a mature protein plus a leader sequence (which
may be referred to as a preprotein), a precursor of a mature
protein having one or more prosequences which are not the leader
sequences of a preprotein, or a preproprotein, which is a precursor
to a proprotein, having a leader sequence and one or more
prosequences, which generally are removed during processing steps
that produce active and mature forms of the polypeptide.
[0115] Deposited Materials
[0116] The cDNA deposit is referred to herein as "the deposited
clone" or as "the cDNA of the deposited clone." The deposited clone
was deposited with the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209, USA, on Nov. 6,
1995 and assigned ATCC Deposit No. 97332.
[0117] The deposited material is a pBluescript SK (-) plasmid
(Stratagene, La Jolla, Calif.) that contains the full length human
Cytostatin III cDNA.
[0118] The deposit has been made under the terms of the Budapest
Treaty on the International Recognition of the Deposit of
Micro-organisms for purposes of Patent Procedure. The deposit is
provided merely as convenience to those of skill in the art and it
is not an indication or an admission that a deposit is required for
enablement, such as that required under 35 U.S.C. .sctn.112.
[0119] The deposit has been made under the terms of the Budapest
Treaty on the international recognition of the deposit of
micro-organisms for purposes of patent procedure. The strain will
be irrevocably and without restriction or condition released to the
public upon the issuance of a patent. The deposit is provided
merely as convenience to those of skill in the art and is not an
admission that a deposit is required for enablement, such as that
required under 35 U.S.C. .sctn.112.
[0120] The sequence of the polynucleotides contained in the
deposited material, as well as the amino acid sequence of the
polypeptide encoded thereby, are controlling in the event of any
conflict with any description of sequences herein.
[0121] A license may be required to make, use or sell the deposited
materials, and no such license is hereby granted.
[0122] Polypeptides
[0123] The present invention further relates to a human Cytostatin
III polypeptide which has the deduced amino acid sequence of FIG. 1
(SEQ ID NO:2) or which has the amino acid sequence encoded by the
deposited clone.
[0124] The invention also relates to fragments, analogs and
derivatives of these polypeptides. The terms "fragment,"
"derivative" and "analog" when referring to the polypeptide of FIG.
1 (SEQ ID NO:2) or that encoded by the deposited cDNA, means a
polypeptide which retains essentially the same biological function
or activity as such polypeptide. Thus, an analog includes a
proprotein which can be activated by cleavage of the proprotein
portion to produce an active mature polypeptide.
[0125] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide. In certain preferred embodiments it is a recombinant
polypeptide.
[0126] The fragment, derivative or analog of the polypeptide of
FIG. 1 (SEQ ID NO:2) or that encoded by the cDNA in the deposited
clone may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0127] Among the particularly preferred embodiments of the
invention in this regard are polypeptides having the amino acid
sequence of Cytostatin III set out in FIG. 1 (SEQ ID NO:2),
variants, analogs, derivatives and fragments thereof, and variants,
analogs and derivatives of the fragments. Alternatively,
particularly preferred embodiments of the invention in this regard
are polypeptides having the amino acid sequence of the Cytostatin
III of the cDNA in the deposited clone, variants, analogs,
derivatives and fragments thereof, and variants, analogs and
derivatives of the fragments.
[0128] Further particularly preferred in this regard are variants,
analogs, derivatives and fragments, and variants, analogs and
derivatives of the fragments, having the amino acid sequence of the
Cytostatin III polypeptide of FIG. 1 (SEQ ID NO:2) or of the cDNA
in the deposited clone, in which several, a few, 5 to 10, 1 to 5, 1
to 3, 2, 1 or no amino acid residues are substituted, deleted or
added, in any combination. Especially preferred among these are
silent substitutions, additions and deletions, which do not alter
the properties and activities of the Cytostatin III. Also
especially preferred in this regard are conservative substitutions.
Most highly preferred are polypeptides having the amino acid
sequence of FIG. 1 (SEQ ID NO:2) or the deposited clone without
substitutions.
[0129] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0130] The term "isolated" means that the material has been altered
from its natural state; e.g., that, if it occurs in nature, it has
been removed from its original environment. For example, a
naturally occurring polynucleotide or polypeptide naturally present
in a living animal in its natural state is not "isolated," but the
same polynucleotide or polypeptide separated from some or all of
the coexisting materials in the natural system is "isolated", as
the term is employed herein.
[0131] As part of or following isolation, such polynucleotides can
be joined to other polynucleotides, such as DNAs, for mutagenesis,
to form fusion proteins, and for propagation or expression in a
host, for instance. The isolated polynucleotides, alone or joined
to other polynucleotides such as vectors, can be introduced into
host cells, in culture or in whole organisms. Introduced into host
cells in culture or in whole organisms, such DNAs still would be
isolated, as the term is used herein, because they would not be in
their naturally occurring form or environment. Similarly, the
polynucleotides and polypeptides may occur in a composition, such
as a media formulation, a solution for introduction into cells, a
composition or solution for chemical or enzymatic reaction, and the
like, which are not naturally compositions, and therein remain
isolated polynucleotides or polypeptides within the meaning of that
term as it is employed herein.
[0132] Fragments
[0133] Also among preferred embodiments of this aspect of the
present invention are polypeptides comprising fragments of
Cytostatin III, most particularly fragments of the Cytostatin III
having the amino acid set out in FIG. 1 (SEQ ID NO:2), or having
the amino acid sequence of the Cytostatin III of the deposited
clone, and fragments of variants and derivatives of the Cytostatin
III of FIG. 1 (SEQ ID NO:2) or of the deposited clone.
[0134] In this regard a fragment is a polypeptide having an amino
acid sequence that entirely is the same as part but not all of the
amino acid sequence of the aforementioned Cytostatin III
polypeptides and variants or derivatives thereof.
[0135] Such fragments may be "free-standing," i.e., not part of or
fused to other amino acids or polypeptides, or they may be
comprised within a larger polypeptide of which they form a part or
region. When comprised within a larger polypeptide, the presently
discussed fragments most preferably form a single continuous
region. However, several fragments may be comprised within a single
larger polypeptide. For instance, certain preferred embodiments
relate to a fragment of a Cytostatin III polypeptide of the present
comprised within a precursor polypeptide designed for expression in
a host and having heterologous pre and pro-polypeptide regions
fused to the amino terminus of the Cytostatin III fragment and an
additional region fused to the carboxyl terminus of the fragment.
Therefore, fragments in one aspect of the meaning intended herein,
refers to the portion or portions of a fusion polypeptide or fusion
protein derived from Cytostatin III.
[0136] Among preferred fragments of Cytostatin III are fragments
about 5-15, 10-20, 15-40, 25-50, 35-60, 50-75, 65-80, 65-90,
65-100, 50-100, 75-100, 90-115, 80-135, 90-130, 100-125 and 110-135
amino acids long.
[0137] In this context about includes the particularly recited
range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or
1 amino acid at either extreme or at both extremes. For instance,
about 65-90 amino acids in this context means a polypeptide
fragment of 65, 65 plus or minus several, a few, 5, 4, 3, 2 or 1
amino acid to 90 or 90 plus or minus several a few, 5, 4, 3, 2 or 1
amino acid residues, i.e., ranges a broad as 65 minus several amino
acids to 90 plus several amino acids to as narrow as 65 plus
several amino acids to 90 minus several amino acids.
[0138] Highly, preferred in this regard are the recited ranges plus
or minus as many as 5 amino acids at either or at both extremes.
Particularly highly preferred are the recited ranges means plus or
minus as many as 3 amino acids at either or at both extremes.
Especially particularly highly preferred are ranges plus or minus 1
amino acid at either or at both extremes. Most highly preferred of
all in this regard are fragments 5-15, 10-20, 15-40, 25-50, 35-60,
50-75, 65-80, 65-90, 65-100, 50-100, 75-100, 90-115, 80-135,
90-130, 100-125 and 110-135 amino acids in length are
preferred.
[0139] Among especially preferred fragments of the invention are
truncation mutants of Cytostatin III. Truncation mutants include
Cytostatin III polypeptides having the amino acid sequence of FIG.
1 (SEQ ID NO:2), or of the deposited clone, or of variants or
derivatives thereof, except for deletion of a continuous series of
residues (that is, a continuous region, part or portion) that
includes the amino terminus, or a continuous series of residues
that includes the carboxyl terminus or, as in double truncation
mutants, deletion of two continuous series of residues, one
including the amino terminus and one including the carboxyl
terminus. Fragments having the size ranges set out about also are
preferred embodiments of truncation fragments, which are especially
preferred among fragments generally.
[0140] Also preferred in this aspect of the invention are fragments
characterized by structural or functional attributes of Cytostatin
III. Preferred embodiments of the invention in this regard include
fragments that comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet-forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions and
high antigenic index regions of Cytostatin III.
[0141] Certain preferred regions in these regards are set out in
FIG. 3, and include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIG. 1 (SEQ ID NO:2). As set out in FIG. 3,
such preferred regions include Gamier-Robson alpha-regions,
beta-regions, turn-regions and coil-regions, Chou-Fasman
alpha-regions, beta-regions and turn-regions, Kyte-Doolittle
hydrophilic regions and hydrophilic regions, Eisenberg alpha and
beta amphipathic regions, Karplus-Schulz flexible regions, Emini
surface-forming regions and Jameson-Wolf high antigenic index
regions.
[0142] Among highly preferred fragments in this regard are those
that comprise regions of Cytostatin III that combine several
structural features, such as several of the features set out above.
In this regard, the regions defined by the residues about 10 to
about 20, about 40 to about 50, about 70 to about 90 and about 100
to about 110 of FIG. 1 (SEQ ID NO:2), which all are characterized
by amino acid compositions highly characteristic of turn-regions,
hydrophilic regions, flexible-regions, surface-forming regions, and
high antigenic index-regions, are especially highly preferred
regions. Such regions may be comprised within a larger polypeptide
or may be by themselves a preferred fragment of the present
invention, as discussed above. It will be appreciated that the term
"about" as used in this paragraph has the meaning set out above
regarding fragments in general.
[0143] Further preferred regions are those that mediate activities
of Cytostatin III. Most highly preferred in this regard are
fragments that have a chemical, biological or other activity of
Cytostatin III, including those with a similar activity or an
improved activity, or with a decreased undesirable activity. Highly
preferred in this regard are fragments that contain regions that
are homologs in sequence, or in position, or in both sequence and
to active regions of related polypeptides, such as the related
polypeptides set out in FIG. 2 (SEQ ID NO:11-15), which include
Mouse MDGI, Bovine MDGI, Human MDGI, Cytostatin I and Cytostatin
II. Among particularly preferred fragments in these regards are
truncation mutants, as discussed above.
[0144] It will be appreciated that the invention also relates to,
among others, polynucleotides encoding the aforementioned
fragments, polynucleotides that hybridize to polynucleotides
encoding the fragments, particularly those that hybridize under
stringent conditions, and polynucleotides, such as PCR primers, for
amplifying polynucleotides that encode the fragments. In these
regards, preferred polynucleotides are those that correspondent to
the preferred fragments, as discussed above.
[0145] Vectors, Host Cells, Expression
[0146] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0147] Host cells can be genetically engineered to incorporate
polynucleotides and express polypeptides of the present invention.
For instance, polynucleotides may be introduced into host cells
using well known techniques of infection, transduction,
transfection, transvection and transformation. The polynucleotides
may be introduced alone or with other polynucleotides. Such other
polynucleotides may be introduced independently, co-introduced or
introduced joined to the polynucleotides of the invention.
[0148] Thus, for instance, polynucleotides of the invention may be
transfected into host cells with another, separate, polynucleotide
encoding a selectable marker, using standard techniques for
co-transfection and selection in, for instance, mammalian cells. In
this case the polynucleotides generally will be stably incorporated
into the host cell genome.
[0149] Alternatively, the polynucleotides may be joined to a vector
containing a selectable marker for propagation in a host. The
vector construct may be introduced into host cells by the
aforementioned techniques. Generally, a plasmid vector is
introduced as DNA in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. Electroporation
also may be used to introduce polynucleotides into a host. If the
vector is a virus, it may be packaged in vitro or introduced into a
packaging cell and the packaged virus may be transduced into cells.
A wide variety of techniques suitable for making polynucleotides
and for introducing polynucleotides into cells in accordance with
this aspect of the invention are well known and routine to those of
skill in the art. Such techniques are reviewed at length in
Sambrook et al. cited above, which is illustrative of the many
laboratory manuals that detail these techniques.
[0150] In accordance with this aspect of the invention the vector
may be, for example, a plasmid vector, a single or double-stranded
phage vector, a single or double-stranded RNA or DNA viral vector.
Such vectors may be introduced into cells as polynucleotides,
preferably RNA, by well known techniques for introducing DNA and
RNA into cells. The vectors, in the case of phage and viral vectors
also may be and preferably are introduced into cells as packaged or
encapsidated virus by well known techniques for infection and
transduction. Viral vectors may be replication competent or
replication defective. In the latter case viral propagation
generally will occur only in complementing host cells.
[0151] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate trans-acting
factors either are supplied by the host, supplied by a
complementing vector or supplied by the vector itself upon
introduction into the host.
[0152] In certain preferred embodiments in this regard, the vectors
provide for specific expression. Such specific expression may be
inducible expression or expression only in certain types of cells
or both inducible and cell-specific. Particularly preferred among
inducible vectors are vectors that can be induced for expression by
environmental factors that are easy to manipulate, such as
temperature and nutrient additives. A variety of vectors suitable
to this aspect of the invention, including constitutive and
inducible expression vectors for use in prokaryotic and eukaryotic
hosts, are well known and employed routinely by those of skill in
the art.
[0153] The engineered host cells can be cultured in conventional
nutrient media, which may be modified as appropriate for, inter
alia, activating promoters, selecting transformants or amplifying
genes. Culture conditions, such as temperature, pH and the like,
previously used with the host cell selected for expression
generally will be suitable for expression of polypeptides of the
present invention as will be apparent to those of skill in the
art.
[0154] A great variety of expression vectors can be used to express
a polypeptide of the invention. Such vectors include chromosomal,
episomal and virus-derived vectors e.g., vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids, all may be used for expression in accordance with this
aspect of the present invention. Generally, any vector suitable to
maintain, propagate or express polynucleotides to express a
polypeptide in a host may be used for expression in this
regard.
[0155] The appropriate DNA sequence may be inserted into the vector
by any of a variety of well-known and routine techniques. In
general, a DNA sequence for expression is joined to an expression
vector by cleaving the DNA sequence and the expression vector with
one or more restriction endonucleases and then joining the
restriction fragments together using T4 DNA ligase. Procedures for
restriction and ligation that can be used to this end are well
known and routine to those of skill. Suitable procedures in this
regard, and for constructing expression vectors using alternative
techniques, which also are well known and routine to those skill,
are set forth in great detail in Sambrook et al. cited elsewhere
herein.
[0156] The DNA sequence in the expression vector is operatively
linked to appropriate expression control sequence(s), including,
for instance, a promoter to direct mRNA transcription.
Representatives of such promoters include the phage lambda PL
promoter, the E. coli lac, trp and tac promoters, the SV40 early
and late promoters and promoters of retroviral LTRs, to name just a
few of the well-known promoters. It will be understood that
numerous promoters not mentioned are suitable for use in this
aspect of the invention are well known and readily may be employed
by those of skill in the manner illustrated by the discussion and
the examples herein.
[0157] In general, expression constructs will contain sites for
transcription initiation and termination, and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the mature transcripts expressed by the constructs will include
a translation initiating AUG at the beginning and a termination
codon appropriately positioned at the end of the polypeptide to be
translated.
[0158] In addition, the constructs may contain control regions that
regulate as well as engender expression. Generally, in accordance
with many commonly practiced procedures, such regions will operate
by controlling transcription, such as repressor binding sites and
enhancers, among others.
[0159] Vectors for propagation and expression generally will
include selectable markers. Such markers also may be suitable for
amplification or the vectors may contain additional markers for
this purpose. In this regard, the expression vectors preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells. Preferred markers
include dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, and tetracycline or ampicillin resistance
genes for culturing E. coli and other bacteria.
[0160] The vector containing the appropriate DNA sequence as
described elsewhere herein, as well as an appropriate promoter, and
other appropriate control sequences, may be introduced into an
appropriate host using a variety of well known techniques suitable
to expression therein of a desired polypeptide. Representative
examples of appropriate hosts include bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Hosts for of a great variety of
expression constructs are well known, and those of skill will be
enabled by the present disclosure readily to select a host for
expressing a polypeptides in accordance with this aspect of the
present invention.
[0161] More particularly, the present invention also includes
recombinant constructs, such as expression constructs, comprising
one or more of the sequences described above. The constructs
comprise a vector, such as a plasmid or viral vector, into which
such a sequence of the invention has been inserted. The sequence
may be inserted in a forward or reverse orientation. In certain
preferred embodiments in this regard, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and there are
many commercially available vectors suitable for use in the present
invention.
[0162] The following vectors, which are commercially available, are
provided by way of example. Among vectors preferred for use in
bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS
vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a,
pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3,
pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG
available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. These vectors are listed solely by way of
illustration of the many commercially available and well known
vectors that are available to those of skill in the art for use in
accordance with this aspect of the present invention. It will be
appreciated that any other plasmid or vector suitable for, for
example, introduction, maintenance, propagation or expression of a
polynucleotide or polypeptide of the invention in a host may be
used in this aspect of the invention.
[0163] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("cat") transcription unit, downstream of restriction site or sites
for introducing a candidate promoter fragment; i.e., a fragment
that may contain a promoter. As is well known, introduction into
the vector of a promoter-containing fragment at the restriction
site upstream of the cat gene engenders production of CAT activity,
which can be detected by standard CAT assays. Vectors suitable to
this end are well known and readily available. Two such vectors are
pKK232-8 and pCM7. Thus, promoters for expression of
polynucleotides of the present invention include not only well
known and readily available promoters, but also promoters that
readily may be obtained by the foregoing technique, using a
reporter gene.
[0164] Among known bacterial promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ and promoters, the T3 and
T7 promoters, the T5 tac promoter, the lambda PR, PL promoters and
the trp promoter. Among known eukaryotic promoters suitable in this
regard are the CMV immediate early promoter, the HSV thymidine
kinase promoter, the early and late SV40 promoters, the promoters
of retroviral LTRs, such as those of the Rous sarcoma virus
("RSV"), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0165] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host and expression in the host
are routine skills in the art.
[0166] The present invention also relates to host cells containing
the above-described constructs discussed above. The host cell can
be a higher eukaryotic cell, such as a mammalian cell, or a lower
eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell.
[0167] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986).
[0168] Constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0169] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989).
[0170] Generally, recombinant expression vectors will include
origins of replication, a promoter derived from a highly-expressed
gene to direct transcription of a downstream structural sequence,
and a selectable marker to permit isolation of vector containing
cells after exposure to the vector.
[0171] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0172] Polynucleotides of the invention, encoding the heterologous
structural sequence of a polypeptide of the invention generally
will be inserted into the vector using standard techniques so that
it is operably linked to the promoter for expression. The
polynucleotide will be positioned so that the transcription start
site is located appropriately 5' to a ribosome binding site. The
ribosome binding site will be 5' to the AUG that initiates
translation of the polypeptide to be expressed. Generally, there
will be no other open reading frames that begin with an initiation
codon, usually AUG, and lie between the ribosome binding site and
the initiating AUG. Also, generally, there will be a translation
stop codon at the end of the polypeptide and there will be a
polyadenylation signal and a transcription termination signal
appropriately disposed at the 3' end of the transcribed region.
[0173] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0174] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals but
also additional heterologous functional regions. Thus, for
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-terminus of the polypeptide to
improve stability and persistence in the host cell, during
purification or during subsequent handling and storage. Also,
region also may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the polypeptide. The addition of peptide moieties to
polypeptides to engender secretion or excretion, to improve
stability and to facilitate purification, among others, are
familiar and routine techniques in the art.
[0175] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0176] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, where the
selected promoter is inducible it is induced by appropriate means
(e.g., temperature shift or exposure to chemical inducer) and cells
are cultured for an additional period.
[0177] Cells typically then are harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0178] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well know to those skilled in the art.
[0179] Various mammalian cell culture systems can be employed for
expression, as well. Examples of mammalian expression systems
include the COS-7 lines of monkey kidney fibroblast, described in
Gluzman et al., Cell 23:175 (1981). Other cell lines capable of
expressing a compatible vector include for example, the C127, 3T3,
CHO, HeLa, human kidney 293 and BHK cell lines.
[0180] The Cytostatin III polypeptide can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Well known techniques for refolding
protein may be employed to regenerate active conformation when the
polypeptide is denatured during isolation and or purification.
[0181] Polypeptides of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a prokaryotic or
eukaryotic host, including, for example, bacterial, yeast, higher
plant, insect and mammalian cells. Depending upon the host employed
in a recombinant production procedure, the polypeptides of the
present invention may be glycosylated or may be non-glycosylated.
In addition, polypeptides of the invention may also include an
initial modified methionine residue, in some cases as a result of
host-mediated processes.
[0182] Cytostatin III polynucleotides and polypeptides may be used
in accordance with the present invention for a variety of
applications, particularly those that make use of the chemical and
biological properties Cytostatin III. Among these are applications
in characterizing cells and organisms and in growing cells and
organisms. Additional applications relate to diagnosis and to
treatment of disorders of cells, tissues and organisms. These
aspects of the invention are illustrated further by the following
discussion.
[0183] Polynucleotide Assays
[0184] This invention is also related to the use of the Cytostatin
III polynucleotides to detect complementary polynucleotides such
as, for example, as a diagnostic reagent. Detection of a mutated
form of Cytostatin III associated with a dysfunction will provide a
diagnostic tool that can add or define a diagnosis of a disease or
susceptibility to a disease which results from under-expression
over-expression or altered expression of Cytostatin III, such as,
for example, breast cancer.
[0185] Individuals carrying mutations in the human Cytostatin III
gene may be detected at the DNA level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from a patient's cells,
such as from blood, urine, saliva, tissue biopsy and autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR prior to analysis. PCR
(Saiki et al., Nature, 324: 163-166 (1986)). RNA or cDNA may also
be used in the same ways. As an example, PCR primers complementary
to the nucleic acid encoding Cytostatin III can be used to identify
and analyze Cytostatin III expression and mutations. For example,
deletions and insertions can be detected by a change in size of the
amplified product in comparison to the normal genotype. Point
mutations can be identified by hybridizing amplified DNA to
radiolabeled Cytostatin III RNA or alternatively, radiolabeled
Cytostatin III antisense DNA sequences. Perfectly matched sequences
can be distinguished from mismatched duplexes by RNase A digestion
or by differences in melting temperatures.
[0186] Sequence differences between a reference gene and genes
having mutations also may be revealed by direct DNA sequencing. In
addition, cloned DNA segments may be employed as probes to detect
specific DNA segments. The sensitivity of such methods can be
greatly enhanced by appropriate use of PCR or another amplification
method. For example, a sequencing primer is used with
double-stranded PCR product or a single-stranded template molecule
generated by a modified PCR. The sequence determination is
performed by conventional procedures with radiolabeled nucleotide
or by automatic sequencing procedures with fluorescent-tags.
[0187] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels, with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230: 1242 (1985)). Sequence changes at specific
locations also may be revealed by nuclease protection assays, such
as RNase and S1 protection or the chemical cleavage method (e.g.,
Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401
(1985)).
[0188] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., restriction fragment length polymorphisms ("RFLP")
and Southern blotting of genomic DNA.
[0189] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations also can be detected by in situ analysis.
[0190] Chromosome Assays
[0191] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0192] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a Cytostatin III
gene. This can be accomplished using a variety of well known
techniques and libraries, which generally are available
commercially. The genomic DNA the is used for in situ chromosome
mapping using well known techniques for this purpose. Typically, in
accordance with routine procedures for chromosome mapping, some
trial and error may be necessary to identify a genomic probe that
gives a good in situ hybridization signal.
[0193] In some cases, in addition, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified fragment.
[0194] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0195] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 500 or 600 bases; however, clones larger than
2,000 bp have a higher likelihood of binding to a unique
chromosomal location with sufficient signal intensity for simple
detection. FISH requires use of the clones from which the express
sequence tag (EST) was derived, and the longer the better. For
example, 2,000 bp is good, 4,000 is better, and more than 4,000 is
probably not necessary to get good results a reasonable percentage
of the time. For a review of this technique, see Verma et al.,
HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press,
New York (1988).
[0196] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, MENDELIAN INHERITANCE IN MAN, available on
line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0197] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is
[0198] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0199] Polypeptide Assays
[0200] The present invention also relates to a diagnostic assays
such as quantitative and diagnostic assays for detecting levels of
Cytostatin III protein in cells and tissues, including
determination of normal and abnormal levels. Thus, for instance, a
diagnostic assay in accordance with the invention for detecting
over-expression of Cytostatin III protein compared to normal
control tissue samples may be used to detect the presence of
myocardial infarction, for example. Assay techniques that can be
used to determine levels of a protein, such as an Cytostatin III
protein of the present invention, in a sample derived from a host
are well-known to those of skill in the art. Such assay methods
include radioimmunoassays, competitive-binding assays, Western Blot
analysis and ELISA assays. Among these ELISAs frequently are
preferred. An ELISA assay initially comprises preparing an antibody
specific to Cytostatin III, preferably a monoclonal antibody. In
addition a reporter antibody generally is prepared which binds to
the monoclonal antibody. The reporter antibody is attached a
detectable reagent such as radioactive, fluorescent or enzymatic
reagent, in this example horseradish peroxidase enzyme.
[0201] To carry out an ELISA a sample is removed from a host and
incubated on a solid support, e.g. a polystyrene dish, that binds
the proteins in the sample. Any free protein binding sites on the
dish are then covered by incubating with a non-specific protein
such as bovine serum albumin. Next, the monoclonal antibody is
incubated in the dish during which time the monoclonal antibodies
attach to any Cytostatin III proteins attached to the polystyrene
dish. Unbound monoclonal antibody is washed out with buffer. The
reporter antibody linked to horseradish peroxidase is placed in the
dish resulting in binding of the reporter antibody to any
monoclonal antibody bound to Cytostatin III. Unattached reporter
antibody is then washed out. Reagents for peroxidase activity,
including a calorimetric substrate are then added to the dish.
Immobilized peroxidase, linked to Cytostatin III through the
primary and secondary antibodies, produces a colored reaction
product. The amount of color developed in a given time period
indicates the amount of Cytostatin III protein present in the
sample. Quantitative results typically are obtained by reference to
a standard curve.
[0202] A competition assay may be employed wherein antibodies
specific to Cytostatin III attached to a solid support and labeled
Cytostatin III and a sample derived from the host are passed over
the solid support and the amount of label detected attached to the
solid support can be correlated to a quantity of Cytostatin III in
the sample.
[0203] Immunoassays and Reagents
[0204] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0205] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0206] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler, G.
and Milstein, C., Nature 256: 495-497 (1975), the trioma technique,
the human B-cell hybridoma technique (Kozbor et al., Immunology
Today 4: 72 (1983) and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
[0207] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice, or other organisms such as other
mammals, may be used to express humanized antibodies to immunogenic
polypeptide products of this invention.
[0208] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or purify the
polypeptide of the present invention by attachment of the antibody
to a solid support for isolation and/or purification by affinity
chromatography.
[0209] Thus, among others, the growth inhibitory and
differentiation stimulating activity of Cytostatin III is useful to
inhibit growth and stimulate differentiation of tumor cells, for
instance in vitro, as for growth of cells for research, industrial
or commercial purposes, for example. The same activities may be
applied to treatment of aberrant cell growth in an organism, such
as growth of cells of a tumor. In these regards, Cytostatin III
polypeptides are preferred, particularly the Cytostatin III having
the amino acid sequence set out in FIG. 1 (SEQ ID NO:2) or the
amino acid sequence of the Cytostatin III of the cDNA of the
deposited clone.
[0210] Similarly, the ability of Cytostatin III to inhibit growth
of Cytostatin III-sensitive cells, such as Cytostatin III-sensitive
endothelial cells, including, for instance, venus endothelial
cells, may be used to prevent, slow or alter cell growth in culture
or in situ.
[0211] Often, tumor cells, such as those at an original tumor and
those at sites of metastasis, must attract new blood vessels to
grow. Cytostatin III may be used to inhibit Cytostatin
III-sensitive cells involved in tumor vascularization, such as
Cytostatin III-sensitive venus endothelial cells for instance, and
may be useful slow tumor growth, or reduce metastatic potential of
tumors or slow progression of metastatic disease.
[0212] Cytostatin III also may be useful to modulate b-adrenergic
activity of certain Cytostatin III-sensitive cells, such as
Cytostatin III-sensitive cardiac myocytes.
[0213] Furthermore, activity of Cytostatin III, such as activity
that modulates mammary gland differentiation or affects the growth
of mammary epithelial cells may be used to promote formation of
alveolar buds, aid development of differentiated lobuloalveoli, and
stimulate the production of milk protein and the accumulation of
fat droplets. Such lactation-stimulating activity may aid milk
production in commercial milk-producing mammals. It also may be
useful to aid milk-production by human mothers, for instance.
[0214] In a related application, modulating activity of Cytostatin
III that affects breast size may be useful to aid return of an
enlarged breast to normal size after parturition. Inhibition of
Cytostatin III activity, for instance, by antisense
phosphorothioates or by antibodies, may be useful for selective
inhibition of endogenous Cytostatin III activity in mammary
epithelial cells to suppress the appearance of alveolar end buds
and to lower the beta-casein level.
[0215] As set out further below, these and other activities and
properties of the Cytostatin III polynucleotides and polypeptides
of the invention have various applications and uses in numerous
fields including applications involving growth of cells in vitro,
commercial production of milk and milk products, and diagnosis and
treatments relating to the fields of oncology, cardiology,
immunology, endocrinology, hematology, metabolic disorders,
musculoskelatal problems and gynecology and obstetrics, to name a
few.
[0216] Cytostatin III Binding Molecules and Assays
[0217] This invention also provides a method for identification of
molecules, such as receptor molecules, that bind Cytostatin III.
Genes encoding proteins that bind Cytostatin III, such as receptor
proteins, can be identified by numerous methods known to those of
skill in the art, for example, ligand panning and FACS sorting.
Such methods are described in many laboratory manuals such as, for
instance, Coligan et al., Current Protocols in Immunology 1(2):
Chapter 5 (1991).
[0218] For instance, expression cloning may be employed for this
purpose. To this end polyadenylated RNA is prepared from a cell
responsive to Cytostatin III, a cDNA library is created from this
RNA, the library is divided into pools and the pools are
transfected individually into cells that are not responsive to
Cytostatin III. The transfected cells then are exposed to labeled
Cytostatin III. (Cytostatin III can be labeled by a variety of
well-known techniques including standard methods of
radio-iodination or inclusion of a recognition site for a
site-specific protein kinase.) Following exposure, the cells are
fixed and binding of cytostatin is determined. These procedures
conveniently are carried out on glass slides.
[0219] Pools are identified of cDNA that produced Cytostatin
III-binding cells. Sub-pools are prepared from these positives,
transfected into host cells and screened as described above. Using
an iterative sub-pooling and re-screening process, one or more
single clones that encode the putative binding molecule, such as a
receptor molecule, can be isolated.
[0220] Alternatively a labeled ligand can be photoaffinity linked
to a cell extract, such as a membrane or a membrane extract,
prepared from cells that express a molecule that it binds, such as
a receptor molecule. Cross-linked material is resolved by
polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray
film. The labeled complex containing the ligand-receptor can be
excised, resolved into peptide fragments, and subjected to protein
microsequencing. The amino acid sequence obtained from
microsequencing can be used to design unique or degenerate
oligonucleotide probes to screen cDNA libraries to identify genes
encoding the putative receptor molecule.
[0221] Polypeptides of the invention also can be used to assess
Cytostatin III binding capacity of Cytostatin III binding
molecules, such as receptor molecules, in cells or in cell-free
preparations.
[0222] Agonists and Antagonists--Assays and Molecules
[0223] The invention also provides a method of screening compounds
to identify those which enhance or block the action of Cytostatin
III on cells, such as its interaction with Cytostatin III-binding
molecules such as receptor molecules. An agonist is a compound
which increases the natural biological functions of Cytostatin III
or which functions in a manner similar to Cytostatin III, while
antagonists decrease or eliminate such functions.
[0224] For example, a cellular compartment, such as a membrane or a
preparation thereof, such as a membrane-preparation, may be
prepared from a cell that expresses a molecule that binds
Cytostatin III, such as a molecule of a signaling or regulatory
pathway modulated by Cytostatin III. The preparation is incubated
with labeled Cytostatin III in the absence or the presence of a
candidate molecule which may be a Cytostatin III agonist or
antagonist. The ability of the candidate molecule to bind the
binding molecule is reflected in decreased binding of the labeled
ligand. Molecules which bind gratuitously, i.e., without inducing
the effects of Cytostatin III on binding the Cytostatin III binding
molecule, are most likely to be good antagonists. Molecules that
bind well and elicit effects that are the same as or closely
[0225] Cytostatin III-like effects of potential agonists and
antagonists may by measured, for instance, by determining activity
of a second messenger system following interaction of the candidate
molecule with a cell or appropriate cell preparation, and comparing
the effect with that of Cytostatin III or molecules that elicit the
same effects as Cytostatin III. Second messenger systems that may
be useful in this regard include but are not limited to AMP
guanylate cyclase, ion channel or phosphoinositide hydrolysis
second messenger systems.
[0226] Another example of an assay for Cytostatin III antagonists
is a competitive assay that combines Cytostatin III and a potential
antagonist with membrane-bound Cytostatin III receptor molecules or
recombinant Cytostatin III receptor molecules under appropriate
conditions for a competitive inhibition assay. Cytostatin III can
be labeled, such as by radioactivity, such that the number of
Cytostatin III molecules bound to a receptor molecule can be
determined accurately to assess the effectiveness of the potential
antagonist.
[0227] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing Cytostatin III-induced
activities, thereby preventing the action of Cytostatin III by
excluding Cytostatin III from binding.
[0228] Potential antagonists include a small molecule which binds
to and occupies the binding site of the polypeptide thereby
preventing binding to cellular binding molecules, such as receptor
molecules, such that normal biological activity is prevented.
Examples of small molecules include but are not limited to small
organic molecules, peptides or peptide-like molecules.
[0229] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed, for example, in--Okano, J. Neurochem. 56:
560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE
EXPRESSION, CRC Press, Boca Raton, FL (1988). Triple helix
formation is discussed in, for instance Lee et al., Nucleic Acids
Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and Dervan et al., Science 251: 1360 (1991). The methods are based
on binding of a polynucleotide to a complementary DNA or RNA. For
example, the 5' coding portion of a polynucleotide that encodes the
mature polypeptide of the present invention may be used to design
an antisense RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is designed to be complementary to
a region of the gene involved in transcription thereby preventing
transcription and the production of Cytostatin III. The antisense
RNA oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into Cytostatin III polypeptide.
The oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of Cytostatin III.
[0230] The antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as hereinafter
described.
[0231] The antagonists may be employed for instance to treat
cardiac myocte hypertrophy or leukemia
[0232] Compositions
[0233] The invention also relates to compositions comprising the
polynucleotide or the polypeptides discussed above or the agonists
or antagonists. Thus, the polypeptides of the present invention may
be employed in combination with a non-sterile or sterile carrier or
carriers for use with cells, tissues or organisms, such as a
pharmaceutical carrier suitable for administration to a subject.
Such compositions comprise, for instance, a media additive or a
therapeutically effective amount of a polypeptide of the invention
and a pharmaceutically acceptable carrier or excipient. Such
carriers may include, but are not limited to, saline, buffered
saline, dextrose, water, glycerol, ethanol and combinations
thereof. The formulation should suit the mode of
administration.
[0234] Kits
[0235] The invention further relates to pharmaceutical packs and
kits comprising one or more containers filled with one or more of
the ingredients of the aforementioned compositions of the
invention. Associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
reflecting approval by the agency of the manufacture, use or sale
of the product for human administration.
[0236] Administration
[0237] Polypeptides of the present invention may be employed alone
or in conjunction with other compounds, such as therapeutic
compounds.
[0238] The pharmaceutical compositions may be administered in any
effective, convenient manner including, for instance,
administration by topical, oral, anal, vaginal, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes among others.
[0239] The pharmaceutical compositions generally are administered
in an amount effective for treatment or prophylaxis of a specific
indication or indications. In general, the compositions are
administered in an amount of at least about 10 mg/kg body weight.
In most cases they will be administered in an amount not in excess
of about 8 mg/kg body weight per day. Preferably, in most cases,
dose is from about 10 mg/kg to about 1 mg/kg body weight, daily. It
will be appreciated that optimum dosage will be determined by
standard methods for each treatment modality and indication, taking
into account the indication, its severity, route of administration,
complicating conditions and the like.
[0240] Gene Therapy
[0241] The Cytostatin III polynucleotides, polypeptides, agonists
and antagonists that are polypeptides may be employed in accordance
with the present invention by expression of such polypeptides in
vivo, in treatment modalities often referred to as "gene
therapy."
[0242] Thus, for example, cells from a patient may be engineered
with a polynucleotide, such as a DNA or RNA, encoding a polypeptide
ex vivo, and the engineered cells then can be provided to a patient
to be treated with the polypeptide. For example, cells may be
engineered ex vivo by the use of a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention.
Such methods are well-known in the art and their use in the present
invention will be apparent from the teachings herein.
[0243] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by procedures known in the art. For example,
a polynucleotide of the invention may be engineered for expression
in a replication defective retroviral vector, as discussed above.
The retroviral expression construct then may be isolated and
introduced into a packaging cell is transduced with a retroviral
plasmid vector containing RNA encoding a polypeptide of the present
invention such that the packaging cell now produces infectious
viral particles containing the gene of interest. These producer
cells may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention.
[0244] Retroviruses from which the retroviral plasmid vectors
herein above mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0245] Such vectors well include one or more promoters for
expressing the polypeptide. Suitable promoters which may be
employed include, but are not limited to, the retroviral LTR; the
SV40 promoter; and the human cytomegalovirus (CMV) promoter
described in Miller et al., Biotechniques 7: 980-990 (1989), or any
other promoter (e.g., cellular promoters such as eukaryotic
cellular promoters including, but not limited to, the histone, RNA
polymerase III, and 13-actin promoters). Other viral promoters
which may be employed include, but are not limited to, adenovirus
promoters, thymidine kinase (TK) promoters, and B19 parvovirus
promoters. The selection of a suitable promoter will be apparent to
those skilled in the art from the teachings contained herein.
[0246] The nucleic acid sequence encoding the polypeptide of the
present invention will be placed under the control of a suitable
promoter. Suitable promoters which may be employed include, but are
not limited to, adenoviral promoters, such as the adenoviral major
late promoter; or heterologous promoters, such as the
cytomegalovirus (CMV) promoter; the respiratory syncytial virus
(RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs (including the modified retroviral
LTRs herein above described); the .beta.-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter which controls the gene encoding the polypeptide.
[0247] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X,
VT-19-17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, A., Human Gene Therapy 1: 5-14 (1990). The
vector may be transduced into the packaging cells through any means
known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO4 precipitation. In
one alternative, the retroviral plasmid vector may be encapsulated
into a liposome, or coupled to a lipid, and then administered to a
host.
[0248] The producer cell line will generate infectious retroviral
vector particles, which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
EXAMPLES
[0249] The present invention is further described by the following
examples. The examples are provided solely to illustrate the
invention by reference to specific embodiments. These
exemplification's, while illustrating certain specific aspects of
the invention, do not portray the limitations or circumscribe the
scope of the disclosed invention.
[0250] Certain terms used herein are explained in the foregoing
glossary.
[0251] All examples were carried out using standard techniques,
which are well known and routine to those of skill in the art,
except where otherwise described in detail. Routine molecular
biology techniques of the following examples can be carried out as
described in standard laboratory manuals, such as Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), herein referred
to as "Sambrook."
[0252] All parts or amounts set out in the following examples are
by weight, unless otherwise specified.
[0253] Unless otherwise stated size separation of fragments in the
examples below was carried out using standard techniques of agarose
and polyacrylamide gel electrophoresis ("PAGE") in Sambrook and
numerous other references such as, for instance, by Goeddel et al.,
Nucleic Acids Res. 8: 4057 (1980).
[0254] Unless described otherwise, ligations were accomplished
using standard buffers, incubation temperatures and times,
approximately equimolar amounts of the DNA fragments to be ligated
and approximately 10 units of T4 DNA ligase ("ligase") per 0.5 mg
of DNA.
Example 1
Expression and Purification of human Cytostatin III using
Bacteria
[0255] The DNA sequence encoding human Cytostatin III in the
deposited polynucleotide was amplified using PCR oligonucleotide
primers specific to the amino acid carboxyl terminal sequence of
the human Cytostatin III protein and to vector sequences 3' to the
gene. Additional nucleotides containing restriction sites to
facilitate cloning were added to the 5' and 3' sequences
respectively.
[0256] The 5' oligonucleotide primer had the sequence 5'CGC GCA TGC
CTC CCA ACC TCA C 3'" (SEQ ID NO:3) containing the underlined SphI
restriction site, which encodes a start AUG, followed by 13
nucleotides of the human Cytostatin III coding sequence set out in
FIG. 1 (SEQ ID NO:1) beginning with the second base of the second
codon.
[0257] The 3' primer had the sequence 5' GCG AAG CTT CTA TCT GAC
CTT CCT G 3' (SEQ ID NO:4) containing the underlined Hind III
restriction site followed by 16 nucleotides complementary to the
last 16 nucleotides of the Cytostatin III coding sequence set out
in FIG. 1 (SEQ ID NO:1), including the stop codon.
[0258] The restrictions sites were convenient to restriction enzyme
sites in the bacterial expression vectors pQE-7, which were used
for bacterial expression in these examples. (Qiagen, Inc. 9259 Eton
Avenue, Chatsworth, Calif., 91311). pQE-7 encodes ampicillin
antibiotic resistance ("Ampr") and contains a bacterial origin of
replication ("ori"), an IPTG inducible promoter, a ribosome binding
site ("RBS"), a 6-His tag and restriction enzyme sites.
[0259] The amplified human Cytostatin III DNA and the vector pQE-7
both were digested with SphI and HindIII, and the digested DNAs
then were ligated together. Insertion of the Cytostatin III DNA
into the SphI/HindIII restricted vector placed the Cytostatin III
coding region downstream of and operably linked to the vector's
IPTG-inducible promoter and in-frame with an initiating AUG
appropriately positioned for translation of Cytostatin III.
[0260] The ligation mixture was transformed into competent E. coli
cells using standard procedures. Such procedures are described in
Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses lac repressor and confers kanamycin
resistance ("Kanr"), was used in carrying out the illustrative
example described here. This strain, which is only one of many that
are suitable for expressing Cytostatin III, is available
commercially from Qiagen.
[0261] Transformants were identified by their ability to grow on LB
plates in the presence of ampicillin. Plasmid DNA was isolated from
resistant colonies and the identity of the cloned DNA was confirmed
by restriction analysis.
[0262] Clones containing the desired constructs were grown
overnight ("O/N") in liquid culture in LB media supplemented with
both ampicillin (100 ug/ml) and kanamycin (25 ug/ml).
[0263] The O/N culture was used to inoculate a large culture, at a
dilution of approximately 1:100 to 1:250. The cells were grown to
an optical density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalactopyranoside ("IPTG") was then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently were incubated further for 3 to 4 hours. Cells
then were harvested by centrifugation and disrupted, by standard
methods. Inclusion bodies were purified from the disrupted cells
using routine collection techniques, and protein was solubilized
from the inclusion bodies into 8M urea. The 8M urea solution
containing the solubilized protein was passed over a PD-10 column
in 2.times. phosphate buffered saline ("PBS"), thereby removing the
urea, exchanging the buffer and refolding the protein. The protein
was purified by a further step of chromatography to remove
endotoxin. Then, it was sterile filtered. The sterile filtered
protein preparation was stored in 2.times. PBS at a concentration
of 95 micrograms per mL.
[0264] Analysis of the preparation by standard methods of
polyacrylamide gel electrophoresis revealed that the preparation
contained about 80% monomer Cytostatin III having the expected
molecular weight of, approximately, 14 kDa.
Example 2
Cloning and Expression of Human Cytostatin III in a Baculovirus
Expression System
[0265] The cDNA sequence encoding the full length human Cytostatin
III protein, in the deposited clone is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of
the gene:
[0266] The 5' primer has the sequence 5' GC GGA TCC TCC CAA CCT CAC
TGG CTA C 3' (SEQ ID NO:5) containing the underlined BamH1
restriction enzyme site followed by 19 bases of the sequence of
Cytostatin III of FIG. 1 (SEQ ID NO:1). Inserted into an expression
vector, as described below, the 5' end of the amplified fragment
encoding human Cytostatin III provides an efficient signal peptide.
An efficient signal for initiation of translation in eukaryotic
cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987)
is appropriately located in the vector portion of the
construct.
[0267] The 3' primer has the sequence 5' GC GGT ACC CTA TCT GAC CTT
CCT G 3' (SEQ ID NO:6) containing the Asp718 restriction followed
by nucleotides complementary to the last 16 nucleotides of the
Cytostatin III coding sequence set out in FIG. 1 (SEQ ID NO:1),
including the stop codon.
[0268] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with BamH1 and Asp718
and again is purified on a 1% agarose gel. This fragment is
designated herein F2.
[0269] The vector pA2-GP is used to express the Cytostatin III
protein in the baculovirus expression system, using standard
methods, such as those described in Summers et al, A MANUAL OF
METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES,
Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
This expression vector contains the strong polyhedrin promoter of
the Autographa californica nuclear polyhedrosis virus (AcMNPV)
followed by convenient restriction sites. The signal peptide of
AcMNPV gp67, including the N-terminal methionine, is located just
upstream of a BamH1 site. The polyadenylation site of the simian
virus 40 ("SV40") is used for efficient polyadenylation. For an
easy selection of recombinant virus the beta-galactosidase gene
from E.coli is inserted in the same orientation as the polyhedrin
promoter and is followed by the polyadenylation signal of the
polyhedrin gene. The polyhedrin sequences are flanked at both sides
by viral sequences for cell-mediated homologous recombination with
wild-type viral DNA to generate viable virus that express the
cloned polynucleotide.
[0270] Many other baculovirus vectors could be used in place of
pA2-GP, such as pAc373, pVL941 and pAcIM1 provided, as those of
skill readily will appreciate, that construction provides
appropriately located signals for transcription, translation,
trafficking and the like, such as an in-frame AUG and a signal
peptide, as required. Such vectors are described in Luckow et al.,
Virology 170: 31-39, among others.
[0271] The plasmid is digested with the restriction enzymes BamH1
and Asp718 and then is dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA
is designated herein "V2".
[0272] Fragment F2 and the dephosphorylated plasmid V2 are ligated
together with T4 DNA ligase. E.coli HB101 cells are transformed
with ligation mix and spread on culture plates. Bacteria are
identified that contain the plasmid with the human Cytostatin III
gene by digesting DNA from individual colonies using BamH1 and
Asp718 and then analyzing the digestion product by gel
electrophoresis. The sequence of the cloned fragment is confirmed
by DNA sequencing. This plasmid is designated herein pBacCytostatin
III.
[0273] 1 mg of the plasmid pBacCytostatin III is co-transfected
with 1.0 mg of a commercially available linearized baculovirus DNA
("BaculoGoldO baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofection method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84: 7413-7417 (1987). 1 mg of BaculoGold virus
DNA and 5 mg of the plasmid pBacCytostatin III are mixed in a
sterile well of a microtiter plate containing 50 ml of serum free
Grace's medium (Life Technologies Inc., Gaithersburg, Md.).
Afterwards 10 ml Lipofectin plus 90 ml Grace's medium are added,
mixed and incubated for 15 minutes at room temperature. Then the
transfection mixture is added drop-wise to Sf9 insect cells (ATCC
CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's
medium without serum. The plate is rocked back and forth to mix the
newly added solution. The plate is then incubated for 5 hours at
27.degree. C. After 5 hours the transfection solution is removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. The plate is put back into an
incubator and cultivation is continued at 27.degree. C. for four
days.
[0274] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, cited above.
An agarose gel with "Blue Gal" (Life Technologies Inc.,
Gaithersburg) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0275] Four days after serial dilution, the virus is added to the
cells. After appropriate incubation, blue stained plaques are
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses is then resuspended in an Eppendorf tube
containing 200 ml of Grace's medium. The agar is removed by a brief
centrifugation and the supernatant containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4.degree. C. A clone
containing properly inserted Cytostatin III is identified by DNA
analysis including restriction mapping and sequencing. This is
designated herein as V-Cytostatin III.
[0276] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-Cytostatin III at a multiplicity of infection ("MOI")
of about 2 (about 1 to about 3). Six hours later the medium is
removed and is replaced with SF900 II medium minus methionine and
cysteine (available from Life Technologies Inc., Gaithersburg). 42
hours later, 5 mCi of 35S-methionine and 5 mCi 35S cysteine
(available from Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by
centrifugation, lysed and the labeled proteins are visualized by
SDS-PAGE and autoradiography. Active proteins are then produced by
dialysis with PBS.
Example 3
Expression of Cytostatin III in COS Cells
[0277] The expression plasmid, Cytostatin III HA, is made by
cloning a cDNA encoding Cytostatin III into the expression vector
pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
[0278] The expression vector pcDNAI/amp contains: (1) an E.coli
origin of replication effective for propagation in E. coli and
other prokaryotic cell; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron, and a polyadenylation
signal arranged so that a cDNA conveniently can be placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
[0279] A DNA fragment encoding the entire Cytostatin III precursor
and a HA tag fused in frame to its 3' end is cloned into the
polylinker region of the vector so that recombinant protein
expression is directed by the CMV promoter. The HA tag corresponds
to an epitope derived from the influenza hemagglutinin protein
described by Wilson et al., Cell 37: 767 (1984). The fusion of the
HA tag to the target protein allows easy detection of the
recombinant protein with an antibody that recognizes the HA
epitope.
[0280] The plasmid construction strategy is as follows.
[0281] The Cytostatin III cDNA of the deposit clone of the
deposited clone is amplified using primers that contained
convenient restriction sites, much as described above regarding the
construction of expression vectors for expression of Cytostatin III
in E. coli and S. fugiperda.
[0282] To facilitate detection, purification and characterization
of the expressed Cytostatin III, one of the primers contains a
hemagglutinin tag ("HA tag") as described above.
[0283] Suitable primers include that following, which are used in
this example.
[0284] The 5' primer, containing the underlined BamHI site, an AUG
start codon and 5 codons thereafter, forming the hexapeptide
haemaglutinin tag, has the following sequence: 5' GC GGA TCC ACC
ATG CCT CCC AAC CTC ACT 3' (SEQ ID NO:7).
[0285] The 3' primer, containing the underlined XbaI site and 15 bp
of 3' coding sequence (at the 3' end) has the following sequence:
5' GC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA TCT GAC CTT
CCT GAA 3' (SEQ ID NO:8).
[0286] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with and then ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037) the transformed culture is plated on ampicillin media plates
which then are incubated to allow growth of ampicillin resistant
colonies. Plasmid DNA is isolated from resistant colonies and
examined by restriction analysis and gel sizing for the presence of
the Cytostatin III-encoding fragment.
[0287] For expression of recombinant Cytostatin III, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989).
[0288] Cells are incubated under conditions for expression of
Cytostatin III by the vector.
[0289] Expression of the Cytostatin III HA fusion protein is
detected by radiolabelling and immunoprecipitation, using methods
described in, for example Harlow et al., ANTIBODIES: A LABORATORY
MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York (1988). To this end, two days after transfection,
the cells are labeled by incubation in media containing
35S-cysteine for 8 hours. The cells and the media are collected,
and the cells are washed and the lysed with detergent-containing
RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC,
50 mM TRIS, pH 7.5, as described by Wilson et al. cited above.
Proteins are precipitated from the cell lysate and from the culture
media using an HA-specific monoclonal antibody. The precipitated
proteins then are analyzed by SDS-PAGE gels and autoradiography. An
expression product of the expected size is seen in the cell lysate,
which is not seen in negative controls.
Example 4
Tissue Distribution of Cytostatin III Expression
[0290] Northern blot analysis is carried out to examine the levels
of expression of Cytostatin III in human tissues, using methods
described by, among others, Sambrook et al, cited above. Total
cellular RNA samples are isolated with RNAzol B system (Biotecx
Laboratories, Inc. 6023 South Loop East, Houston, Tex. 77033).
[0291] About 10 mg of Total RNA is isolated from tissue samples.
The RNA is size resolved by electrophoresis through a 1% agarose
gel under strongly denaturing conditions. RNA is blotted from the
gel onto a nylon filter, and the filter then is prepared for
hybridization to a detectably labeled polynucleotide probe.
[0292] As a probe to detect mRNA that encodes Cytostatin III, the
antisense strand of the coding region of the cDNA insert in the
deposited clone is labeled to a high specific activity. The cDNA is
labeled by primer extension, using the Prime-It kit, available from
Stratagene. The reaction is carried out using 50 ng of the cDNA,
following the standard reaction protocol as recommended by the
supplier. The labeled polynucleotide is purified away from other
labeled reaction components by column chromatography using a
Select-G-50 column, obtained from 5-Prime -3-Prime, Inc. of 5603
Arapahoe Road, Boulder, Colo. 80303.
[0293] The labeled probe is hybridized to the filter, at a
concentration of 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5
M NaPO4, pH 7.4 at 65.degree. C., overnight.
[0294] Thereafter the probe solution is drained and the filter is
washed twice at room temperature and twice at 60.degree. C. with
0.5.times. SSC, 0.1% SDS. The filter then is dried and exposed to
film at -70.degree. C. overnight with an intensifying screen.
[0295] Autoradiography shows that mRNA for Cytostatin III is
abundant in breast lymph node cells.
Example 5
Gene Therapeutic Expression of Human Cytostatin III
[0296] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature overnight. After 24 hours at room
temperature, the flask is inverted--the chunks of tissue remain
fixed to the bottom of the flask--and fresh media is added (e.g.,
Ham's F12 media, with 10% FBS, penicillin and streptomycin). The
tissue is then incubated at 37.degree. C. for approximately one
week. At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerges. The monolayer is trypsinized and
scaled into larger flasks.
[0297] A vector for gene therapy is digested with restriction
enzymes for cloning a fragment to be expressed. The digested vector
is treated with calf intestinal phosphatase to prevent
self-ligation. The dephosphorylated, linear vector is fractionated
on an agarose gel and purified.
[0298] Cytostatin cDNA capable of expressing active Cytostatin III,
is isolated. The ends of the fragment are modified, if necessary,
for cloning into the vector. For instance, 5" overhanging may be
treated with DNA polymerase to create blunt ends. 3' overhanging
ends may be removed using S1 nuclease. Linkers may be ligated to
blunt ends with T4 DNA ligase.
[0299] Equal quantities of the Moloney murine leukemia virus linear
backbone and the Cytostatin III fragment are mixed together and
joined using T4 DNA ligase. The ligation mixture is used to
transform E. Coli and the bacteria are then plated onto
agar-containing kanamycin. Kanamycin phenotype and restriction
analysis confirm that the vector has the properly inserted
gene.
[0300] Packaging cells are grown in tissue culture to confluent
density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf
serum (CS), penicillin and streptomycin. The vector containing the
Cytostatin III gene is introduced into the packaging cells by
standard techniques. Infectious viral particles containing the
Cytostatin III gene are collected from the packaging cells, which
now are called producer cells.
[0301] Fresh media is added to the producer cells, and after an
appropriate incubation period media is harvested from the plates of
confluent producer cells. The media, containing the infectious
viral particles, is filtered through a Millipore filter to remove
detached producer cells. The filtered media then is used to infect
fibroblast cells. Media is removed from a sub-confluent plate of
fibroblasts and quickly replaced with the filtered media. Polybrene
(Aldrich) may be included in the media to facilitate transduction.
After appropriate incubation, the media is removed and replaced
with fresh media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is low, then it is necessary to use a retroviral vector that
has a selectable marker, such as neo or his, to select out
transduced cells for expansion.
[0302] Engineered fibroblasts then may be injected into rats,
either alone or after having been grown to confluence on
microcarrier beads, such as cytodex 3 beads. The injected
fibroblasts produce Cytostatin III product, and the biological
actions of the protein are conveyed to the host.
[0303] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
Example 6
[0304] Expression of Recombinant Cytostatin III in CHO Cells
[0305] The vector pC1 is used for the expression of Cytostatin III
protein. Plasmid pC1 is a derivative of the plasmid pSV2-dhfr [ATCC
Accession No. 37146]. Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary- or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Lift Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J.
R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin,
J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenham, M .A. 1991, Biotechnology Vol. 9:64-68).
Cells grown in increasing concentrations of MTX develop resistance
to the drug by overproducing the target enzyme, DHFR, as a result
of amplification of the DHFR gene. If a second gene is linked to
the DHFR gene it is usually co-amplified and over-expressed. It is
state of the art to develop cell lines carrying more than 1,000
copies of the genes. Subsequently, when the methotrexate is
withdrawn, cell lines contain the amplified gene integrated into
the chromosome(s).
[0306] Plasmid pC1 contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular
Biology, March 1985, 438-4470) plus a fragment isolated from the
enhancer of the immediate early gene of human cytomegalovirus (CMV)
(Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter
are the following single restriction enzyme cleavage sites that
allow the integration of the genes: BamHI, Pvull, and Nrul. Behind
these cloning sites the plasmid contains translational stop codons
in all three reading frames followed by the 3' intron and the
polyadenylation site of the rat preproinsulin gene. Other high
efficient promoters can also be used for the expression, e.g., the
human .beta.-actin promoter, the SV40 early or late promoters or
the long terminal repeats from other retroviruses, e.g., HIV and
HTLVI. For the polyadenylation of the mRNA other signals, e.g.,
from the human growth hormone or globin genes can be used as
well.
[0307] Stable cell lines carrying a gene of interest integrated
into the chromosome can also be selected upon co-transfection with
a selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g. G418 plus methotrexate.
[0308] The plasmid pC1 is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphatase
by procedures known in the art. The vector is then isolated from a
1% agarose gel.
[0309] The DNA sequence encoding cytostatin III, ATCC #97332, is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene:
[0310] The 5' primer has the sequence 5' GC GGA TCC TCC CAA CCT CAC
TGG CTA C 3' (SEQ ID NO:9) containing the underlined BamH1
restriction enzyme site followed by 19 bases of the sequence of
Cytostatin III of FIG. 1 (SEQ ID NO:1). Inserted into an expression
vector, as described below, the 5' end of the amplified fragment
encoding human Cytostatin III provides an efficient signal peptide.
An efficient signal for initiation of translation in eukaryotic
cells, as described by Kozak, M., J. Mol. Biol. 196: 947-950 (1987)
is appropriately located in the vector portion of the
construct.
[0311] The 3' primer has the sequence 5' GC GGT ACC CTA TCT GAC CTT
CCT G 3' (SEQ ID NO:10) containing the Asp718 restriction followed
by nucleotides complementary to the last 16 nucleotides of the
Cytostatin III coding sequence set out in FIG. 1 (SEQ ID NO:1),
including the stop codon.
[0312] The amplified fragments are isolated from a 1% agarose gel
as described above and then digested with the endonuclease BamHI
and then purified again on a 1% agarose gel. The isolated fragment
and the dephosphorylated vector are then ligated with T4 DNA
ligase. E.coli HB101 cells are then transformed and bacteria
identified that contained the plasmid pC1 inserted in the correct
orientation using the restriction enzyme BamHI. The sequence of the
inserted gene is confirmed by DNA sequencing.
[0313] Transfection of CHO-DHFR-Cells
[0314] Chinese hamster ovary cells lacking an active DHFR enzyme
are used for transfection. 5 mg of the expression plasmid C1 are
cotransfected with 0.5 mg of the plasmid pSVneo using the
lipofectin method (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the gene neo from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) and cultivated from 10-14 days. After this period, single
clones are trypsinized and then seeded in 6-well petri dishes using
different concentrations of methotrexate (25, 50 nm, 100 nm, 200
nm, 400 nm). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing
even higher concentrations of methotrexate (500 nM, 1 mM, 2 mM, 5
mM). The same procedure is repeated until clones grow at a
concentration of 100 mM.
[0315] The expression of the desired gene product is analyzed by
Western blot analysis and SDS-PAGE.
[0316] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
Sequence CWU 1
1
* * * * *