U.S. patent application number 09/291144 was filed with the patent office on 2002-08-01 for methods for use for integrin b1c cell growth inhibitor.
Invention is credited to LANGUINO, LUCIA, MEREDITH, JERE E. JR., SCHWARTZ, MARTIN A., TAKADA, YOSHIKAZU.
Application Number | 20020102240 09/291144 |
Document ID | / |
Family ID | 23275233 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102240 |
Kind Code |
A1 |
SCHWARTZ, MARTIN A. ; et
al. |
August 1, 2002 |
METHODS FOR USE FOR INTEGRIN B1C CELL GROWTH INHIBITOR
Abstract
The present invention provides a method for inhibiting cell
proliferation in a cell comprising contacting the cell with a
nucleic acid sequence or a polypeptide having essentially the
sequence of the .beta..sub.1C integrin. Also included in the
invention are peptides consisting of amino acid residues which are
the size of or fewer than the sequence of the cytoplasmic domain of
the .beta..sub.1C integrin consisting essentially of the amino acid
sequence of SEQ ID NO:1 and SEQ ID NO:2, and functional fragments
thereof which are useful for inhibiting cellular proliferation.
Peptides, polynucleotides, and antibodies immunoreactive with the
peptides, and methods of use for inhibiting cell growth are also
provided.
Inventors: |
SCHWARTZ, MARTIN A.; (POWAY,
CA) ; MEREDITH, JERE E. JR.; (SAN DIEGO, CA) ;
TAKADA, YOSHIKAZU; (SAN DIEGO, CA) ; LANGUINO,
LUCIA; (MILFORD, CT) |
Correspondence
Address: |
FISH & RICHARDSON, PC
4350 LA JOLLA VILLAGE DRIVE
SUITE 500
SAN DIEGO
CA
92122
US
|
Family ID: |
23275233 |
Appl. No.: |
09/291144 |
Filed: |
April 12, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09291144 |
Apr 12, 1999 |
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08951200 |
Oct 14, 1997 |
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08951200 |
Oct 14, 1997 |
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08327118 |
Oct 21, 1994 |
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Current U.S.
Class: |
424/93.21 ;
435/320.1; 435/325; 435/455; 435/69.1; 514/44R |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/7055 20130101 |
Class at
Publication: |
424/93.21 ;
514/44; 435/455; 435/320.1; 435/325; 435/69.1 |
International
Class: |
A61K 048/00; C12N
015/00; C12P 021/02; C12N 005/08 |
Claims
What is claimed:
1. A method for treating a cellular proliferative disorder in a
subject, comprising administering to the subject with the disorder
a therapeutically effective amount of .beta..sub.1C integrin
reagent which inhibits cellular proliferation.
2. The method of claim 1, wherein the .beta..sub.1C integrin
reagent comprises a nucleic acid sequence encoding .beta..sub.1C
integrin.
3. The method of claim 1, wherein the .beta..sub.1C integrin
reagent comprises a polypeptide having an amino acid sequence of
.beta..sub.1C integrin.
4. The method of claim 1, wherein the reagent is an amino acid
sequence comprising of SEQ ID NO:1.
5. The method of claim 1, wherein the reagent is a nucleic acid
sequence comprising of a nucleic acid encoding SEQ ID NO:1.
6. The method of claim 1, wherein the reagent is an anti-idiotype
antibody which binds to a paratope of an antibody which binds to
the amino acid sequence of SEQ ID NO:1.
7. The method of claim 1, wherein the reagent is a peptide as set
forth in SEQ ID NO:2.
8. The method of claim 1, wherein the reagent is a nucleic acid
sequence encoding SEQ ID NO:2.
9. The method of claim 1, wherein the reagent is an anti-idiotype
antibody which binds to a paratope of an antibody which binds to
the amino acid sequence of SEQ ID NO:2.
10. The method of claim 1, wherein the cell proliferative disorder
is a hematopoietic cell disorder.
11. A method of inhibiting cell proliferation in a cell comprising
contacting the cell with a nucleic acid encoding integrin
.beta..sub.1C, SEQ ID NO: 1 or SEQ ID NO:2, or encoding functional
fragments thereof.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to the field of
cell-surface glycoproteins and specifically to the use of integrin
.beta..sub.1C and cytoplasmic peptides thereof, for inhibition of
cell growth.
[0003] 2. Description of Related Art
[0004] Integrins are a family of cell surface glycoproteins which
mediate cell-cell and cell-extracellular matrix interactions and
play an important role in processes such as cell migration, tissue
repair and tumor invasion (Hynes, R., Cell 48:549, 1987; Ruoslahti
and Giancotti, Cancer Cells 1:119,989). They are heterodimers
composed of non-covalently linked .alpha. and .beta. subunits which
associate in different combinations generating several receptor
complexes with distinct binding specificities.
[0005] The .beta.1 class of integrins is found in various
combinations to form integrins with different functions. For
example, the .alpha.1/.beta.1 and the .alpha.2/.beta.1 are
receptors for collagens; the .alpha.3/.beta.1 has broad specificity
and binds to collagen, fibronectin and laminin; the
.alpha.4/.beta.1 is a receptor that mediates lymphocytes-target
adhesion during cytolysis as well as the interaction of lymphocytes
with endothelial cells; the .alpha.5/.beta.1 and the
.alpha.6/.beta.1 complexes are receptors specific for fibronectin
and laminin, respectively. Both .alpha. and .beta. subunits are
transmembrane proteins that provide a linkage between the
extracellular matrix protein and actin, and are located in
specialized areas of the plasma membrane called focal contacts
(Damsky, et al., J. Cell Biol., 100: 1528, 1985).
[0006] The cytoplasmic domains of the integrins play an important
role in integrin functions. First, recent studies have shown that
the cytoplasmic domain of the .beta.2 subunit modulates the
affinity of the .alpha.L.beta.3 integrin (LFA-1) for its ligand,
ICAM-1 (Hibbs, et al., Science, 251:1611, 1991). Second, tyrosine
phosphorylation of the .beta.1 subunit cytoplasmic domain has been
found to reduce the binding of the fibronectin receptor to
fibronectin extracellularly and to talin inside the cell (Tapley,
et al., Oncogene 4:325, 1989). Third, truncation of the .beta.1
subunit cytoplasmic domain can abolish the ability of the .beta.1
integrins to localize in adhesion plaques (Marcantonio, et al.,
Cell Reg., 1:597, 1990).
[0007] Recently, several cytoplasmic terminal variants for the
human .beta.1 integrin (.beta..sub.1A) (Argraves, et al., J. Cell
Biol., 105:1183, 1987) have been identified. One such variant,
.beta..sub.1B described by Altruda, et al., (Gene, 95:261, 1990),
is 9 amino acids shorter than the corresponding domain of .beta.1
and contains unique C-terminal sequences. A second variant,
.beta..sub.1C, was identified as an alternative splice variant of
.beta.1 that contains an insert of 116 nucleotides which produces a
frame shift in the native .beta.1 nucleotide sequence and codes for
a unique 48-amino acid C-terminus (Languino and Ruoslahti, J. Biol.
Chem., 267:7116, 1992).
[0008] Although various .beta.1 integrin variants have been
identified, the function for these polypeptides has been unclear.
The present invention provides a biological activity for the
.beta..sub.1C variant, including methods of use for the full length
polypeptide as well as functional cytoplasmic fragments of the
polypeptide.
SUMMARY OF THE INVENTION
[0009] The present invention is based on the seminal discovery of
the biological activity of .beta..sub.1C integrin as a cell growth
inhibitor. .beta..sub.1C is an alternative splice variant of the
.beta..sub.1 integrin and differs from .beta..sub.1 by including a
unique cytoplasmic region. In contrast to native .beta..sub.1, the
cytoplasmic domain of .beta..sub.1C imparts a cell proliferative
inhibitor activity on the polypeptide.
[0010] Thus, in one embodiment, the invention provides a method for
treating a cellular proliferative disorder in a subject, comprising
administering to the subject with the disorder a therapeutically
effective amount of .beta..sub.1C integrin reagent which inhibits
cellular proliferation. A .beta..sub.1C integrin reagent includes
polynucleotide or polypeptide sequences encoding .beta..sub.1C
integrin and cytoplasmic fragments thereof. In addition, a
.beta..sub.1C reagent as used herein includes anti-idiotype
antibodies which bind to a paratope of an antibody which binds to
the amino acid sequence of the peptide of SEQ ID NO:1.
[0011] In another embodiment, the invention provides a method of
inhibiting cell proliferation in a cell comprising contacting the
cell with a nucleic acid having essentially the nucleic acid
sequence encoding integrin .beta..sub.1C, SEQ ID NO:1 or SEQ ID
NO:2, or functional fragments thereof and a method of inhibiting
cell proliferation in a cell comprising contacting the cell with a
polypeptide having essentially the amino acid sequence of integrin
.beta..sub.1C, SEQ ID NO:1 or SEQ ID NO:2, or functional fragments
thereof.
[0012] The invention also includes isolated peptides consisting
essentially of the unique 48 amino acids of pIc (SEQ ID NO:1) and
the last 14 C-terminal amino acids of .beta..sub.1C (SEQ ID NO:2),
and polynucleotide sequences encoding the peptides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1A shows a comparison of the amino acid sequence of the
C-termini of .beta..sub.1 and .beta..sub.1C integrins. (numbers
shown indicate the amino acid number-/nucleotide numbers).
Sequences are shown NH.sub.2 to COOH and 5' to 3'.
[0014] FIG. 1B shows a comparison of the nucleotide and amino acid
sequence of the C-terminii of .beta..sub.1 and .beta..sub.1C
integrins. The asterisk indicates the alternatively spliced exon.
The 116-bp insert in .beta..sub.1C produces a frame shift in the
3'end of the .beta..sub.1 sequence. Sequences are shown NH.sub.2 to
COOH and 5' to 3'.
[0015] FIG. 2 shows a vector map for pBJ-1 plasmid vector.
[0016] FIG. 3 shows immunostaining for expression and localization
of .beta..sub.1 and .beta..sub.1C. Cells were injected with cDNAs
coding for integrin .beta..sub.1 (Panel A and B) or .beta..sub.1C
(C and D). After 24 hours, cells were fixed and stained with a
rabbit polyclonal antibody against human .beta..sub.1 (A and C) or
a mouse monoclonal against vinculin (B and D). The same cell is
shown in A and B and in C and D.
[0017] FIG. 4 shows the effect of .beta..sub.1C on DNA synthesis.
Cells were injected with cDNA coding for wild type .beta..sub.1,
.beta..sub.1 with a truncated cytoplasmic domain, or .beta..sub.1C.
10% serum and BdrU were added and after 24 hr, cells were fixed and
labeled to detect incorporation of BdrU into nuclei.
[0018] FIG. 5 shows the reversal of cell growth inhibition by
antibody. Cells were injected with wild type .beta..sub.1 or
.beta..sub.1S cDNA. Two hours later cells were injected with IgG
against .beta..sub.1C.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a method of use for the
alternative splice variant of integrin .beta..sub.1 subunit,
.beta..sub.1C. For the first time, the present invention shows that
this splice variant, which contains 48 unique amino acids in the
cytoplasmic domain of the wild type .beta..sub.1 integrin, is
involved in the regulation of proliferation in a cell, and
specifically, the .beta..sub.1C polypeptide inhibits DNA synthesis.
Based on this discovery, synthetic peptides from the unique
cytoplasmic region of the polypeptide can be used to inhibit DNA
synthesis in a cell. The discovery of the functional properties of
the .beta..sub.1C integrin has led to the development of novel
methods and compositions for inhibiting cellular proliferation.
[0020] As used herein, the term "synthetic peptide" denotes a
peptide which does not comprise an entire naturally occurring
protein molecule. These peptides are "synthetic" in that they may
be produced by human intervention using such techniques as chemical
synthesis, recombinant genetic techniques, or fragmentation of
whole receptor or the like.
[0021] The peptides of the invention range in length from about 10
to about 100 amino acids and include amino acid sequences which
correspond to amino acid residues 778-825 of the .beta..sub.1C
subunit, or SLSVAQPGVQWCDISSLQPLTSRFQQFSCLSLPSTWDYRVKILFIRVP (SEQ
ID NO:1) or amino acid 812-825 of the .beta..sub.1C subunit, or
TWDYRVKILFIRVP (SEQ ID NO:2). These sequences represent surface
oriented peptides of the cytoplasmic domain of the .beta..sub.1C
subunit. Therefore, the peptide of SEQ ID NO:1 contains a critical
domain (SEQ ID NO:2) for the inhibition of DNA synthesis and cell
proliferation. A deletion mutant of .beta..sub.1C integrin
polypeptide lacking SEQ ID NO:2, is unable to inhibit DNA synthesis
in a cell.
[0022] The peptides of the invention include "functional fragments"
from the cytoplasmic domain of .beta..sub.1C subunit, as long as
the DNA synthesis inhibitory activity of the peptide, as noted
above, remains. Smaller peptides containing the biological activity
of SEQ ID NO:1 are included in the invention. For example, SEQ ID
NO:2 is one such peptide. Other peptides can be readily identified
by those of skill using the routine screening methods described
herein without resorting to undue experimentation. For example, the
peptides can be assayed by standard DNA synthesis assays, such as
labeled 5-bromodeoxyuridine (BdrU) incorporation or incorporation
of radiolabeled tritiated thymidine to determine whether the
peptide of interest contains the DNA synthesis inhibition activity
of .beta..sub.1C. Other DNA synthesis assays will be known to those
of skill in the art. These peptides can be as few as 5, preferably
as few as 10 amino acids in length.
[0023] Minor modifications of the primary amino acid sequence of
the .beta..sub.1C polypeptide or peptides of the invention may
result in peptides which have substantially equivalent activity as
compared to the specific peptides described herein. Such
modifications may be deliberate, as by site-directed mutagenesis,
or may be spontaneous. All of the peptides produced by these
modifications are included herein as long as the biological
activity of the original peptide still exists.
[0024] Further, deletion of one or more amino acids can also result
in a modification of the structure of the resultant molecule
without significantly altering its biological activity. This can
lead to the development of a smaller active molecule which would
also have utility. For example, one of skill in the art can use
standard techniques to remove amino or carboxy terminal amino acids
from SEQ ID NO:1, as embodied by SEQ ID NO:2, as long as the amino
acids are not required for biological activity of the particular
peptide. Therefore, as demonstrated by SEQ ID NO:2, the last 14
amino acids of the peptide of SEQ ID NO:1 may still act as an
inhibitor of DNA synthesis.
[0025] The term "conservative variation" as used herein denotes the
replacement of an amino acid residue by another, biologically
similar residue. Examples of conservative variations include the
substitution of one hydrophobic residue such as isoleucine, valine,
leucine or methionine for another, or the substitution of one polar
residue for another, such as the substitution of arginine for
lysine, glutamic for aspartic acids, or glutamine for asparagine,
and the like. The term "coservative variation" also includes the
use of a substituted amino acid in place of an unsubstituted parent
amino acid provided that antibodies raised to the substituted
polypeptide also immunoreact with the unsubstituted polypeptide.
Peptides which contain a biological activity of a peptide of the
invention can be easily identified using standard assays, such as
the BdrU DNA synthesis assay exemplified herein.
[0026] Peptides of the invention can be synthesized by such
commonly used methods as t-BOC or FMOC protection of alpha-amino
groups. Both methods involve stepwise syntheses whereby a single
amino acid is added at each step starting from the C terminus of
the peptide (See, Coligan, et al., Current Protocols in Immunology,
Wiley Interscience, 1991, Unit 9). Peptides of the invention can
also be synthesized by the well known solid phase peptide synthesis
methods described by Merrifield, (J. Am. Chem. Soc., 85:2149,
1962), and Stewart and Young, (Solid Phase Peptides Synthesis,
Freeman, San Francisco, 1969, pp.27-62), using a
copoly(styrene-divinylbenzene) containing 0.1- 1.0 mmol amines/g
polymer. On completion of chemical synthesis, the peptides can be
deprotected and cleaved from the polymer by treatment with liquid
HF-10% anisole for about 1/4-1 hours at 0.degree. C. After
evaporation of the reagents, the peptides are extracted from the
polymer with 1% acetic acid solution which is then lyophilized to
yield the crude material. This can normally be purified by such
techniques as gel filtration on Sephadex G-15 using 5% acetic acid
as a solvent. Lyophilization of appropriate fractions of the column
will yield the homogeneous peptide or peptide derivatives, which
can then be characterized by such standard techniques as amino acid
analysis, thin layer chromatography, high performance liquid
chromatography, ultraviolet absorption spectroscopy, molar
rotation, solubility, and quantitated by the solid phase Edman
degradation.
[0027] The peptides of the invention can be used singularly, in
mixtures, or as multimers such as aggregates, polymers, and the
like. Thus, the invention embraces synthetic peptides which
comprise one or more of the same, or different, peptides of the
invention to produce a homogeneous or heterogeneous polymer with
respect to the particular peptides of the invention which are
contained therein. Appropriate techniques for producing various
mixtures, aggregates, multimers and the like will be known to those
of skill in the art. For example, the invention would include a
peptide comprising SEQ ID NO:1 and SEQ ID NO:2 or other DNA
synthesis inhibitory peptides, wherein SEQ ID NO:1, SEQ ID NO:2
and/or other peptides are linked directly or indirectly, for
example, by using a spacer or linker moiety. Such moieties and
their use are well known to those of skill in the art.
[0028] The invention also provides isolated nucleic acid sequences
or polynucleotides which encode the .beta..sub.1C polypeptide or
.beta..sub.1C cytoplasmic peptides of the invention. As used
herein, "polynucleotide" refers to a polymer of
deoxyribonucleotides or ribonucleotides, in the form of a separate
fragment or as a component of a larger construct. DNA encoding a
peptide of the invention can be assembled from cDNA fragments or
from oligonucleotides which provide a synthetic gene which is
capable of being expressed in a recombinant transcriptional unit.
Polynucleotide sequences of the invention include DNA, RNA and cDNA
sequences. A polynucleotide sequence can be deduced from the
genetic code, however, the degeneracy of the code must be taken
into account. Polynucleotides of the invention include sequences
which are degenerate as a result of the genetic code.
[0029] The polynucleotide encoding the peptides of the invention
includes a polynucleotide that encodes SEQ ID NO:1, or fill length
.beta..sub.1C, as well as complementary nucleic acid sequences. A
complementary sequence may include an antisense nucleotide. When
the sequence is RNA, the deoxynucleotides A, G, C, and T of SEQ ID
NO:1 are replaced by ribonucleotides A, G, C, and U, respectively.
Also included in the invention are fragments of the above-described
nucleic acid sequences that are at least 15 bases in length, which
is sufficient to permit the fragment to selectively hybridize to
DNA that encodes the peptides of the invention under physiological
conditions.
[0030] Polynucleotide sequences encoding the peptides of the
invention can be expressed in either prokaryotes or eukaryotes.
Hosts can include microbial, yeast, insect and mammalian organisms.
Methods of expressing DNA sequences having eukaryotic or viral
sequences in prokaryotes are well known in the art. Biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host are known in the art. Such vectors are used
to incorporate DNA sequences of the invention.
[0031] The development of specific DNA sequences encoding the
peptides of the invention can also be obtained by: 1) isolation of
double-stranded DNA sequences from the genomic DNA; 2) chemical
manufacture of a DNA sequence to provide the necessary codons for
the polypeptide of interest; and 3) in vitro synthesis of a
double-stranded DNA sequence by reverse transcription of mRNA
isolated from a eukaryotic donor cell. In the latter case, a
double-stranded DNA complement of mRNA is eventually formed which
is generally referred to as cDNA. In addition, the peptides of the
invention can be obtained by polymerase chain reaction (PCR).
[0032] Of the above-noted methods for developing specific DNA
sequences for use in recombinant procedures, the isolation of
genomic DNA isolates is the least common. This is especially true
when it is desirable to obtain the microbial expression of
mammalian polypeptides due to the presence of introns.
[0033] The synthesis of DNA sequences is frequently the method of
choice when the entire sequence of amino acid residues of the
desired polypeptide product is known. When the entire sequence of
amino acid residues of the desired polypeptide is not known, the
direct synthesis of DNA sequences is not possible and the method of
choice is the synthesis of cDNA sequences. Among the standard
procedures for isolating cDNA sequences of interest is the
formation of plasmid- or phage-carrying cDNA libraries which are
derived from reverse transcription of mRNA which is abundant in
donor cells that have a high level of genetic expression. When used
in combination with polymerase chain reaction technology, even rare
expression products can be cloned. In those cases where significant
portions of the amino acid sequence of the polypeptide are known,
the production of labeled single or double-stranded DNA or RNA
probe sequences duplicating a sequence putatively present in the
target cDNA may be employed in DNA/DNA hybridization procedures
which are carried out on cloned copies of the cDNA which have been
denatured into a single-stranded form (Jay, et al., Nucl. Acid
Res., 11:2325, 1983).
[0034] A cDNA expression library, such as lambda gt11, can be
screened indirectly for .beta..sub.1C peptides having at least one
epitope, using antibodies specific for this region. Such antibodies
can be either polyclonally or monoclonally derived and used to
detect expression product indicative of the presence of
.beta..sub.1C cDNA.
[0035] DNA sequences encoding the peptides of the invention can be
expressed in vitro by DNA transfer into a suitable host cell. "Host
cells" are cells in which a vector can be propagated and its DNA
expressed. The term also includes any progeny of the subject host
cell. It is understood that all progeny may not be identical to the
parental cell since there may be mutations that occur during
replication. However, such progeny are included when the term "host
cell" is used. Methods of stable transfer, meaning that the foreign
DNA is continuously maintained in the host, are known in the
art.
[0036] In the present invention, the .beta..sub.1C peptide
nucleotide sequences may be inserted into a recombinant expression
vector. The term "recombinant expression vector" refers to a
plasmid, virus or other vehicle known in the art that has been
manipulated by insertion or incorporation of the .beta..sub.1C
peptide genetic sequences. Such expression vectors contain a
promoter sequence which facilitates the efficient transcription of
the inserted genetic sequence of the host. The expression vector
typically contains an origin of replication, a promoter, as well as
specific genes which allow phenotypic selection of the transformed
cells. Vectors suitable for use in the present invention include,
but are not limited to the T7-based expression vector for
expression in bacteria (Rosenberg, et al., Gene, 56:125, 1987), the
pMSXND expression vector for expression in mammalian cells (Lee and
Nathans, J. Biol. Chem., 263:3521, 1988) and baculovirus-derived
vectors for expression in insect cells. The DNA segment can be
present in the vector operably linked to regulatory elements, for
example, a promoter (e.g., T7, metallothionein I, or polyhedrin
promoters). Promoter sequences include both inducible and
constituitive promoters, as well as tissue specific promoters. Such
promoters will be known to those of skill in the art and are used
depending on the tissue or cell type desired to be contacted.
[0037] Polynucleotide sequences encoding .beta..sub.1C polypeptide
or peptides of the invention can be expressed in either prokaryotes
or eukaryotes. Hosts can include microbial, yeast, insect and
mammalian organisms. Methods of expressing DNA sequences having
eukaryotic or viral sequences in prokaryotes are well known in the
art. Biologically functional viral and plasmid DNA vectors capable
of expression and replication in a host are known in the art. Such
vectors are used to incorporate DNA sequences of the invention.
[0038] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbC1 can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired.
[0039] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or virus vectors may
be used. Eukaryotic cells can also be cotransformed with DNA
sequences encoding the peptides of the invention, and a second
foreign DNA molecule encoding a selectable phenotype, such as the
herpes simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (see for example, Eukaryotic Viral
Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
[0040] Isolation and purification of microbial expressed
polypeptide, or fragments thereof, provided by the invention, may
be carried out by conventional means including preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies. In addition, the use of a carrier/fusion
protein system, such as glutathione-S-transferase (GST) or other
carriers known in the art, can be used to purify recombinantly
produced proteins of the invention.
[0041] In another embodiment, the invention provides a method for
inhibiting cell proliferation in a cell comprising contacting the
cell with a nucleic acid having essentially the nucleic acid
sequence encoding integrin .beta..sub.1C, SEQ ID NO:1 or SEQ ID
NO:2, or functional fragments thereof, or contacting the cell with
a polypeptide having essentially the amino acid sequence of
integrin .beta..sub.1C, SEQ ID NO:1 or SEQ ID NO:2, or functional
fragments thereof. The method of the invention can be performed in
vitro, in vivo, or ex vivo.
[0042] The term "contacting" refers to means of introducing the
particular .beta..sub.1C reagent to the cell. For example,
contacting includes physical/mechanical or chemical means.
Physical/mechanical means refers to microinjection or
electroporation, for example. Preferably, the .beta..sub.1C
polynucleotides of the invention are microinjected into cell
nuclei. Chemical means, such as transformation, are described
above.
[0043] The present invention provides a method for treating a
cellular proliferative disorder in a subject, comprising
administering to the subject with the disorder therapeutically
effective amount of .beta..sub.1C integrin reagent which inhibits
cellular proliferation. In the method of the invention, the
.beta..sub.1C integrin reagent comprises a nucleic acid sequence
essentially encoding .beta..sub.1C integrin or a polypeptide having
essentially an amino acid sequence of .beta..sub.1C integrin (for
sequences, see Argraves, et al., supra; Languino & Ruoslahti,
supra). The reagent may be an amino acid sequence consisting
essentially of SEQ ID NO:1, or a nucleic acid sequence consisting
essentially of a nucleic acid encoding SEQ ID NO:1 or an
anti-idiotype antibody which binds to a paratope of an antibody
which binds to the amino acid sequence of SEQ ID NO:1. In addition,
the reagent may be a peptide consisting essentially of SEQ ID NO:2,
a nucleic acid sequence consisting essentially of a nucleic acid
encoding SEQ ID NO:2, or an anti-idiotype antibody which binds to a
paratope of an antibody which binds to the amino acid sequence of
SEQ ID NO:2.
[0044] The term "therapeutically effective amount" as used herein
refers to the amount of .beta..sub.1C reagent of the invention, as
described above, administered in sufficient quantity to inhibit
cellular proliferation and decrease the symptoms of the cellular
proliferative disorder. The dosage ranges for the administration of
the polynucleotide, polypeptide, peptide, or anti-idiotype antibody
of the invention are those large enough to produce the desired
effect. Generally, the dosage will vary with the age, condition,
sex, and extent of the infection with bacteria or other agent as
described above, in the patient and can be determined by one
skilled in the art. The dosage can be adjusted by the individual
physician in the event of any contraindications.
[0045] The term "cell-proliferative disorder" denotes malignant as
well as non-malignant cell populations which often appear to differ
from the surrounding tissue both morphologically and genotypically.
For example, the .beta..sub.1C reagents described above are useful
in treating malignancies of the various organ systems, such as, for
example, lung, breast, lymphoid, gastrointestinal, and
genito-urinary tract as well as adenocarcinomas which include
malignancies such as most colon cancers, renal-cell carcinoma,
prostate cancer, non-small cell carcinoma of the lung, cancer of
the small intestine, and cancer of the esophagus. The .beta..sub.1C
reagents are also useful in treating non-malignant
cell-proliferative diseases such as psoriasis, pemphigus vulgaris,
Behcet's syndrome, and lipid histiocytosis. In addition, the method
of the invention is useful for inhibiting pathological
angiogenesis, for example in disorders of the retina, in arthritis,
or in tumors. Essentially, any disorder which is etiologically
linked to cellular proliferation would be considered susceptible to
treatment with the .beta..sub.1C reagents described.
[0046] The present invention also provides gene therapy for the
treatment of cell proliferative disorders. Such therapy would
achieve its therapeutic effect by introduction of the appropriate
.beta..sub.1C polynucleotide, polypeptide/peptide or anti-idiotype
antibody into cells of subjects having the proliferative
disorder.
[0047] Delivery of sense .beta..sub.1C polynucleotide constructs
can be achieved using a recombinant expression vector such as a
chimeric virus or a colloidal dispersion system.
[0048] Various viral vectors which can be utilized for gene therapy
as taught herein include adenovirus, herpes virus, vaccinia, or,
preferably, an RNA virus such as a retrovirus. Preferably, the
retroviral vector is a derivative of a murine or avian retrovirus.
Examples of retroviral vectors in which a single foreign gene can
be inserted include, but are not limited to: Moloney murine
leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),
murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
Most preferably, a non-human primate retroviral vector is employed,
such as the gibbon ape leukemia virus (GaLV), thereby providing a
broader host range than murine vectors, for example. A number of
additional retroviral vectors can incorporate multiple genes. All
of these vectors can transfer or incorporate a gene for a
selectable marker so that transduced cells can be identified and
generated. By inserting a .beta..sub.1C polypeptide or peptide
nucleotide sequence (including promoter region) of interest into
the viral vector, along with another gene which encodes the ligand
for a receptor on a specific target cell, for example, the vector
is now target specific. Retroviral vectors can be made target
specific by inserting, for example, a polynucleotide encoding a
sugar, a glycolipid, or a protein. Preferred targeting is
accomplished by using an antibody to target the retroviral vector.
Those of skill in the art will know of, or can readily ascertain
without undue experimentation, methods which allow target specific
delivery of the retroviral vector containing the .beta..sub.1C
polynucleotide.
[0049] Since recombinant retroviruses are defective, they require
assistance in order to produce infectious vector particles. This
assistance can be provided, for example, by using helper cell lines
that contain plasmids encoding all of the structural genes of the
retrovirus under the control of regulatory sequences within the
LTR. These plasmids are missing a nucleotide sequence which enables
the packaging mechanism to recognize an RNA transcript for
encapsidation. Helper cell lines which have deletions of the
packaging signal include but are not limited to .PSI.2, PA317 and
PA12, for example. These cell lines produce empty virions, since no
genome is packaged. If a retroviral vector is introduced into such
cells in which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector can be
packaged and vector virion produced.
[0050] Another targeted delivery system for pIc polynucleotide,
polypeptide/peptide, or anti-idiotype antibody is a colloidal
dispersion system. Colloidal dispersion systems include
macromolecule complexes, nanocapsules, microspheres, beads, and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. The preferred colloidal system of
this invention is a liposome. Liposomes are artificial membrane
vesicles which are useful as delivery vehicles in vitro and in
vivo. It has been shown that large unilamellar vesicles (LUV),
which range in size from 0.2-4.0 um can encapsulate a substantial
percentage of an aqueous buffer containing large macromolecules.
RNA, DNA and intact virions can be encapsulated within the aqueous
interior and be delivered to cells in a biologically active form
(Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition to
mammalian cells, liposomes have been used for delivery of
polynucleotides in plant, yeast and bacterial cells. In order for a
liposome to be an efficient gene transfer vehicle, the following
characteristics should be present: (1) encapsulation of the genes
of interest at high efficiency while not compromising their
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques, 6:682,
1988).
[0051] The composition of the liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids may also be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0052] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0053] The targeting of liposomes has been classified based on
anatomical and mechanistic factors. Anatomical classification is
based on the level of selectivity, for example, organ-specific,
cell-specific, and organelle-specific. Mechanistic targeting can be
distinguished based upon whether it is passive or active. Passive
targeting utilizes the natural tendency of liposomes to distribute
to cells of the reticulo-endothelial system (RES) in organs which
contain sinusoidal capillaries. Active targeting, on the other
hand, involves alteration of the liposome by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein, or by changing the composition or size of
the liposome in order to achieve targeting to organs and cell types
other than the naturally occurring sites of localization.
[0054] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand.
[0055] In general, the compounds bound to the surface of the
targeted delivery system will be ligands and receptors which will
allow the targeted delivery system to find and "home in" on the
desired cells. A ligand may be any compound of interest which will
bind to another compound, such as a receptor. In general, surface
membrane proteins which bind to specific effector molecules are
referred to as receptors. In the present invention, antibodies are
preferred receptors. Antibodies can be used to target liposomes to
specific cell-surface ligands. For example, certain antigens
expressed specifically on tumor cells, referred to as
tumor-associated antigens (TAAs), may be exploited for the purpose
of targeting .beta..sub.1Canti-id antibody-containing liposomes
directly to the malignant tumor. Since the .beta..sub.1C gene
product may be indiscriminate with respect to cell type in its
action, a targeted delivery system offers a significant improvement
over randomly injecting non-specific liposomes. A number of
procedures can be used to covalently attach either polyclonal or
monoclonal antibodies to a liposome bilayer. Antibody-targeted
liposomes can include monoclonal or polyclonal antibodies or
fragments thereof such as Fab, or F(ab').sub.2, as long as they
bind efficiently to an antigenic epitope on the target cells.
Liposomes may also be targeted to cells expressing receptors for
hormones or other serum factors.
[0056] In another aspect, the present invention is directed to
polyclonal and monoclonal antibodies which bind to the peptides of
the invention. Antibody which consists essentially of pooled
monoclonal antibodies with different epitopic specificities, as
well as distinct monoclonal antibody preparations are provided.
Monoclonal antibodies are made from antigen containing fragments of
the protein by methods well known in the art (Kohler, et al.,
Nature, 256:495, 1975; Current Protocols in Molecular Biology,
Ausubel, et al., ed., 1989). The term "antibody" as used in this
invention includes intact molecules as well as fragments thereof,
such as Fab, F(ab').sub.2, and Fv which are capable of binding the
epitopic determinant.
[0057] As used in this invention, the term "epitope" means any
antigenic determinant on an antigen to which the paratope of an
antibody binds. Epitopic determinants usually consist of chemically
active surface groupings of molecules such as amino acids or sugar
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0058] Antibodies which bind to the peptides of the invention can
be prepared using an intact or full-length .beta..sub.1C
polypeptide or cytoplasmic fragments containing the peptides of
interest as the immunizing antigen. A peptide, such as SEQ ID NO:1,
used to immunize an animal can be derived from translated cDNA or
chemical synthesis and is purified and conjugated to a carrier
protein, if desired. Such commonly used carriers which are
chemically coupled to the peptide include keyhole limpet hemocyanin
(KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus
toxoid. The coupled peptide is then used to immunize the
animal.
[0059] If desired, polyclonal antibodies can be further purified,
for example, by binding to and elution from a matrix to which the
peptide to which the antibodies were raised against is bound. Those
of skill in the art will know of various techniques common in the
immunology arts for purification and/or concentration of polyclonal
antibodies, as well as monoclonal antibodies (See for example,
Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley
Interscience, 1991, incorporated by reference).
[0060] Another method for the identification and isolation of an
antibody binding domain which exhibits binding with a peptide of
the invention is the bacteriophage .lambda. vector system. This
vector system has been used to express a combinatorial library of
Fab fragments from the mouse antibody repertoire in Escherichia
coli (Huse, et al., Science, 246:1275-1281, 1989) and from the
human antibody repertoire (Mullinax, et al., Proc. Natl. Acad.
Sci., 87:8095-8099, 1990). As described therein, antibody
exhibiting binding for a preselected ligand were identified and
isolated from these antibody expression libraries. This methodology
can also be applied to hybridoma cell lines expressing monoclonal
antibodies with binding for a preselected ligand. Hybridomas which
secrete a desired monoclonal antibody can be produced in various
ways using techniques well understood by those having ordinary
skill in the art and will not be repeated here. For example, where
an animal was immunized with peptide/carrier conjugate and
hybridomas are later prepared from lymphocytes of the animal,
hybridoma producing monoclonal antibody specific for the peptide
will bind to peptide, but not carrier, in a standard immunoassay
screen. Details of these techniques are described in such
references as Monoclonal Antibodies-Hybridomas: A New Dimension in
Biological Analysis, Edited by Roger H. Kennett, et al., Plenum
Press, 1980; and U.S. Pat. No. 4,172,124.
[0061] In addition, methods of producing chimeric antibody
molecules with various combinations of "humanized" antibodies are
known in the art and include combining murine variable regions with
human constant regions (Cabily, et al Proc.Natl.Acad.Sci. USA,
81:3273, 1984), or by grafting the murine-antibody complementary
determining regions (CDRs) onto the human framework (Riechmann, et
al., Nature 332:323, 1988).
[0062] The antibodies of the invention are immunoreactive and bind
with the peptides of the invention. It is also possible to use
anti-idiotype technology to produce monoclonal antibodies which
mimic an epitope. For example, an anti-idiotypic monoclonal
antibody made to a first monoclonal antibody will have a binding
domain in the hypervariable region which is the "image" of the
epitope bound by the first monoclonal antibody. In the present
invention, an anti-idiotype antibody, for example, for SEQ ID NO:1,
would be the image of amino acid residues which are present in
residues 778-825 of the cytoplasmic domain of .beta..sub.1C and
would inhibit cellular DNA synthesis and proliferation.
[0063] The antibodies and peptides of the invention are suited for
in vitro use, for example, in immunoassays in which they can be
utilized in liquid phase or bound to a solid phase carrier. In
addition, the monoclonal antibodies or peptides in these
immunoassays can be detectably labeled in various ways. Examples of
types of immunoassays which can utilize monoclonal antibodies or
peptides of the invention are competitive and non-competitive
immunoassays in either a direct or indirect format. Examples of
such immunoassays are the radioimmunoassay (RIA) and the sandwich
(immunometric) assay. Detection of the antigens using the
monoclonal antibodies or antibodies using peptides of the invention
can be done utilizing immunoassays which are run in either the
forward, reverse, or simultaneous modes, including
immunohistochemical assays on physiological samples. Those of skill
in the art will know, or can readily discern, other immunoassay
formats without undue experimentation.
[0064] The monoclonal antibodies or peptides of the invention can
be bound to many different carriers and used to detect the presence
of .beta..sub.1C in a cell, for example. Examples of well-known
carriers include glass, polystyrene, polypropylene, polyethylene,
dextran, nylon, amylases, natural and modified celluloses,
polyacrylamides, agaroses and magnetite. The nature of the carrier
can be either soluble or insoluble for purposes of the invention.
Those skilled in the art will know of other suitable carriers for
binding monoclonal antibodies or peptides, or will be able to
ascertain such, using routine experimentation.
[0065] There are many different labels and methods of labeling
known to those of ordinary skill in the art. Examples of the types
of labels which can be used in the present invention include
enzymes, radioisotopes, fluorescent compounds, colloidal metals,
chemiluminescent compounds, and bio-luminescent compounds. Those of
ordinary skill in the art will know of other suitable labels for
binding to the monoclonal antibodies or peptides of the invention,
or will be able to ascertain such, using routine experimentation.
Furthermore, the binding of these labels to the monoclonal
antibodies or peptides of the invention can be done using standard
techniques common to those of ordinary skill in the art.
[0066] For purposes of the invention, .beta..sub.1C may be detected
by the monoclonal antibodies of the invention when present in
biological fluids and tissues. Any sample containing a detectable
amount of cells expressing .beta..sub.1C can be used. A sample can
be a liquid such as urine, saliva, cerebrospinal fluid, blood,
serum and the like, or a solid or semi-solid such as tissues,
feces, and the like, or, alternatively, a solid tissue such as
those commonly used in histological diagnosis. Preferably, the
sample is serum.
[0067] It is desirable to use the methods described above, for
example, in order to detect .beta..sub.1C expressing cells.
Identification of .beta..sub.1C positive cells by antibodies as
described herein, may be useful in determining which cells are
involved in the cell proliferative disorder or which may be
susceptible to treatment by the methods of the invention. The
inhibitory ability of peptides described herein, derived from the
.beta..sub.1C polypeptide, or synthetic equivalents of the peptide,
may be used to inhibit undesirable mitotic activity of
hematopoietic or platelet cells. Significantly, .beta..sub.1C
reagents described herein are useful as inhibitors for preventing
endothelial cell growth, for example in pathological angiogenesis,
or for inhibiting cell proliferation associated with
inflammation.
[0068] A therapeutic method in accordance with this invention
entails the administration of a therapeutic agent of the invention
by injection or infusion. The therapeutic agent may be a peptide,
polynucleotide, or polypeptide of the invention. In addition, an
anti-idiotype antibody which binds to a monoclonal antibody which
binds a peptide of the invention may also be used in the
therapeutic method of the invention. The amount of therapeutic
agent required to inhibit DNA synthesis in a cell depends on such
factors as the type and severity of the disorder or infection, the
size and weight of the infected subject, and the effectiveness of
other concomitantly employed modes of prophylaxis or therapy. The
therapeutic method of the invention includes treatment of a subject
with a .beta..sub.1C reagent following surgical reduction of the
tumor burden.
[0069] The invention also includes a therapeutic pharmaceutical
composition comprising an isolated peptide consisting essentially
of the amino sequence of integrin .beta..sub.1C, SEQ ID NO:1 or SEQ
ID NO:2, in combination with a pharmaceutically acceptable carrier
and a therapeutic pharmaceutical composition comprising a
nucleotide sequence encoding a polypeptide consisting essentially
of the amino sequence of .beta..sub.1C SEQ ID NO:1 or SEQ ID NO:2,
in combination with a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carrier preparations for parenteral
administration include sterile or aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils. The
active therapeutic ingredient is often mixed with excipients which
are pharmaceutically acceptable and compatible with the active
ingredient. Suitable excipients include water, saline, dextrose,
glycerol and ethanol, or combinations thereof. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers,
such as those based on Ringer's dextrose, and the like.
Preservatives and other additives may also be present such as, for
example, anti-microbials, anti-oxidants, chelating agents, and
inert gases and the like.
[0070] A peptide or antibody of the invention can be formulated
into the therapeutic composition as neutralized pharmaceutically
acceptable salt forms. These include the acid addition salts
(formed with the free amino groups of the polypeptide or antibody
molecule) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acid, or organic acids such as
acetic, oxalic, tartaric and the like. Salts also include those
formed from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium or ferric hydroxides, and organic
bases such as isopropylamine, trimethylamine, histidine, procaine
and the like.
[0071] The invention also envisions the use of antisense or
ribozyme sequences for the inhibition of .beta..sub.1C gene
expression when appropriate. For example, when it is desirable to
increase cell proliferation in a cell that expresses .beta..sub.1C,
antisense is introduced into the cell to inhibit .beta..sub.1C
expression. Therefore, the polynucleotide sequence for
.beta..sub.1C of the invention, including .beta..sub.1C peptides,
also includes sequences complementary to the polynucleotide
encoding (antisense sequences). Antisense nucleic acids are DNA or
RNA molecules that are complementary to at least a portion of a
specific mRNA molecule (Weintraub, Scientific American, 262:40,
1990). The invention embraces all antisense polynucleotides capable
of inhibiting production of polypeptide. In the cell, the antisense
nucleic acids hybridize to the corresponding mRNA, forming a
double-stranded molecule. The antisense nucleic acids interfere
with the translation of the mRNA since the cell will not translate
a mRNA that is double-stranded. Antisense oligomers of about 15
nucleotides are preferred, since they are easily synthesized and
are less likely to cause problems than larger molecules when
introduced into the target--producing cell. The use of antisense
methods to inhibit the translation of genes is well known in the
art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).
[0072] In addition, ribozyme nucleotide sequences for are included
in the invention. Ribozymes are RNA molecules possessing the
ability to specifically cleave other single-stranded RNA in a
manner analogous to DNA restriction endonucleases. Through the
modification of nucleotide sequences which encode these RNAs, it is
possible to engineer molecules that recognize specific nucleotide
sequences in an RNA molecule and cleave it (Cech, J.Amer.Med.Assn.,
260:3030, 1988). A major advantage of this approach is that,
because they are sequence-specific, only mRNAs with particular
sequences are inactivated.
[0073] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
which are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that that sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-based
recognition sequences are preferable to shorter recognition
sequences.
[0074] The following examples are intended to illustrate but not
limit the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
[0075] The present invention shows, for the first time, that an
alternatively spliced .beta.1 integrin functions as a potent
inhibitor of cell growth. This integrin, named .beta.1C, was
originally identified by Languino and Ruoslahti (supra, 1992) using
a PCR strategy. This integrin has a unique 48 amino acid sequence
that replaces much of the .beta..sub.1C cytoplasmic domain (FIG.
1). The 48 aa alternative cytoplasmic domain has 29% homology to
the C-terminal half of the src SH2 domain. .beta..sub.1C has been
found at low levels in placenta, platelets, hematopoetic cell
lines, endothelial cells exposed to TNF, and in some myelomas
(Languino and Ruoslahti, supra, 1992).
[0076] The small size of the .beta..sub.1C cytoplasmic domain (48
aa) allows the precise structural and functional studies to be
undertaken to map the active regions, and that ultimately small
molecules could be designed to have similar effects. Inhibitors of
cell growth have potential as anti-cancer drugs, either directly by
inhibiting tumor cell growth, or indirectly by inhibiting the
endothelial cell growth that is required for tumor
angiogenesis.
Example 1
Preparation of .beta..sub.1C Expression Vector
[0077] For expression of the .beta..sub.1C variant protein of this
invention in cells that do not endogenously express the protein, an
expression vector containing the cDNA for encoding the
.beta..sub.1C variant was constructed as described below. The
expression vector designated pBJ-1, into which the complete cDNA
sequence for encoding the .beta..sub.1C variant protein was cloned,
was derived from the Sr.alpha. promoter-based cDNA expression
cloning vectors, referred to as pcD-Sr.alpha., as described by
Takebe, et al., (Mol. Cell. Biol., 8:466-472, 1988), the disclosure
of which is hereby incorporated by reference. The pcD-SR.alpha.
vector utilized a promoter system having the Sr.alpha. promoter and
the SV40 late-gene splicing junction, a vector-primer segment
having a KpnI-oligo(dT) priming site, SV40 late-region
polyadenylation signal (polyA) and the pUC plasmid-derived vector
sequence (the pBR322 replication origin and .beta.-lactamase [AmpR]
gene, and also contained nucleotide sequences of the SV40 DNA
fragment flanked by the Eco RI and KpnI sites from plasmid pcDV1
(Okayama, et al, Mol. Cell. biol., 2:161-170, 1982) and the short
DNA fragment from plasmid pL1 (Okayama and Berg, Mol. Cell. Biol,
3:280-289,1983).
[0078] A polylinker comprising the restriction cloning sites XhoI,
XbaI, SfiI, NotI, Eco RI, Eco RV, HindIII, ClaI, SalI/XhoI and
having the nucleotide sequence
5-CTAGTGGCCTCCGCGGCCGCGAATTCGATATCAAGCTTATCGATCCAGTA-- 3'(SEQ ID
NO:3) was cloned into the XhoI-digested pcD-SR.alpha. to form the
pBJ-1 expression vector, the schematic of which is shown in FIG. 2.
The XhoI digest of pcD-SR.alpha. resulted in the deletion of the
SV40 late gene splicing junction and the SV40 DNA fragment. The
resulting pBJ-1 expression vector was approximately 3.2 kilobases
(kb).
[0079] To construct a pBJ-1 expression vector from which the
.beta..sub.1C variant protein was expressed, the pBJ-1 vector was
first linearized in the polylinker by digestion with XbaI/NotI. The
cDNA encoding the .beta..sub.1C (GenBank Accession No. M84237) was
cloned into the pBJ-1 linearized vector with two separate fragments
having cohesive ends to allow for directional ligation of the 5'
end of wild-type .beta..sub.1 cDNA in-frame with the 3' end of
wild-type .beta..sub.1 cDNA and the .beta..sub.1C cDNA sequence.
The 5' end of wild-type .beta..sub.1 DNA was isolated from a
pBluescript vector that contained the complete human wild-type
.beta..sub.1 cDNA sequence. The construction of the wild-type
.beta..sub.1 cDNA pBluescript vector was described by Takada,
etal., (J. Cell Biol., 119:913-921, 1992), the disclosure of which
is hereby incorporated by reference. From pBluescript, a
XbaI/HindIII fragment of approximately 2.3 kb was isolated. The
XbaI site was located in the pBluescript vector sequence while the
HindIII site cut at nucleotide position 2357 of the wild-type
.beta..sub.1 cDNA sequence. The latter sequence has been described
by Argraves, et al., (J. Cell. Biol., 105:1183-1190, 1987), the
disclosure of which is hereby incorporated by reference.
[0080] The 3'end of the wild-type .beta..sub.1 cDNA sequence along
with the cDNA sequence encoding the .beta..sub.1C variant protein
was isolated from the PCR-1000 vector (Invitrogen, San Diego,
Calif.) into which a PCR amplified region of .beta..sub.1C cDNA had
been previously cloned as described by Languino, et al., (J. Biol.
Chem., 2677116-7120, 1992), the disclosure of which is hereby
incorporated by reference. The amplified fragment having 474 base
pairs (bp) began at nucleotide position 2139 in the extracellular
domain corresponding to wild-type .beta..sub.1 DNA and extended to
the 3' end of the .beta..sub.1C variant cytoplasmic domain. The
PCR-1000 vector containing the .beta..sub.1C cDNA was then digested
with HindIII/EagI to isolate a DNA fragment of approximately 250
bp. The isolated fragment contained the 3'end wild-type p DNA
beginning at nucleotide position 2358 and extending to 2434
followed by a 116 base pair segment encoding the .beta..sub.1C
variant cytoplasmic domain starting at nucleotide 2435. The
.beta..sub.1C variant cytoplasmic 116 bp cDNA and 48 amino acid
residue sequence encoded by the cDNA are shown in FIG. 1B. The
untranslated wild-type .beta..sub.1 DNA followed by PCR-1000 vector
sequence ending at EagI polylinker cloning site comprised the rest
of the nucleotide sequence in the isolated HindIII/EagI fragment.
The complete .beta..sub.1C variant cDNA sequence is available under
GenBank.TM./EMBL Data Bank with Accession Number M84237.
[0081] The two isolated fragments described above, the Xba/HindIII
fragment containing the 5' end of wild-type .beta..sub.1 cDNA and
the HindIII/EagI fragment containing the 3' end of wild-type
.beta..sub.1 and .beta..sub.1C variant cDNA encoding the variant
cytoplasmic domain, were then directionally ligated by annealing of
cohesive ends into the XbaI/NotI-linearized pBJ-1 vector to form
the .beta..sub.1C-pBJ-1 expression vector. NotI and EagI are
compatible restriction sites. The .beta..sub.1C-pBJ-1 vector in E.
coli strain DH5.alpha. was deposited with American Type Culture
Collection (ATCC), Rockville, Md., on Oct. 20, 1994.
Example 2
Transient Expression of .beta..sub.1C in 10T1/2 Cells
[0082] cDNA coding for human .beta..sub.1C (Languino and Ruoslahti,
supra) or .beta..sub.1 (Argraves, et al., supra) was cloned into
the pBJ-1 vector (Takebe, et al., Mol. Cell Biol., 8:466, 1988) and
the resulting vector (ATCC X, Rockville, Md.) (FIG. 2) was injected
directly into the nucleus of quiescent C3H 10T 1/2 murine
fibroblasts (ATCC CCL226, Rockville, Md.) by standard
microinjection techniques known in the art. This method gives
efficient expression of foreign genes even in cells that are not
readily transfectable.
[0083] Briefly, C3H10T1/2 cells were injected with either pBJ-1
containing .beta..sub.1C or .beta..sub.1 cDNA. Wild type human
.beta.1 cDNA was injected into cells as a control.
[0084] FIG. 3 shows the results of cells that were injected with
cDNAs coding for integrin .beta..sub.1 (Panel A and B) or
.beta..sub.1C (C and D). After 24 hours, cells were fixed and
stained with a rabbit polyclonal antibody produced against purified
human .beta..sub.1 (A and C) or a mouse monoclonal against vinculin
(SIGMA) (B and D). The same cell is shown in A and B and in C and
D.
[0085] Using standard immunological techniques, both .beta..sub.1C
and .beta..sub.1 were detected on the surfaces of injected cells
within 2-3 hours (see, for example, Current Protocols in
Immunology, Coligan, et al., Wiley Interscience, Inc., 1994). Human
.beta..sub.1 was recognized by staining the cells with an
anti-human polyclonal antibody that does not react with the
endogenous mouse integrin. The .beta..sub.1C was found diffusely
distributed on the cell surface, in contrast to .beta..sub.1, which
was found localized to focal adhesions (FIG. 3). This pattern was
not unexpected, since previous work has shown that the integrin
cytoplasmic domain is required for localization to focal adhesions
(Hayashi, et al., J Cell Biol, 110:175, 1990). At 24-48 hours after
injection, .beta..sub.1C and .beta..sub.1 injected cells showed no
obvious change in morphology or appearance, and no change in the
structure of the actin cytoskeleton or of focal adhesions when
cells were immunostained for vinculin (FIG. 3) or actin. Expression
levels of human .beta..sub.1C and .beta..sub.1 were equivalent in
these experiments, as determined by quantitation of fluorescence
intensity in surface-stained cells by methods commonly used in the
art, and were low compared to endogenous integrins (as determined
by surface staining).
Example 3
Inhibition of DNA Synthesis by .beta.1C
[0086] To assess cell cycle progression, 10% serum and the
thymidine analog 5-bromodeoxyuridine (BrdU) were added to injected
cultures, and after 24 hr, the cells were fixed and labeled for
BrdU incorporation into nuclei by standard methods (see for
example, Ausubel, et al., Current Protocols in Molecular Biology,
John Wiley & Sons, Inc., 1994). Cells were injected with cDNA
encoding wild type .beta..sub.1, .beta..sub.1 with a truncated
cytoplasmic domain, or .beta..sub.1C.
[0087] Whereas .beta..sub.1C had no significant effect,
.beta..sub.1C-positive cells showed a 95% inhibition of DNA
synthesis (FIG. 4). As a further control to determine whether
expression of any integrin with an altered cytoplasmic domain is
growth inhibitory, cells were injected with a cDNA coding for a
mutant .beta..sub.1 lacking the majority of the cytoplasmic domain.
This mutation was previously shown to prevent localization to focal
adhesions. Expression of this receptor at levels about 50% higher
than those for .beta..sub.1C produced only slight inhibition of
growth (FIG. 4). This result shows that the .beta..sub.1C
cytoplasmic domain acts in a dominant fashion to inhibit cell
growth. In addition, a truncated .beta..sub.1C cDNA having 14aa
(SEQ ID NO:2) deleted from the cytoplasmic domain was injected into
cells and had no effect on DNA synthesis.
[0088] To determine whether binding of .beta..sub.1C to
extracellular matrix proteins is required for growth inhibition,
two methods were used to block ligand binding. First cells were
treated with anti-human .beta..sub.1 monoclonal antibodies (rabbit
polyclonal antibodies produced against purified .beta..sub.1) that
block ligand binding. Second, .beta..sub.1C cDNA containing a point
mutation in the extracellular domain that blocks ligand binding was
injected into the cells. No reversal of growth inhibition was
observed indicating that expression of .beta..sub.1C is sufficient
to inhibit cell growth.
Example 4
Reversal of DNA Synthesis by .beta..sub.1C Antibody
[0089] To further test whether .beta..sub.1C cytoplasmic domain
actively inhibits growth, cells injected with .beta..sub.1C or
.beta.1 cDNA were injected 2 hours later with an affinity purified
IgG antibody raised against the .beta..sub.1C peptide,
KKSCLSLPSTWDYRVKILFIRVP, as described in Languino and Ruoslahti,
supra. DNA synthesis was measured as described above by stimulating
cells with 10% serum and addition of BrdU. Cells were analyzed for
percent labeled nuclei.
[0090] Injection of the antibody increased the level of DNA
synthesis by about 6-8 fold in .beta..sub.1C expressing cells (FIG.
5). The anti-.beta..sub.1C IgG restored DNA synthesis to
approximately 50% of control levels (relative to cells expressing
human .beta..sub.1 integrin).
Example 5
Chimeric .beta..sub.1C Variants
[0091] Chimeric receptors are constructed consisting of the
transmembrane and extracellular portion of the IL-2 receptor, fused
to the .beta..sub.1C cytoplasmic domain. A chimeric IL-2
receptor/integrin cytoplasmic domain is already available for the
integrin .beta..sub.1 subunit (La flamme, et al., J. Cell Biol.,
117:437, 1992). Second, the .beta..sub.1C cytoplasmic domain is
expressed as a soluble cytoplasmic protein. These constructs are
prepared, and cloned into the BJ-1 expression vector. cDNA's are
injected into 10T 1/2 cells and the effects on DNA synthesis are
assayed as described above. Analogous constructs with the normal
.beta..sub.1 cytoplasmic domain are used as controls.
[0092] If the IL-2/.beta..sub.1C chimera is not growth inhibitory,
receptor ligation or clustering is most likely required. Therefore,
anti-IL-2 receptor antibodies are used to induce receptor
clustering. Antibodies are adsorbed to the coverslips prior to
plating cells, or added in solution (with or without second
antibody). DNA synthesis is again be assayed. These experiments
allow determination of whether the .beta..sub.1C cytoplasmic domain
is growth inhibitory when soluble in the cytoplasm, when anchored
to the inner surface of the plasma membrane, or when clustered in
the plasma membrane.
Example 6
.beta..sub.1C Amino Acid Variants
[0093] 1. Deletion Variants
[0094] To determine if the entire .beta..sub.1C cytoplasmic domain
is required for growth inhibition, first, receptors with C-terminal
deletions are prepared using site-directed mutagenesis by methods
known to those of skill in the art. Segments of approximately 8
amino acids are successively deleted, to yield a set of 5 nested
deletions. Second, receptors are prepared in which segments from
the N-terminus of the alternatively spliced region are deleted.
Mutated receptors are then expressed in 10.sub.1/2 cells and DNA
synthesis assayed. These experiments enable determination of the
critical amino acids .beta..sub.1C that are sufficient to inhibit
cell growth.
[0095] 2. Point Mutation Variants
[0096] Using the smallest growth inhibitory fragment of
.beta..sub.1C as a starting point (either a truncated transmembrane
receptor or a soluble protein), the effects of point mutations will
be analyzed. The "alanine scanning" approach is preferably used in
which residues are mutated to alanine, in order to assess the
functional role of each residue without disrupting overall
structure. Mutants are constructed using standard site-directed
mutagenesis techniques known to those of skill in the art;
alternatively, if a sufficiently short soluble peptide proved to be
growth inhibitory, peptides are prepared synthetically. Peptides or
cDNAs are injected and DNA synthesis assayed as above.
Example 7
Expression of .beta..sub.1C Under Control of an Inducible
Promoter
[0097] The microinjection method cannot be used for biochemical
studies that require large numbers of cells. Because constitutive
expression of often prevents cell growth, cells are injected with
.beta..sub.1C under control of an inducible promoter. Wild-type
.beta..sub.1, and .beta..sub.1C cDNAs are inserted into a vector
containing the LacSwitch System (Stratagene, La Jolla, Calif.). The
LacSwitch expression system gives extremely low levels of basal
expression (reported to be .about.20 molecules/cell) due to
repression by the lac repressor. Upon treatment with the lactose
analog IPTG, expression is greatly increased. These cDNAs are
transfected into 10.sub.1/2 cells together with a plasmid
containing the lactose repressor gene. Cells are selected for G418
resistance, and clones isolated.
[0098] Cell lines are treated with IPTG and .beta..sub.1 expression
analyzed by flow cytometry. Lines that show no expression in the
absence of IPTG and good expression in response to IPTG are used
for further biochemical studies of .beta..sub.1C.
[0099] Deposit of Materials
[0100] The following cell line has been deposited with the American
Type Culture Collection, 1301 Parklawn Drive, Rockville, Md., USA
(ATCC) on Oct. 20, 1994:
1 Cell Line/Vector ATCC Accession No. E coli. DH5.alpha.
.beta..sub.1C-pBJ-1 X
[0101] The deposit was made under the provisions of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of viable cultures for 30 years from the date of deposit. The
organisms will be made available by ATCC under the terms of the
Budapest Treaty which assures permanent and unrestricted
availability of the progeny of the culture to the public upon
issuance of the pertinent U.S. patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes
first, and assures availability of the progeny to one determined by
the U.S. Commissioner of Patents and Trademarks to be entitled
thereto according to 35 USC .sctn.122 and the Commissioner's rules
pursuant thereto (including 37 CFR .sctn.1.14 with particular
reference to 886 OG 638).
[0102] If the culture deposit should die or be lost or destroyed
when cultivated under suitable conditions, it will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of a deposited strain is not to be construed
as a license to practice the invention in contravention of the
rights granted under the authority of any government in accordance
with its patent laws.
[0103] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made without departing from the spirit or
scope of the invention.
Sequence CWU 1
1
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