Note: Descriptions are shown in the official language in which they were submitted.
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MULTIDRUG RESISTANCE GENE
Cmss reference to related applications. This application is a C1P of
application 08/029,344 filed March 8,1993, which is a CIP of application
07/966,923
filed October 27, 1992.
BACKGROUND OF THE INVENTION
It is well known that many types of cancer regress initially in response to
currently available drugs. However, if the disease should recur, as it does
with
variable frequency, it is often refractory to further treatment with either
the agent
originally used for treatment or agents to which the patient has not been
previously
exposed. Currently there is little that can be done for patients whose tumors
display
this form of multidrug resistance.
One mechanism by which cancer cells can simultaneously develop resistance
to an array of structurally diverse drugs has been elucidated over the last 15
years
with the characterization of P-glycoprotein.
P-glycoprotein is a member of a superfamily of membrane proteins that serve
to transport a variety of molecules, ragging from ions to proteins, across
cell
membranes. This superfamiiy is known as the ATP-binding cassette (ABC)
superfamily of membrane transport proteins. For a review see C. F. Higgins,
Ann.
Rev. Cell Biol. 8, 67 (1992). For example, in addition to P-glycoprotein which
transports chemotherapeutic drugs, this family includes the rystic fibrosis
transmembrane conductance regulator, which controls chloride ion fluxes, as
well as
insect proteins that mediate resistance to antimalarial drugs. P-glycoprotein
is
believed to confer resistance to multiple anticancer drugs by acting as an
energy
dependent efflux pump that limits the intracellular accumulation of a wide
range of
cytotoxie agenu and other xenobiotia. Compounds that are excluded from
mammalian cells by P-glycoprotein are frequently natural product-type drugs
but
other large heterocyclic molecules are also "subsuates" for this efflux pump.
The discovery of P-glycoprotein and its occurrence in a variety of tumor types
has stimulated the search for compounds that are capable of blocking its
function
and consequently, of reversing resistance. These investigations have resulted
in
identification of a large number of so-called chemosensitizers or reversing
agents.
Some of these compounds act by inhibiting the pumping action of P-glycoprotein
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while the mechanism of action of others is still undetermined. A select group
of
these agents are currently under intensive clinical investigation and they
show
considerable promise as adjuncts to conventional chemotherapy.
Chemosensitizers
which can reverse P-glycoprotein-mediated multidrug resistance include
verapaznil
and cyclosporin A. -
Unfortunately, overexpression of P-glycoprotein does not explain the high
frequency of multidrug resistance in some of the more prevalent forms of
cancer,
such as lung cancer. In the Western world, lung cancer accounts for
approximately
30% of total cancer deaths. There are four major histological categories of
Lung
tumors: epidermoid or squamous cell adenocarcinomas, large cell carcinomas,
adeaocarcinomas and small cell carcinomas. The first three categories, known
collectively as non-small cell lung cancers, differ from the last in their
initial
response to chemotherapy and radiotherapy. Non-small cell lung cancers are
relatively resistant to both forms of treatment from the outset. In contrast,
small cell
Lung cancer, which accounts for 20% of all lung tumors, exhibits a high
initial
response rate (80-90% in limited disease) to chemotherapy. However, almost all
patients relapse with a multidrug resistant form of the disease and two year
survival
rates are less than 10%. Although the drug resistance profile displayed in
relapsed
small cell lung cancer patients is similar to that conferred by P-
glycoprotein,
P-glycoprotein appears not to be involved. In addition, limited studies in
cell culture
and in patients indicate that multidrug resistance in small cell lung cancer
does not
respond to chemosensitizers, such as verapamil and cyclosporin A, that show
promise
with other types of drug resistant tumors.
Survival rates in Lung cancer have not improved significantly in forty years
and, because of its common occurrence, there is clearly a great need for
improved
therapy for this disease.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a
nucleic
acid which encodes a protein which can confer multidrug resistance on a drug ~
sensitive mammalian cell when expressed in the cell and which is overexpressed
in
certain multidrug resistant cancer cell Lines. The nucleic acid of the
invention was .
isolated from a multidrug resistant cancer cell line which does not
overexpress P-
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giyooprotein and whose resistance is not substantially reversed by
chemosensitizers
which inhibit P-glycoprotein. The nucleic acid and encoded protein of the
present
invention represent molecules which can be targeted therapeutically in
multidrug
resistant tumors expressing the nucleic acid and protein.
The present invention provides an isolated nucleic acid having a nucleotide
sequence which encodes a protein associated with multidrug resistance which is
overexpressed in multidrug resistant cells independently of overexpression of
P-glycoprotein. The protein has been named multidrug resistance-associated
protein
(referred to as MRP). The protein of the invention differs in amino acid
sequence
from P-glycoprotein. The isolated nucleic acid, when expressed in a cell which
is not
multidrug resistant, can confer on the cell multidrug resistance.
In a preferred embodiment, an isolated nucleic acid is provided having a
sequence which codes for a protein associated with multidrug resistance having
as
amino acid sequence which has substantial sequence homology with the amino
acid
sequence shown in SEQ ID N0:2. Most preferably the isolated nucleic aad has a
sequence having substantial sequence homology with the nucleotide sequence
shown
in SEQ ID NO:1. The invention further provides an isolated nucleic acid which
is
antisense to a nucleic acid having substantial sequence homology with the
nucleotide
sequence shown in SEQ ID NO:1.
The invention further provides a recombinant expression vector adapted for
transformation of a host cell comprising the nucleic acid of the invention
operatively
linked to a regulatory sequence. The invention also provides a recombinant
expression vector adapted for transformation of a host cell comprising a DNA
molecule operatively linked to a regulatory sequence to allow expression of an
RNA
molecule which is antisense to a nucleotide sequence of SEQ ID NO: 1.
The invention also provides a method of preparing a protein capable of
conferring multidrug resistance utilizing the nucleic aad of the invention The
method comprises culturing a transformant host cell including a recombinant
expression vector comprising a nucleic acid of the invention and an regulatory
sequence operatively linked to nucleic acid in a suitable medium until a
multidrug
resistance protein is formed and thereafter isolating the protein.
' The invention further provides an isolated protein having the biological
activity of MRP, which can confer multidrug resistance on a drug sensitive
cell when
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the protein is expressed in the cell, said resistance not being reversed by
chemosensitizers of P-glyooprotein. The isolated protein of the invention is
associated with mulydrug resistance in tumor cells and is overexpressed in
multidrug
resistant cells which may or may not overexpress P-glycoprotein. In a
preferred
embodiment the protein has an amino acid sequence which has substantial
homology .
with the amino aad sequence shown in SEQ ID NO: 2.
The invention further provides an antibody specific for an epitope of a
protein '
of the invention. Preferably the antibody is a monoclonal antibody. The
anybody
can be coupled to a detectable substance or a substance having toxic or
therapeuyc
activity.
The invention also provides a bispeciyc antibody capable of binding to a
tumor cell which expresses a protein of the invention and to a detectable
substance,
or a substance having toxic or therapeuyc activity. Preferably, the toxic
substance
is a cytotoxic cell and the bispecific antibody is capable of crosslinking the
tumor cell
and the cytotoxic cell thereby facilitating Iysis of the tumor cell. TT~e
invenyon
further provides a tetrameric antibody complex of a first monoclonal antibody
which
is capable of binding to a tumor cell expressing a protein of the invention
and a
second monoclonal antibody which is capable of binding to a detectable
substance
or a substance having toxic or therapeutic activity wherein the first and
second
anybody are from a first animal speaes, conjugated to form a cyclic tetramer
with
two monoclonal anybodies of a second animal speaes directed against the Fc
fragment of the anybodies of the first animal speaes.
The antibodies, bispecific antibodies or tetrameric antibody complexes can be
incorporated in composiyons suitable for administration in a pharmaceutically
acceptable carrier.
Molecules which bind to a protein of the invention, including the antibodies,
bispeciyc anybodies and tetrameric antibody complexes of the invenyon, can be
used
in a method for identifying multidrug resistant tumor cells by labelling the
molecule
with a detectable substance, contacting the molecule with tumor cells and
detecting
the detectable substance bound to the tumor cells. A molecule which binds to a
protein of the invenyon can further be used in a method for inhibiting
mulydrug
resistance of a cell by blocking activity of an MRP protein. A molecule which
binds
to a protein of the invention can further be used to kill a multidrug
resistant cell
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which expresses the protein by contacting the molecule, coupled to a toxic or
therapeutic substance, with the multidrug resistant cell. Nucleic acids of the
invention can be used in a method for protecting a drug sensitive cell from
cytotoxiaty due to exposure to a drug by transfecting the cell with a nucleic
acid in
a form suitable for expression of the protein encoded by the nucleic acid in
the cell,
thereby conferring drug resistance on the cell.
The recombinant molecules of the invention can be used to produce
transformant host cells expressing the protein of the invention. The
recombinant
molecules of the invention can be also used to produce transgenic nonhuman
animals and nonhuman knockout animals. The transfected cells, transgenic
animals
and knockout animals can be used to test substances for their effect on
multidrug
resistance. A method for identifying a substance which is a chemosensitizer of
a
therapeutic agent and a method for identifying a cytotoxic substance for
multidrug
resistant cells, using transformant host cells or animals of the invention,
are
provided.
The invention also relates to a cell line which is multidrug resistant, does
not
overexpress P-glycoprotein and is substantially resistant to hydrophobic
drugs. The
cell line may be derived from small cell lung cancer cells, preferably the
cell line
NCI-H69. Most preferably the multidrug resistant cell line is H69AR (ATCC CRL
11351). A revertant drug sensitive cell Iine may be obtained from the
multidrug
resistant cell line by culturing the multidrug resistant cell line in the
absence of a
drug for a period of time sufficient to produce a revertant drug sensitive
cell line.
Preferably the revertant drug sensitive cell line is H69PR (ATCC CRL 11350).
BRIEF DESCRIPTION OF DRAWINGS
Figure 1A is a Northern blot of poly(A*)RNA from H69, H69AR and H69PR
cells hybridized with a 1.8 kb EvoRl cDNA fragment of the multidrug resistance
protein of the invention.
Figure IB is a Southern blot analysis of EcoRI - digested genomic DNA from
Hb9, H69AR and H69PR cells hybridized with a l.8kb EcoRl cDNA fragment of
the multidrug resistance protein of the invention.
Figure 1C is a Northern blot of sensitive and resistant HeLa cell poly
(A*)RNA hybridized with a 1.8 kb EcoRI cDNA fragment of the multidrug
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resistance protein of the invention.
Figure 2 is a cluster analysis of the relative similarity of the multidrug
resistance protein of the invention to selected members of the ATP-binding
cassette
transporter superfamily that contain hydrophobic transmembrane regions
followed
by nucleotide binding folds.
Figures 3A & B are the complete amino acid sequence of the multidrug re-
sistance protein of the invention aligned with the complete amino acid
sequence of
itPgpA {Ixi/PgpA).
Figure 3C is a diagram showing the alignment of the extended nucleotide
binding regions of the multidrug resistance protein of the invention, human
CFTR
and leishmania ltPgpA and human P-glycoprotein (Hum/Mdrl).
Figure 4 is a Northern blot of total RNA from normal tissues hybridized with
a 0.9 kb F,coRI cDNA fragment of the multidrug resistance protein of the
invention
Figure 5 is an ISCN-derived idiogram of the human karyotype showing silver
grain distribution following in situ hybridization of a 1.8 kb l:,coRI cDNA
fragment
of the multidrug resistance protein of the invention to metaphase chromosomes.
Figure 6 is a graph depicting the relative cytotoxicity of doxorubicin on
MRP-transfected HeLa cell populations ('T2, TS), a clone of the TS papulation
(TS-5), untransfected HeLa cells and HeLa cells transfected with the parental
expression vector (C1).
Figure 7A is a Northern blot of poly(A)+ RNA from transfected and control
HeLa cells hybridized with a 4 kb MRP cDNA fragment which hybridizes with
endogenous MRP mRNA (e) and expression vector-derived MRP mRNA (v).
Hybridization with a GAPDH cDNA demonstrates the relative amounts of poty(A) +
RNA in each lane.
Figure 7B is a Northern blot of poly(A)+ RNA from uansferted HeLa cells
and control cells hybridized with a DNA fragment from the pRc/CMV vector which
hybridizes only to expression vector-derned MRP mRNA (v). Hybridization with
a GAPDH cDNA demonstrates the relative amounts of poly(A)+ RNA in each lane.
Figure 7G is a Northern blot (MRP mRNA) and immunobIots (MRP protein)
depicting the relative levels of expression vector-derived MRP mRNA and
protein
in transfected HeLa cells and endogenous MRP mRNA and protein in the H69AR
cell tine.
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Figure 8A is a Northern blot of poly(A)+ RNA from transfected HeLa cells
and control cells hybridized with cDNA probes for topoisomerase II {Topo II)
alpha
and beta mRNAs. Hybridization with a GAPDH cDNA demonstrates the relative
amounts of poly(A)+ RNA in each lane.
Figure 8B is a Northern blot of poly(A)+ RNA from transfected HeLa cells
and control cells hybridized with a cDNA probe for annexin II mRNA.
Hybridization with a GAPDH cDNA demonstrates the relative amounts of poly(A) +
RNA in each lane.
Figure 9 is a graph depicting the relative cytotoxiaty of VP-16, vinctistine
and
cisplatin on MRP-transfected HeLa cell populations (T2, TS), a clone of the TS
population (TS-5), untransfected HeLa cells and HeLa cells transfected with
the
parental expression vector (C1).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Multidrug resistant mammalian cell lines have been derived from a number
of tumor types and have provided in vitro models for the study of acquired
resistance. Although seleMed by a single natural product-type drug, these cell
Lines
are cross-resistant to a wide range of chemically unrelated xenobiotia with
multiple
subcellular targets. Typically, these cells are resistant to anthracyclines
(e.g.
doxorubicin (DOX), epipodophylIotoxins (e.g. VP-16) and the Vinca alkaloids
(e.g.
vinblastine)] but not to antimetabolites such as 5-fluorouracil, or to
platinum-containing drugs. Multidrug resistant cells also frequently exhibit a
collateral sensitivity to certain hydrophobic drugs including local
anesthetics and
steroid hormones.
The most commonly reported alteration in multidrug resistant tumor cells has
been the increased expression of the 170 lcDa plasma membrane glycoprotein,
P-glycaprotein (P-gp), which is encoded by the MDRI gene. Studies carried out
in
several laboratories with clinical samples and cell lines representing many
tumor
types have lead to the conclusion that P-gp, while clinically relevant in some
malignancies, is unlikely to be important in others. Overexpression of P-gp is
an
infrequent occurrence in both small ceD lung cancer (SCLC) and non small cell
lung
cancer (NSCLC).
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One of the most widely used cell lines in experimental studies of SCLC is
NCI-H69 {H69) (Gazdar et al., Cancer Res. 44, 3502-3507 (1980)) (ATCC HTB
119). This cell line was treated repeatedly with an anthracycline, such as
daunorubicin or epixubicin and preferably DOX, and step-wise selected to a
Onal
concentration of 0.8 ~M, to produce a multidrug resistant cell line,
designated as
H69AR. A description of the procedures which can be used to produce a
multidrug
resistant cell line such as H69AR is found in Cole, Cancer Chemother
Pharmacol.
17, 259-2b3 (I986) and in Mirski et al., Cancer Research 47, 2594-2598 (1987).
The H69AR cell line (ATCC CRL 11351 ) is about 50-fold resistant to DOX
as compared to the parental H69 cell line. H69AR is also cross-resistant to a
wide
variety of natural product-type drugs. On the other hand, drugs such as
carboplatin,
5-fluorouracil and bIeomycin are equally toxic to both sensitive H69 and
resistant
H69AR cells. Although the cross-resistance pattern of H69AR cells is typical
of
resistance associated with increased levels of P-gp, these cells are different
in that
they display little or no collateral sensitivity to hydrophobic drugs such as
steroids
or local anaesthetics. Another distinguishing feature of H69AR of potential
clinical
relevance that distinguishes it from P-gp overexpressing cell lines is the
limited
ability of verapamil, cyclosporin A and other chemosensitizing agents that
interact
with P-gp, to reverse DOX resistance in these cells. The absence of P-gp
overexpression supports the suggestion that H69AR provides a clinically
relevant
model of drug resistance in lung cancer.
A revenant drug sensitive cell line H69PR (Cole et al., Br J. Cancer 65,
498-502, 1992) (ATCC CRL 11350) was isolated by culturing the H69AR cell line
in the absence of drugs such as DOX for a sufficient time to produce a
revenant cell
line. Preferably the cell Line H69PR is cultured in the absence of drugs for
at least
3 months and up to about 48 months, most preferably 42 months.
The cell lines of the invention may be used to assay for a substance that
affects a multidrug resistant tumor cell. Cells from a cell line of the
invention may
be incubated with a test substance which is suspected of affecting multidrug
resistance. The effect of the substance can be determined by analyzing the
drug
resistance pattern of the cells and comparing the results to a control. As
discussed
above, the multidrug resistant cell line of the invention is resistant to
anthracylines,
epipodophyllotoxins, Vinca alkaloids and other natural-product type drugs.
Thus,
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it is possible to screen for an agonist or antagonist substance of multidrug
resistance
or an antagonist that inhibits the effects of an agonist.
In an embodiment of the invention, a substance that is suspected of being
cytotoxic to a multidrug resistant tumor cell can be identified. Therefore, it
is
possible using the above described method to identify substances which may be
useful in the treatment of multidrug resistant tumors.
As described in the Examples, the H69AR cell line has been used to identify
a cDNA encoding a novel protein associated with multidrug resistance
designated
MRP. The DNA sequence and deduced amino acid sequence of MRP are shown
in SEQ ID N0:1 and SEQ ID NO. 2, respectively. MRP mRNA is overexpressed
in certain multidrug resistant tumor cell lines, including H69AR. Furthermore,
expression of MRP protein in a drug sensitive mammalian cell line confers
multidrug
resistance on the cell line. A protein described herein as "having biological
activity
of MItP" can confer on a mammalian cell multidrug resistance to
anthracyclines,
epipodophyllotoxins and Yinka alkaloids when the protein is expressed in the
mammalian cell, and this resistance is not substantially reversed by
chemosensitizers
which reverse P-glycoprotein-mediated multidmg resistance, such as verapamil
or
cyclosporin A.
The terms "drug resistant" or "drug resistance" as used herein to describe a
property of a cell refer to the ability of the cell to withstand without
cytotoxiaty
increased concentrations of a drug as compared to an appropriate control cell.
An
appropriate control cell for a cell which has been made drug resistant by
continued
exposure to a drug is the parental cell from which the drug resistant cell was
derived.
An appropriate control cell for a cell which has been made drug resistant by
expression in the cell of a protein which confers drug resistance on the cell
is the
same cell without the protein expressed. Appropriate control cells for
naturally
occurring tumor cells in vivo made drug resistant by continued exposure to a
drug
are the same tumor cells at the time of initial exposure to the drug.
The invention provides isolated nucleic acids encoding proteins having
biological acitivity of MRP. In a preferred embodiment, the nucleic acid is a
cDNA
comprising a nucleotide sequence shown in SEQ ID NO: 1. The invention further
provides antisense nucleic acids of nucleic acids encoding proteins having
biological
acitivity of MRP. The invention further provides recombinant expression
vectors
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comprising the nucleic acids and antisense nucleic acids of the invention and
transformant host cells containing recombinant nucleic acids of the invention.
The invention provides isolated proteins having biological acitivity of MRP
and a method for preparing such proteins. In a preferred embodiment, the
isolated
protein having biological activity of MRP comprises an amino acid sequence
shown
in SEQ ID NO: 2. The protein comprising the amino acid sequence of SEQ ID NO:
2 is a member of the ABC superfamily of membrane transport proteins. The
invention further provides antibodies specific for the isolated proteins of
the
invention and compositions suitable for administration comprising such
antibodies.
The invention further provides transgenic and knockout nonhuman animals
produced
using the nucleic acids of the im~ention.
The invention provides a method for identifying multidrug resistant cell using
the nucleic acids and antibodies of the invention. The invention further
provides
methods for inhibiting multidrug resistance of a multidrug resistant cell and
for
killing a multidrug resistant cell using the nucleic acids and antibodies of
the
invention. The invention further provides methods for identifying substances
which
are chemosensitizers of therapeutic agents or cytotoxic to drug resistant
cells using
the transformant host cells and animals of the invention. Furthermore, the
invention
provides diagnostic kits for identifying drug resistant tumor cells.
These and other aspects of this invention are described in detail in the
following subsections.
I. Isolated Nucleic Aads
The invention provides isolated nucleic acids encoding proteins having
biological acitivity activity of MRP. The term "isolated" refers to a nucleic
acid
substantially free of cellular material or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other chemicals when
chemically synthesized. An "isolated" nucleic acid is also free of sequences
which
naturally flank the nucleic acid (i.e., sequences located at the 5' and 3'
ends of the
nucleic acid) in the organism from which the nucleic acid is derived. The term
"nucleic acid" is intended to include DNA and RNA and can be either double
stranded or single stranded. In a preferred embodiment, the nucleic acid is a
cDNA
comprising a nucleotide sequence shown in SEQ ID NO:1. In another embodiment,
the nucleic acid is a cDNA comprising the coding region of the nucleotide
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shown in SEQ ID NO: 1. In another embodiment, the nucleic acid encodes a
protein comprising an amino acid sequence shown in SEQ ID NO: 2.
It will be appreciated that the invention includes nucleic acids having
substantial sequence homology with the nucleotide sequence shown in SEQ ID NO:
1 or encoding proteins having substantial homology to the amino aad sequence
shown in SEQ ID NO: 2. Homology refers to sequence similarity between
sequences and can be determined by comparing a position in each sequence which
may be aligned for purposes of comparison. When a position in the compared
sequence is occupied by the same nucleotide base or amino aad, then the
molecules
are homologous at that position. A degree of homology between sequences is a
function of the number of matching or homologous positions shared by the
sequences.
The term "sequences having substantial sequence homology" means those
nucleotide and amino acid sequences which have slight or inconsequential
sequence
variations from the sequences disclosed in SEQ ID NO:1 and SEQ ID NO: 2, i.e.
the
homologous nucleic acids function in substantially the same manner to produce
substantially the same polypeptides as the actual sequences. The variations
may be
attributable to local mutations or structural modifications. It is expected
that
substitutions or alterations can be made in various regions of the nucleotide
or
amino acid sequence without affecting protein function, particularly if they
lie
outside the regions predicted to be of functional significance.
Analysis of the protein encoded by SEQ ID NO:1, comprising the amino acid
sequence of SEQ ID NO: 2, reveals 12 hydrophobic stretches predicted to be
membrane-spanning regions and of functional importance. These amino acid
residues correspond to positions 99-115,137-153,175-191, 365-381, 444-460, 466-
482,
555-571, 591-607, 969-985, 1028-1044, 1102-1118 and 1205-1221 of SEQ ID NO: 2.
Nucleotide substitutions that result in amino acid sequence changes within
these
regions, especially those that reduce the hybrophobic nature of these regions,
are not
likely to be translated into a functional protein.
Analysis of the protein encoded by SEQ ID NO:1, comprising the amino acid
sequence of SEQ ID NO: 2, reveals two regions having the structural
characteristics
of nucleotide binding folds (NBFs) typical of ATP-binding cassette domains
(ABC
domains). See Hyde, S.C. et al., Nature 346, 362-365 (1990). Elements
comprising
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part of the structure of these NBFs are conserved in other members of the ABC
superfamily of membrane transport proteins and the domains have been shown to
bind nucleotides and to be functionally important. For example see Higgins,
C.F.,
Ann. Rev. Cell Biol. 8, 67-113 (1992). In the protein comprising the amino
acid
sequence shown in SEQ ID NO: 2, the two NBFs are located between about amino
acid residues 661-810 and 1310-1469 of SEQ ID NO:2. Nucleotide and
corresponding amino acid substitutions which decrease the degree of homology
of
these regions compared to ocher members of the ABC superfamily of membrane
transport proteins are likely not to be tolerated in a functional protein.
Alternatively, nucleotide and corresponding amino acid substitutions which
maintain
the structure of an NBF are likely to be tolerated. For example, it has been
demonstrated that nucleotides encoding an NBF of one member of the ABC
superfamily of membrane transport proteins can be substituted for the
homologous
domain of another member while maintaining function of the protein. See
Buschman, F. and Gros, P. MoL Cell. Biol. I1, 595-603 (1991). Accordingly, the
invention provides for a nucleic acid encoding a protein comprising an amino
acid
sequence represented by V-W-X-Y-Z, wherein V are amino aad residues
corresponding to amino acid residues from about 1 to 660 of SEQ iD NO: 2, W
are
amino acid residues of an NBF substantially homologous with amino acid
residues
from about 661 to 810 of SEQ ID NO: 2, X are amino acid residues corresponding
to amino acid residues from about 811 to 1309 of SEQ ID NO: 2, Y are amino
acid
residues of an NBF substantially homologous with amino aad residues from about
1310 to 1469 of SEQ ID NO: 2 and Z are amino aad residues corresponding to
amino acid residues from about 1470 to 1531 of SEQ ID NO: 2. The term "from
about" is intended to mean that the junction between two regions of the
protein (e.g.
between V and W) may vary by a few amino acids from those specifically
indicated.
It is anticipated that, outside of the regions specified above, a nucleic acid
encoding a protein comprising an amino acid sequence which is about 50%
similar
with the amino acid sequence shown SEQ ID N0:2 will provide functional
proteins.
Alternatively, proteins comprising an amino acid sequence which is 60%, 70%,
80%
or 90% homologous with the amino acid sequence shown SEQ ID N0:2 may provide
proteins having MRP activity. The invention encompasses a nucleic acid
encoding
a protein having biological activity of MRP which is at least 50% homologous
with
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the amino acid sequence of SEQ ID NO: 2.
It will further be appreciated that variant forms of the nucleic acids of the
invention which arise by alternative splicing of an mRNA corresponding to a
cDNA
of the invention are encompassed by the invention. Hybridization of a cDNA of
the
invention, containing all or part of SEQ ID NO: 1, to cellular RNA identifies
an
mRNA of approximately 6.Skb with an extended open reading frame of 1531 amino
acids. Several cDNA clones have been isolated that contain internal deletions
which
maintain the original reading frame, suggesting that they may be produced by
alternative splicing. The existence of mRNA species containing these deletions
was
confirmed by reverse PCR of RNA from both mulddrug resistant and sensitive
cells.
In most cases, the variant mItNAs represent minor components of 10% or less.
However, some comprise more than 20% of total MRP mRNA. Alternative splice
forms have been identified which remove nucleotides 657 to 783 of SEQ ID NO: 1
(amino acids 155-196 inclusive of SEQ ID NO: 2), 1845 to 1992 (amino acids
S51-599 inclusive), 2287 to 2463 (amino acids 698-756 inclusive), 2287 to 2628
(amino acids 698-811 inclusive) and 4230 to about 4818 (amino-aads 1346 to
1531
inclusive). Two of the more common variants lack segments of the NH2 proximal
NBF. Both begin at the same site (amino acid 698) and they affect regions of
the
cassette that are very near and GOOH proximal to the common exon 9 splicing
variant of the cystic fibrosis transmembrane conductance regulator (CFTR)
mRNA.
See Chu, C-S. et al., EMBOJournal I0, 1355-1363 (1991). The shorter of the two
(amino aads 698-756) eliminates a phenylalanine at a position corresponding to
F508 of CFTR. The longer one (amino acids 698-811) removes the active
transport
family signature that includes the conserved LSGGQ sequence and the Walker B
motif. Another of the more common variants (amino acids 1346-1531) lacks a
region specifying a segment of the protein close to the COOH terminus, similar
to
the location affected by alternative splicing of exon 23 of CFTR mRNA. See
Yoshimura. K., et al. J. Biol. Chem. 268, 686-690 (1993). In addition, two
other
deletions have been identified, one of which eliminates two of the
transmembrane
domains in the NH2 proximal half of the molecule (amino acids 551-599), and
another which removes a potential secretory signal cleavage site located
between
amino acids 189/190 (amino acids 155-196).
Another aspect of the invention provides a nucleic acid which hybridizes
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under high or low stringency conditions to a nucleic acid which encodes a
protein
having all or a portion of an amino acid sequence shown SEQ ID N0:2.
Appropriate stringency conditions which promote DNA hybridization, for
example,
6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a
wash of 2.0
x SSC at 50°C are known to those skilled in the art or can be found in
Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-b3.b.
For
example, the salt concentration in the wash step can be selected from a low
stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2
x SSC at
SO°C. In addition, the temperature in the wash step can be increased
from low
stringency conditions at room temperature, about 22°C to high
stringency conditions,
at about 65°C.
Isolated nucleic aads encoding a protein having the biological activity of
MRP, as described herein, and having a sequence which differs from a
nucleotide
sequence shown in SEQ ID NO:1 due to degeneracy in the genetic code are also
within the scope of the invention. Such nucleic acids encode functionally
equivalent
proteins {e.g., a protein having MRP activity) but differ in sequence from the
sequence of SEQ ID NO: 1 due to degeneracy in the genetic code. For example,
a number of amino aads are designated by more than one triplet. Colons that
specify the same amino acid, or synonyms (for example, CAU and CAC are
synonyms for histidine) may occur due to degenerary in the genetic code. As
one
example, DNA sequence polymorphisms within the nucleotide sequence of an MRP
protein {especially those within the third base of a colon) may result in
"silent"
mutations in the DNA which do not affect the amino acid encoded. However, it
is
expected that DNA sequence polymorphisms that do lead to changes in the amino
acid sequences of an MRP protein will exist within a population. It will be
appreciated by one skilled in the art that these variations in one or more
nucleotides
(up to about 3-4% of the nucleotides) of the nucleic acids encoding proteins
having
the biological activity of MRP may exist among individuals within a population
due
to natural allelic variation. Any and all such nucleotide variations and
resulting
amino acid polymorphisms are within the scope of the invention. Furthermore,
there
may be one or more isoforms or related, cross-reacting family members of MRP
described herein. Such isoforms or family members are defined as proteins
related
in biological activity and amino acid sequence to MRP, but encoded by genes at
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different loci.
An isolated nucleic acid of the present imrention encoding a protein having
the biological activity of MRP can be isolated from a multidrug resistant cell
line
which displays resistance to such drugs as anthracyclines, epipodophyllotoxins
and
Vinka alkaloids that is not substantially reversed by chemosensitizers which
reverse
P-glycoprotein-mediated multidrug resistance, such as verapamil or cyclosporin
A.
One example of such a cell line is H69AR. Other suitable cell lines can be
produced by stepwise selection of a non-resistant cell line in the presence of
increasing concentrations of a drug for which resistance is to be acquired
over a
period of several months to years. For example, a cell line is cultured in the
presence of an anthracycline, preferably doxorubicin, for about 14 months.
Multidrug resistance is then assessed by exposing the selected cell line to
other
drugs, e.g. an epipodo-phyllotoxin such as VP-16 and a Vinca alkaloid such as
vincristine, and determining the cytotoxicity of the drug for the cell line.
The ability
of chemosensitizers which reverse P-glycoprotein-mediated multidrug
resistance, such
as verapamil and cyclosporin A, to reverse the multidrug resistance is then
assessed
by exposing the selected cell line to these agents in the presence of the
resistant
drugs. A detailed description of the procedures which can be used to produce
appropriate multidrug resistant cell line such as Hb9AR is found in Cole,
Cancer
Chemother Pharmacol. i7, 259-263 (1986) and in Mirski et al., Cancer Research
47,
2594-2598 (1987).
An appropriate multidrug resistant cell line (e.g. a multidrug resistant cell
line
which displays resistance to anthracyclines, epipodophyllotoxins and Vinca
alkaloids
that is not substantially reversed by verapamil or cyclosporin A) is used to
isolate a
nucleic acid of the invention by preparing a cDNA library from this cell line
by
standard techniques and screening this library with cDNA produced from total
mRNA isolated from the multidrug resistant cell line and its drug sensitive
parental
cell line. For example, a cDNA library constructed from total mRNA from H69AR
cells is prepared. The library is plated and two sets of replica filters are
prepared
by standard methods. One set of filters is then screened with cDNA prepared
from
H69AR mRNA and the other set of filters is screened with a comparable amount
of cDNA prepared from H69 mRNA. The cDNA used for screening the library is
labelled, typically with a radioactive label. Following visualization of the
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hybridization results by standard procedures, eDNA clones displaying increased
hybridization with H60AR cDNA when compared to H69 cDNA can be selected
from the library. These cDNAs are derived from mRNAs overexpressed in H69AR
cells when compared with H69 cells. For descriptions of differential cDNA
library
screening see King, C.R., et al. J. Bio. Chem 254, 6781 (1979); Van tier
Bliek, A.M.,
et al., Mol. Cell. Biol. 6, 1671 (1986).
Determination of whether a cDNA so isolated has the biological activity of
MRP can be accomplished by expressing the cDNA in a nonresistant mammalian
cell, by standard techniques, and assessing whether expression in the cell of
the
protein encoded by the eDNA confers on the cell multidrug resistance to
anthracyclines, epipodophyllotoxins and Vinca alkaloids that is not
substantially
reversed by verapamil or cyclosporin A. A eDNA having the biological activity
of
MRP ~ so isolated can be sequenced by standard techniques, such as
dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to
determine the nucleic acid sequence and the predicted amino acid sequence of
the
encoded protein.
An isolated nucleic acid of the irnention which is DNA can also be isolated
by preparing a labelled nucleic aad probe encompassing atl or part of the
nucleotide
sequence shown in SEQ ID NO: 1 and using this labelled nucleic acid probe to
screen an appropriate DNA library (e.g. a cDNA or genomic DNA library). For
instance, a cDNA library made from a multi-drug resistant cell line as
described
above can be used to isolate a cDNA encoding a protein having MRP activity by
screening the library with the labelled probe using standard techniques.
Preferably,
an H69AR cDNA library is used. Alternatively, a genomic DNA library can be
similarly screened to isolate a genomic clone encompassing a gene encoding a
protein having MRP activity. As demonstrated in Example 4, a human MRP gene
has been mapped to chromosome 16. Therefore, a chromosome 16 library rather
than a total genomic DNA library can also be used to isolate a human MRP gene.
Nucleic acids isolated by screening of a cDNA or genomic DNA library can be
sequenced by standard techniques.
An isolated nucleic acid of the invention which is DNA can also be isolated
by selectively amplifying a nucleic acid encoding a protein having MRP
activity using
the polymerase chain reaction (PCR) method and genomic DNA or mRNA. ~To
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prepare cDNA from mRNA, total cellular mRNA can be isolated, for instance from
a multidrug resistant cell line, by a variety of techniques, e.g., by using
the
guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry,
18,
5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse
transcriptase. Moloney MLV reverse transcriptase available from Gibco/BRL,
Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America,
' Inc., St. Petersburg, FT., are preferably employed. It is possible to design
synthetic
oligonucleotide primers from the nucleotide sequence shown in SEQ ID NO:l for
use in PCR. A nucleic aad can be amplified from eDNA or genomic DNA using
these oligonucleotide primers and standard PCR amplification techniques. The
nucleic acid so amplified can be cloned into an appropriate vector and
characterized
by DNA sequence analysis.
A isolated nucleic acid of the invention which is RNA can be be isolated by
cloning a cDNA of the invention into an appropriate vector which allows for
traaSCripdon of the cDNA to produce an RNA molea~le which encodes a protein
having MRP activity. For example, a cDNA can be cloned downstream of a
bacteriophage promoter, e.g. a T7 promoter, in a vector and the eDNA can be
transcribed in vitro with T7 polymerase. A resultant RNA can be isolated by
standard techniques.
A nucleic aad of the invention, for instance an oligonucleotide, can also be
chemically synthesized using standard techniques. Various methods of
chemically
synthesizing polydeoxynucleotides are known, including solid-phase synthesis
which,
like peptide synthesis, has been fully automated in commercially available DNA
synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et
al. U.S.
Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
Analysis of the nucleotide sequence of SEQ ID NO: 1 using currently
available computer software designed for the purpose, such as PC/Gene -
IntelliGenetics Inc., Calif., permits the identification of the initiation
codon and
untranslated sequences of an MRP. The cDNA coding strand, depicted as SEQ D?
. NO: 1, contains a 4593 nucleotide open reading frame encoding 1531 amino
acids,
as well as 195 5' untranslated nucleotides and 223 3' untranslated
nucleotides. The
' intron-exon structure and the transcription regulatory sequences of the gene
encoding the MRP cDNA can .be identified by using a nucleic acid of the
invention
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to probe a genomic DNA clone library. Regulatory elements, such as promoter
and
enhancers necessary for expression of the gene encoding the MRP in various
tissues,
can be identified using cornrentional techniques. The function of the elements
can
be confirmed by using them to express a reporter gene such as the bacterial
gene
lacZ which is operatively linked to the fragments. Such a construct can be
introduced into cultured cells using standard procedures or into non-human
transgenic animal models. In addition to identifying regulatory elements in
DNA,
such constructs can also be used to identify nuclear proteins interacting with
said
elements, using techniques known in the art.
A number of unique restriction sites for restriction enzymes are present in
the
nucleic acid comprising the nucleotide sequence shown in SEQ ID NO:1 These
restriction sites provide acxess to nucleotide fragments which code for
polypeptides
unique to the protein encoded by SEQ ID NO:1 (i.e. a protein of the
invention).
The isolated nucleic acids of the invention or oligonucleotide fragments of
the
isolated nucleic ands, allow those skilled in the art to construct nucleotide
probes
for use in the detection of nucleotide sequences in biological materials, such
as
tumor cell samples. A nucleotide probe can be labelled with a radioactive
element
which provides for an adequate signal as a means for detection and has
sufficient
half life to be useful for detection, such as ~P, 3H, '4C or the like. Other
materials
which can be used to label the probe include antigens that are recognized by a
specific labelled antibody, fluorescent compounds, enzymes, antibodies
specific for
a labelled antigen, and chemiluminescent compounds. An appropriate label can
be
selected having regard to the rate of hybridization and binding of the probe
to the
nucleotide to be detected and the amount of nucleotide available for
hybridization.
II. Antisense Nucleic Acids
The invention also relates to an antisense nucleic acid, or oligonucleotide
fragment thereof, of a nucleic acid of the invention. An antisense nucleic
acid can
comprise a nucleotide sequence which is complementary to a coding strand of a
nucleic acid, e.g. complementary to an mRNA sequence, constructed according to
the rules of Watson and Crick base pairing, and can hydrogen bond to the
coding
strand of the nucleic acid. The antisense sequence complementary to a sequence
of
an mRNA can be complementary to a sequence found in the coding region of the
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CA 02448557 2003-11-25
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mRNA or can be complementary to a 5' or 3' untranslated region of the mRNA.
Furthermore, an antisense nucleic acid can be complementary in sequence to a
regulatory region of the gene encoding the mRNA, for instance a transcription
initiation sequence or regulatory element. Preferably, an antisense nucleic
aad
complementary to a region preceding or spanning the initiation codon or in the
3'
untranslated region of an mRNA is used. An antisense nucleic acid can be
designed
based upon the nucleotide sequence shown in SEQ ID N0: 1. A nucleic acid is
designed which has a sequence compementary to a sequence of the coding or
untranslated region of the shown nucleic acid. Alternatively, an antisense
nucleic
acid can be designed based upon sequences of an MRP gene, identified by
screening
a genomic library as described above. For example, the sequence of an
important
regulatory element can be determined as described above and a sequence which
is
antisense to the regulatory element can be designed.
The antisense nucleic acids and oligonucleoddes of the invention can be
constructed using chemical synthesis and enzymatic ligation reactions using
procedures known in the art. The antisense nucleic acid or oligonucleotide can
be
chemically synthesized using naturally occurring nucleotides or variously
mod~ed
nucleotides designed to increase the biological stability of the molecules or
to
increase the physical stability of the duplex formed between the antisense and
sense
nucleic acids e.g. phosphorothioate derivatives and acridine substituted
nucleotides
can be used. Alternatively, the antisense nucleic acids and oligonucleotides
can be
produced biologically using an expression vector into which a nucleic acid has
been
subcloned in an antisense orientation {i.e. nucleic acid transcribed from the
inserted
nucleic acid will be of an antisense orientation to a target nucleic acid of
interest).
The antisense expression vector is introduced into cells in the form of a
re~mbinant
plasmid, phagemid or attenuated virus in which antisense nucleic acids are
produced
under the control of a high efficiency regulatory region, the activity of
which can be
determined by the cell type into which the vector is introduced. For a
discussion of
the regulation of gene expression using antisense genes see Weintraub, H. et
al.,
Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
Genetics, Vol. 1(1) 1986.
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III. Recombinant Expression Vectors
The nucleic acids of the present invention which encode proteins having MRP
activity can be incorporated in a known manner into a recombinant expression
vector
which ensures good expression of the encoded protein or part thereof. The
recombinant expression vectors are "suitable for transformation of a host
cell", which
means that the recombinant expression vectors contain a nucleic acid or an
oligonucleotide fragment thereof of the invention and a regulatory sequence,
selected on the basis of the host cells to be used for expression, which is
operatively
linked to the nucleic acid or oligonucleotide fragment. Operatively linked is
intended to mean that the nucleic acid is linked to a regulatory sequence in a
manner which allows expression of the nucleic acid. Regulatory sequences are
art-recognized and are selected to direct expression of the desired protein in
an
appropriate host cell. Accordingly, the term regulatory sequence includes
promoters,
enhancers and other expression control elements. Such regulatory sequences are
known to those skilled in the art or one described in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)
can be used. It should be understood that the design of the expression vector
may
depend on such factors as the choice of the host cell to be transfected and/or
the
type of protein desired to be expressed. Such expression vectors can be used
to
transfect cells to thereby produce proteins or peptides encoded by nucleic
acids as
described herein.
The recombinant expression vectors of the invention can be designed for
expression of encoded proteins in prokaryotic or eukaryotic cells. For
example,
proteins can be expressed in bacterial cells such as E. coli, insect cells
(using
baculovirus), yeast cells or mammalian cells. Other suitable host cells can be
found
in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, CA (1990).
Expression in prokaryotes is most often carried out in E. coli with vectors
containing constitutive or inducible promotors directing the expression of
either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids
usually
to the amino terminus of the expressed target gene. Such fusion vectors
typically
serve three purposes: l) to increase expression of recombinant protein; 2) to
increase
the solubility of the target recombinant protein; and 3) to aid in the
purification of
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the target recombinant protein by acting as a ligand in affinity purification.
Often,
in fusion expression vectors, a proteolytic cleavage site is introduced at the
junction
of the fusion moiety and the target recombinant protein to enable separation
of the
target recombinant protein from the fusion moiety subsequent to purification
of the
fusion protein. Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression vectors
include
pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly,
MA) and pRITS (Pharmacia, Piscataway, NJ) which fuse glutathione S-tranferase,
maltose E binding protein, or protein A, respectively, to the target
recombinant
protein.
Inducible non-fusion expression vectors include pTrc (Amann et al., (1988)
Gene b9:30I-315) and pET lld (Studier et al., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 60-
89).
While target gene expression relies on host RNA polymerase transcription from
the
hybrid trp-lac fusion promoter in pTrc, expression of target genes inserted
into pET
lld relies on transcription from the T7 gn10-lac 0 fusion promoter mediated by
coexpressed viral RNA polymerise ('I7 gnl). This viral polymerise is supplied
by
host strains BL21(DE3) or HMS174(DE3) from a resident ~, prophage harboring a
T! gnl under the transcriptional control of the lactlV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically
cleave the recombinant protein (Gottesman, S., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 119-
128).
Another strategy is to alter the nucleic acid sequence of the nucleic acid to
be
inserted into an expression vector (e.g. a nucleic acid encoding an MRP
protein) so
that the individual codons for each amino acid would be those preferentially
utilized
in highly expressed E. coli proteins (Wada et al., (1992) Nuc. Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the invention
could be
carried out by standard DNA synthesis techniques.
Examples of vectors for expression in yeast S.cerivisae include pYepSecl
(Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz,
(1982)
Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2
(Invitrogen Corporation, San Diego, CA).
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Baculovirus vectors available for expression of proteins in cultured insect
sells
(SF 9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.
3:2156-2165)
and the pVLseries (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-
39).
Expression of an MRP protein is mammalian cells is accomplished using a
mammalian expression vector. Examples of mammalian expression vectors include
pCDMB (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987),
EMBOJ. 6:187-195). When used in mammalian cells, the expression vector's
control
functions are often provided by viral material. For example, commonly used
promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and most
frequently, Simian Virus 40. Preferably, the pRc/CMV expression vector
(Invitrogen) is used. In the pRc/CMV vector, nucleic acid introduced into the
vector to be expressed is under the control of the enhancer/promoter sequence
from
the immediate early gene of human cytomegalovirus. Additionally, a gene
conferring
neomycin resistance is ended by the vector. In one embodiment, the recombinant
expression vector is capable of directing expression of the nucleic acid
preferentially
in a particular cell type. This means that the expression vector's control
functions
are provided by regulatory sequences which allow for preferential expression
of a
nucleic acid contained in the vector in a particular cell type, thereby
allowing for
tissue or cell-type specific expression of an encoded protein. For example, a
nucleic
acid encoding a protein with MRP activity can be preferentially expressed in
cardiac
muscle cells using promoter and enhancer sequences from a gene which is
expressed
preferentially in cardiac muscle cells, such as a cardiac myosin gene or a
cardiac
actin gene.
The recombinant expression vector of the invention can be a plasmid. The
recombinant expression vector of the invention further can be a virus, or
portion
thereof, which allows for expression of a nucleic acid introduced into the
viral
nucleic acid. For example, replication defective retroviruses, adenoviruses
and
adeno-associated viruses can be used.
The invention further provides a recombinant expression vector comprising
a DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in a manner which allows for expression, by transcription of the DNA
molecule, of an RNA molecule which is antisense to the nucleotide sequence of
SEQ
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1D NO:1. Regulatory sequences operatively linked to the antisense nucleic acid
can
be chosen which direct the continuous expression of the antisense RNA molecule
in
a variety of cell types, for instance a viral promoter and/or enhancer, or
regulatory
sequences can be chosen which direct tissue or cell type specific expression
of
antisense RNA, as described above.
IV. Transformant Host Cells
The recombinant expression vectors of the invention can be used to make a
transformant host cell including the recombinant expression vector. The term
"transformant host cell" is intended to include prokaryotic and eukaryotic
cell which
have been transformed or transfected with a recombinant expression vector of
the
invention. The terms "transformed with", "transfected with", "transformation"
and
"transfection" are intended to encompass introduction of nucleic acid (e.g. a
vector)
into a cell by one of many possible techniques known in the art. Prokaryotic
cells
can be transformed with nucleic acid by, for example, electroporation or
calcium-chloride mediated transformation. Nucleic acid can be introduced into
mammalian cells via conventional techniques such as calcium phosphate or
calcium
chloride co-precipitation, DEAF-dextran-mediated transfection, lipofectin,
electroporation or microinjection. Suitable methods for transforming and
transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)),
and
other laboratory textbooks.
The number of host cells transformed with a recombinant expression vector
of the invention by techniques such as those described above will depend upon
the
type of recombinant expression vector used and the type of transformation
technique
used. Plasmid vectors introduced into mammalian cells are integrated into host
cell
DNA at only a low frequency. In order to identify these integrants, a gene
that
contains a selectable marker (i.e., resistance to antibiotics) is generally
introduced
into the host cells along with the gene of interest. Preferred selectable
markers
include those which confer resistance to certain drugs, such as 6418 and
hygromycin.
Selectable markers can be introduced on a separate plasmid from the nucleic
acid
' of interest or, preferably, are introduced on a the same plasmid. Host cells
transformed with a one or more recombinant expression vectors containing a
nucleic
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acid of the invention and a gene for a selectable marker can be identified by
selecting for cells using the selectable marker. For example, if the
selectable marker
encoded a gene conferring neomycin resistance (such as pRc/CMV), transformant
cells can be selected with 6418. Cells that have incorporated the selectable
marker
gene will survive, while the other cells die.
As demonstrated in Examples 5 and 6, the nucleic acids of the invention can
confer multidrug resistance to drugs including anthracyclines,
epipodophyllotoxins
and Vinka alkaloids on a drug sensitive cell when transfected into the cell.
Thus,
these drugs can be used as selecting agents when preparing a transformant host
cell
rather than using an independent selectable marker (such as neomycin
resistance).
Therefore, the nucleoc acids of the invention are useful not only for
conferring
multidrug resistance on a cell but also as selectable markers for cells into
which the
nucelic acid has been introduced. See for example Pastan et al. U.S. Patent
No.
5,166,059; Croop et al. U.S. Patent No. 5,198,344. Cells are selected by
exposure to
one or more drugs for which resistance is conferred by the nucleic acid. An
MRP-
encoding nucleic acid in a recombinant expression vector can be introduced
into a
cell together with a second nucleic acid comprising a gene of interest, either
in the
same vector or in separate vectors, and transformant cells can be selected
based
upon their acquired drug resistance. Drug resistant cells which are selected
will
contain the MRP-encoding nucleic acid often cointegrated with the gene of
interest.
Furthermore, by increasing stepwise the concentration of drug used in
selecting the
cells, it is possible to obtain transformant cells with a higher number of
copies of the
introduced nucleic acid, including both the MRP-encoding nucleic acid and a
gene
of interest. Therefore, the nucleic acids of the invention are also useful as
amplifiable markers.
The nucleic acids of the invention encode proteins "having biological activity
of MRP". The biological activity of MRP is defined as the ability of the
protein,
when expressed in a drug sensitive mammalian cell, to confer on the cell
multidrug
resistance to such drugs as anthracyclines, epipodophyllotoxins and Vinca
alkaloids
that is not substantially reversed by chemosensitizers which reverse
P-glycoprotein-mediated multidrug resistance, such as verapamil or cyclosporin
A.
An isolated nucleic acid of the invention can be tested for MRP activity by
incorporating the nucleic acid into a recombinant expression vector of the
invention,
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transforming a drug sensitive mammalian cell with the recombinant expression
vector
to make a transformant host cell of the invention as described above and
testing the
multidrug resistance of the transformant host cell. The multidrug resistance
of the
transformant host cell is tested by determining the cytotoxicity of the drugs
to be
, tested (i.e. anthracyclines, epipodophyllotoxins and Vinca alkaloids) for
the
transformed cell as compared to the untransformed cell, and the ability of
other
drugs (i.e. verapamil and cyclosporin A) to reverse multidrug resistance. For
example, in a preferred embodiment, the transformant host cell is a HeLa cell,
and
the multidrug resistance of transfected HeLa cells is compared to that of
untraasfected HeLa cells or preferably to HeLa cells transfected with the
parental
expression vector lacking the nucleic acid encoding a protein having MRP
activity.
V. Isolated Proteins
The invention provides isolated proteins having biological activity of MRP.
The term "isolated" refers to a protein substantially free of cellular
material or
culture medium when produced by recombinant DNA techniques, or chemical
precursors or other chemicals when chemically synthesized. In a preferred
embodiment the protein having biological activity of MRP comprises an amino
acid
sequence shown in SEQ >D NO: 2. Alternatively, the protein can be encoded by a
nucleic acid comprising the nucleotide sequence of SEQ iD NO: 1. Proteins
having
biological activity of MRP which have substantial sequence homology to the
amino
acid sequence of SEQ ID NO: 2, as defined above, are also encompassed by the
invention. Furthermore, proteins having biological activity of MRP that are
encoded
by nucleic acids which hybridize under high or low stringency conditions, as
defined
above, to a nucleic acid comprising a nucleotide sequence shown in SEQ ID NO:
1
are encompassed by the invention. Additionally, immunogenic portions of MRP
proteins are within the scope of the invention. As demonstrated in Example 7,
two
immunogenic portions of a protein rnmprising an amino acid sequence shown in
SEQ ID NO: 2 correspond to amino acid residues 932-943 shown in SEQ ID NO:
- 2 (residues AELQKAEAKKEE) and amino acid residues 1427-1441 (residues
GENLSVGQRQLVCLA). Preferred immunogenic portions correspond to regions
' of the protein not conserved in other ABC superfamily members, i.e. outside
of the
two NBF domains (amino acid residues 661-810 and 1310-14b9), and include
regions
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between the 12 membrane spanning regions. An immunogenic portion will be of at
least about eight amino acids in length.
The MRP protein, or isofonms or parts thereof, of the invention can be
isolated by expression in a suitable host cell using techniques known in the
art.
Suitable host cells include prokaryotic or eukaryotic organisms or cell lines,
for
example, yeast, E. coli and insect cells. The recombinant expression vectors
of the
invention, described above, can be used to express a protein having MRP
activity in
a host cell in order to isolate the protein. The invention provides a method
of
preparing an isolated protein of the invention comprising introducing into a
host cell
a recombinant nucleic acid encoding the protein, allowing the protein to be
expressed in the host cell and isolating the protein. Preferably, the
recombinant
nucleic acid is a recombinant expression vector. Proteins can be isolated from
a host
cell expressing the protein according to standard procedures of the art,
including
ammonium sulfate precipitation, fractionation column chromatography (e.g. ion
exchange, gel filtration, electrophoresis, affinity chromatography, etc.) and
ultimately,
crystallization (see generally, "Enzyme Purification and Related
Techniques°,
Methods in Enzymology, 22, 233-577 (1971)).
Alternatively, the protein or parts thereof can be prepared by chemical
synthesis using techniques well known in the chemistry of proteins such as
solid
phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or
synthesis
in homogeneous solution (Houbenweyl,1987, Methods of Organic Chemistry, ed. E.
Wansch, Vol. 15 I and II, Thieme, Stuttgart).
VI. Antibodies
The proteins of the invention, or portions thereof, can be used to prepare
antibodies specific for the proteins. Antibodies can be prepared which bind a
distinct epitope in an unconserved region of the protein. An unconserved
region of
the protein is one which does not have substantial sequence homology to other
proteins, for example other members of the ABC superfamily of membrane
transport
proteins. For example, unconserved regions encompassing sequences between the
twelve membrane spanning regions, excluding the NBF domains, can be used.
Alternatively, a region from one of the two NBF domains can be used to prepare
an
antibody to a conserved region of an MRP protein. An antibody to a conserved
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region may be capable of reacting with other members of the ABC family of
membrane transport proteins. Conventional methods can be used to prepare the
antibodies. For example, by using a peptide of an MRP protein, polyclonal
antisera
or monoclonal antibodies can be made using standard methods. As demonstrated
in Example 7, a mammal, (e.g., a mouse, hamster, or rabbit) can be immunized
with
an immunogenic form of the peptide which elicits an antibody response in the
mammal. Techniques for conferring immunogenicity on a peptide include
conjugation to carriers or other techniques well known in the art. For
example, the
peptide can be administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in plasma or
serum.
Standard ELISA or other immunoassay can be used with the immunogen as antigen
to assess the levels of antibodies. Following immunization, andsera can be
obtained
and, if desired, polyclonal antibodies isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes)
can be harvested from an immunized animal and fused with myeloma cells by
standard somatic cell fusion procedures thus immortalizing these cells and
yielding
hybridoma cells. Such techniques are well known in the art. For example, the
hybridoma technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human B-cell hybridoma
technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et al. Monoclonal
Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and
screening of combinatorial antibody libraries (Huse et al., Science 246,1275
(1989)).
Hybridoma cells can be screened immunochemically for production of antibodies
specifically reactive with the peptide and monoclonal antibodies isolated.
The term antibody as used herein is intended to include fragments thereof
which are also specifically reactive with a protein, or peptide thereof,
having the
biological activity of MRP. Antibodies can be fragmented using conventional
techniques and the fragments screened for utility in the same manner as
described
above for whole antibodies. For example, F{ab')2 fragments can be generated by
treating antibody with pepsin. The resulting F(ab')2 fragment can be treated
to
reduce disulfide bridges to produce Fab' fragments.
When antibodies produced in non-human subjects are used therapeutically in
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humans, they are recognized to varying degrees as foreign and an immune
response
may be generated in the patient. One approach for minimizing or eliminating
this
problem, which is preferable to general immunosuppression, is to produce
chimeric
antibody derivatives, i.e., antibody molecules that combine a non-human animal
variable region and a human constant region. Chimeric antibody molecules can
include, for example, the antigen binding domain from an antibody of a mouse,
rat,
or other species, with human constant regions. A variety of approaches for
making
chimeric antibodies have been described and can be used to make chimeric
antibodies containing the immunoglobulin variable region which recognizes the
gene
product of the novel B lymphocyte antigens of the invention. See, for example,
Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al.,
Nature
314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S.
Patent No.
4,816,397; Tanaguchi et al., European Patent Publication EP171496; European
Patent Publication 0173494, United Kingdom Patent GB 2177096B. It is expected
that such chimeric antibodies would be less immunogenic in a human subject
than
the corresponding non-chimeric antibody.
For human therapeutic purposes the monoclonal or chimeric antibodies
specifically reactive with a protein, or peptide thereof:, having the
biological activity
of a MRP as described herein can be further humanized by producing human
constant region chimeras, in which parts of the variable regions, especially
the
conserved framework regions of the antigen-binding domain, are of human origin
and only the hypervariable regions are of non-human origin. Such altered
immunoglobulin molecules may be made by any of several techniques known in the
art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983);
Kozbor et
al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16
(1982)), and are preferably made according to the teachings of PCT Publication
W092/06193 or EP 0239400. Humanized antibodies can be commercially produced
by, for example, Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great
Britain. Humanized antibodies which have reduced immunogenicity are preferred
for immunotherapy in human subjects. Immunotherapy with a humanized antibody
will likely reduce the necessity for any concomitant immunosuppression and may
result in increased long term effectiveness for the treatment of chronic
disease
situations or situations requiring repeated antibody treatments.
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Another method of generating specific antibodies, or antibody fragments,
reactive against protein, or peptide thereof, having the biological activity
of a MRP
is to screen expression libraries enrnding immunogiobulin genes, or portions
thereof,
expressed in bacteria with peptides produced from the nucleic acid molecules
of the
present invention For example, complete Fab fragments, VH regions and FV
regions can be expressed in bacteria using phage expression libraries. See for
example Ward et al., Nature 341, S44-546: (1989); Huse et al., Science 246,
1275-1281 (1989); and McCafferty et al. Nature 348, SS2-SS4 (1994). Screening
such
libraries with, for example, a B7-2 peptide can identify imunoglobulin
fragments
reactive with B7-2. Alternatively, the SCID-hu mouse developed by Genpharm can
be used to produce anri'bodies, or fragments thereof.
The polyclonal, monoclonal or chimeric monoclonal antibodies can be used
to detect the proteins of the invention, portions thereof or closely related
isoforms
in various biological materials, for exampie they can be used in an ELISA,
radioimmunoassay or histochemical tests. Thus, the antibodies can be used to
quantify the amount of an MRP protein of the invention, portions thereof or
closely
related isoforms in a sample in order to diagnose multidrug resistance, and to
determine the role of MRP proteins in particular cellular events or
pathological
states, particularly its role in multidrug resistance. Using methods described
hereinbefore, polyclonal, monoclonal antibodies, or chimeric monoclonal
antibodies
can be raised to nonconserved regions of MRP and used to distinguish MRP from
closely related isoforms and other proteins that share a common conserved
epitope.
The polyclonal or monoclonal antibodies can be coupled to a detectable
substance. The term "coupled" is used to mean that the detectable substance is
physically linked to the antibody. Suitable detectable substances include
various
enzymes, prosthetic groups, fluorescent materials, luminescent materials and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
alkaline ghosphatase, B-galactosidase, or acetylcholinesterase; examples of
suitable
prosthetic group complexes include streptavidin/biotiwand avidin/biotin;
examples
of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; and
examples
of suitable radioactive material include '~I, '~'I, ~S or 3H.
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The present invention allows the skilled artisan to prepare bispecific
antibodies and tetrameric antibody complexes. Bispecific antibodies can be
prepared
by forming hybrid hybridomas. The hybrid hybridomas can be prepared using the
procedures known in the art such as those disclosed in Staerz & Bevan, (PNAS
(USA) 83: 1453, 1986 and Immunology Today, 7:241, 1986). In general, a hybrid
,
hybridoma is formed by fusing a first cell line which produces a first
monoclonal
antibody which is capable of binding to a tumor cell expressing a protein of
the
invention and a second cell line which produces a second monoclonal antibody
which
is capable of binding to a detectable substance, or a substance having toxic
or
therapeutic activity. The bispecific antibodies can also be constructed by
chemical
means using procedures such as those described by Staerz et al., (Nature,
314:628,
1985) and Perez et al., (Nature 316:354, 1985).
Bispecific monoclonal antibodies containing a variable region of an antibody,
preferably a human antibody, specific for an MRP protein of the invention or
portion thereof, a variable region of an antibody which is capable of binding
to a
detectable substance, or a substance having toxic or therapeutic activity and
the
constant regions of human immunoglobulins such as human IgGI, IgG2, IgG3 and
IgG4 can also be constructed as described above. Bispecific chimeric
monoclonal
antibodies can also be constructed as described above.
A tetrameric antibody rnmplex can be prepared by preparing a first
monoclonal antibody which is capable of binding to a tumor cell expressing a
protein
of the invention and a second monoclonal antibody which is capable of binding
to
a detectable substance or a substance having toxic or therapeutic activity.
The first
and second antibody are from a first animal speaes. The first and second
antibody
are reaMed with an about equimolar amount of antibodies of a second animal
species or Fab fragments thereof, which are directed against the Fc-fragments
of the
antibodies of the first animal species. The tetrameric complex formed is then
isolated. (See U.S. Patent No. 4,868,109 to Iansdorp for a description of
methods
for preparing tetrameric antibody complexes).
Examples of detectable substances are enzymes, such as horseradish
peroxidase, alkaline phosphatase, glucose oxidase and galactosidase. Examples
of
substances having toxic activity are cytotoxic cells such as macrophages,
neutrophils,
eosinophils, NK cells, LAK cells, and large granular lymphocytes or substances
which
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are toxic to tumor cells such as radionuclides, and toxins such as diptheria
toxin and
ricin or attenuated derivatives thereof. It will be appreciated that the
antibody can
be directed against the Fc receptor on cytotoxic cells. Examples of substances
having therapeutic activity are chemotherapeutic agents such as carboplatin
and
methotrexate. Preferably, the chemotherapeutic agent is not a drug to which a
protein having MRP activity confers resistance.
The antibodies, bispecific antibodies and tetrameric antibody complexes of
the invention directed against a substance having toxic or therapeutic
activity coupled
with the substance having toxic or therapeutic activity can be used to treat
multidrug
resistant tumors. Accordingly, the invention provides a composition comprising
antibodies, bispecific antibodies or tetrameric antibody complexes in a
pharmaceutically acceptable carrier. Preferably, the antibodies, bispecific
antibodies
or tetrameric antibody complexes are coupled to or capable of binding to a
substance having toxic or therapeutic activity and to a tumor cell expressing
a protein
of the invention.
The impositions of the invention are administered to subjects in a
biologically compatible form suitable for pharmaceutical administration in
vivo. By
"biologically compatible form suitable for administration in vivo" is meant a
form of
the antibody to be administered in which any toxic effects are outweighed by
the
therapeutic effects of the antibody. The term subject is intended to include
living
organisms in which an immune response can be elicited, e.g., mammals. Examples
of subjects include humans, dogs, cats, mice, rats, and transgenic species
thereof.
Administration of a therapeutically active amount of the therapeutic
compositions
of the present invention is defined as an amount effective, at dosages and for
periods
of time necessay to achieve the desired result. For example, a therapeutically
active
amount of an antibody reactive with an MRP protein of the invention may vary
according to factors such as the disease state, age, sex, and weight of the
individual,
and the ability of antibody to elicit a desired response in the individual.
Dosage
regima may be adjusted to provide the optimum therapeutic response. For
example,
several divided doses may be administered daily or the dose may be
proportionally
reduced as indicated by the exigencies of the therapeutic situation.
The active compound (e.g., antibody) may be administered in a convenient
manner such as by injection (subcutaneous, intravenous, etc.), oral
administration,
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inhalation, transdermal application, or rectal administration. Depending on
the
route of administration, the active compound may be coated in a material to
protect
the compound from the action of enzymes, acids and other natural conditions
which
may inactivate the compound.
An antibody composition can be administered to a subject in an appropriate ,
carrier or diluent, co-administered with enzyme inhibitors or in an
appropriate
carrier such as liposomes. The term "pharmaceutically acceptable carrier" as
used '
herein is intended to include diluents such as saline and aqueous buffer
solutions.
To administer an antibody reactive with an MRP protein by other than
parenteral.
administration, it may be necessary to coat the antibody with, or co-
administer the
antibody with, a material to prevent its inactivation. Enzyme inhibitors
include
pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
Liposomes include water-in-oil-in-water emulsions as well as conventional
liposomes
(Strejan et al., (1984) J. Neuroimmunol ?:27). The active compound may also be
administered parenterally or intraperitoneally. Dispersions can also be
prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary conditions of storage and use, these preparations may contain a
preservative
to prevent the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. In
all cases,
the composition must be sterile and must be fluid to the extent that easy
syringability
exists. It must be stable under the conditions of manufacture and storage and
must
be preserved against the contaminating action of microorganisms such as
bacteria
and fungi. The pharmaceutically acceptable carrier can be a solvent or
dispersion
medium containing, for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyetheylene glycol, and the like), and suitable
mixtures
thereof. The proper fluidity can be maintained, for example, by the use of a
coating
such as lecithin, by the maintenance of the required particle size in the case
of
dispersion and by the use of surfactants. Prevention of the action of
microorganisms .
can be achieved by various antibacterial and antifungal agents, for example,
parabens, chtorobutanol, phenol, asorbic aad, thimerosal, and the like. In
many '
cases, it will be preferable to include isotonic agents, for example, sugars,
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golyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for example,
aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating active compound
(e.g., antibody reactive against an MRP protein) in the required amount in an
appropriate solvent with one or a combination of ingredients enumerated above,
as
required, followed by filtered sterilization. Generally, dispersions are
prepared by
incorporating the active compound into a sterile vehicle which contains a
basic
dispersion medium and the required other ingredients from those enumerated
above.
In the case of sterile powders for the preparation of sterile injectable
solutions, the
preferred methods of preparation are vacuum drying and freeze-drying which
yields
a powder of the active ingredient (e.g., antibody) plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
When the active compound is suitably protected, as described above, the
composition may be orally administered, for example, with an inert diluent or
an
assimilable edible carrier. As used herein "pharmaceutically acceptable
carrier"
includes arty and all solvents, dispersion media, coatings, antibacterial and
antifungal
agents, isotonic and absorption delaying agents, and the like. The use of such
media
and agents for pharmaceutically active substances is well known in the art.
Except
insofar as any conventional media or agent is incompatible with the active
compound, use thereof in the therapeutic compositions is contemplated,
Supplementary active compounds can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as
used herein refers to physically discrete units suited as unitary dosages for
the
mammalian subjects to be treated; each unit containing a predetermined
quantity of
active compound calculated to produce the desired therapeutic effect in
association
with the required pharmaceutical carrier. The specification for the dosage
unit
forms of the im~ention are dictated by and directly dependent on (a) the
unique
characteristics of the active compound and the particular therapeutic effect
to be
achieved, and (b) the limitations inherent in the art of compounding such an
active
compound for the therapeutic treatment of individuals.
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VII. Transgenic and Knockout Animals
Nucleic aads which encode proteins having biological activity of MRP can be
used to generate either transgenic animals or "knock out" animals which, in
turn, are
useful in the development and screening of therapeutically useful reagents. A
transgenic animal (e.g., a mouse) is an animal having cells that contain a
transgene,
which transgene was introduced into the animal or an ancestor of the animal at
a
prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated
into
the genome of a cell from which a transgenic animal develops. In one
embodiment,
a human MRP cDNA, comprising the nucleotide sequence shown in SEQ ID NO:
l,or an appropriate sequence thereoil can be used to clone a marine MRP gene
in
accordance with established techniques and the genomic nucleic acid used to
generate transgenic animals that contain cells which express MRP protein,
Methods
for generating transgenic animals, particularly animals such as mice, have
become
conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866
and 4,870,009. In a preferred embodiment, plasmids containing recombinant
molecules of the invention are microinjected into mouse embryos. In
particular, the
plasmids are microinjected into the male pronuclei of fertilized one-cell
mouse eggs;
the injected eggs are transferred to pseudo-pregnant foster females; and, the
eggs
in the foster females are allowed to develop to term. [Hogan, B, et al.,
(1986) A
Laboratory Manual, Cold Spring Harbor, New York, Cold Spring Harbor
Laboratory). Alternatively, an embryonal stem cell line can be transfected
with an
expression vector containing nucleic acid encoding a protein having MRP
activity
and cells containing the nucleic acid can be used to form aggregation chimeras
with
embryos from a suitable recipient mouse strain The chimeric embryos can then
be
implanted into a suitable pseudopregnant female mouse of the appropriate
strain
and the embryo brought to term. Progeny harbouring the transfected DNA in
their
germ cells can be used to breed uniformly transgenic mice.
Typically, particular cells would be targeted for MRP transgene incorporation
by use of tissue specific enhancers operatively linked to the MRP-encoding
gene.
For example, promoters and/or enhancers which direct expression of a gene to
which they are operatively linked preferentially in cardiac muscle cells can
be used
to create a transgenic animal which expresses an MRP protein preferentially in
cardiac muscle tissue. Examples of suitable promoters and enhancers include
those
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which regulate the expression of the genes for cardiac myosin and cardiac
actin.
Transgenic animals that include a copy of an MRP transgene introduced into the
germ line of the animal at an embryonic stage can also be used to examine the
effect
of increased MRP expression in various tissues.
The pattern and extent of expression of a recombinant molecule of the
invention in a transgenic mouse is facilitated by fusing a reporter gene to
the
recombinant molecule such that both genes are co-transcribed to form a
polycistronic
mRNA. The reporter gene can be introduced into the recombinant molecule using
conventional methods such as those described in Sambrook et al., 1989,
Molecular
Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press. E~cient
expression of both cistrons of the polycistronic mRNA encoding the protein of
the
invention and the reporter protein can be achieved by inclusion of a known
internal
translational initiation sequence such as that present in poliovirus mRNA. The
reporter gene should be under the control of the regulatory sequence of the
recombinant molecule of the imrention and the pattern and extent of expression
of
the gene encoding a protein of the invention can accordingly be determined by
assaying for the phenotype of the reporter gene. Preferably the reporter gene
codes
for a phenotype not displayed by the host cell and the phenotype can be
assayed
quantitatively. Examples of suitable reporter genes include lacZ (B-
galactosidase),
neo (neomycin phosphotransferase), CAT (chloramphenicol acetyltransferase)
dhfr
(dihydrofolate reductase), aphIV (hygromycin phosphotransferase), lux
(luciferase),
uidA (B-glucuronidase). Preferably, the reporter gene is lacZ which codes for
B-
galactosidase. 8-galactosidase can be assayed using the lactose analogue
X-gal(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) which is broken down
by
13-galactosidase to a product that is blue in color. (See for example Old R.W.
&
Primrose S.B., Principles of Gene Manipulation. An Introduction to Genetic
Engineering, 4th ed. Oxford University Press at pages 63-66 for a discussion
of
procedures for screening for recombinants).
Although experimental animals used in the preferred embodiment disclosed
are mice, the invention should not be limited thereto. It can be desirable to
use
other species such as rats, hamsters and rabbits.
The transgenic animals of the invention can be used to investigate the
molecular basis of multidrug resistance. The transgenic animals of the
irnrention can
CA 02448557 2003-11-25
WO 94!10303 PCTlCA93100439
also be used to test substances for the ability to prevent, slow or reverse
the
development of multidrug resistance. A transgenic animal can be treated with
the
substance in parallel with an untreated control transgenic animal.
Cells from the transgenic animals of the invention can be cultured using
standard tissue culture techniques. In particular, cells carrying the
recombinant
molecule of the invention can be cultured and used to test substances for the
ability
to prevent, slow or reverse multidrug resistance.
Additionally, the non-human homologues of genes encoding proteins having
MRP activity can be used to construct an MRP "knock out" animal which has a
defective or altered MRP gene. For example, a human MRP cDNA, comprising the
nucleotide sequence shown in SEQ ID NO: 1, or an appropriate sequence thereof,
can be used to clone a murine MRP gene in accordance with established
techniques.
A portion of the genomic MRP DNA (e.g., an exon) can be deleted or replaced
with
another gene, such as a gene encoding a selectable marker which can be used to
monitor integration. The altered MRP DNA can then be transfected into an
embryonal stem cell line. The altered MRP DNA will homologously recombine with
the endogenous MRP gene in certain cells and clones containing the altered
gene
can be selected. Cells containing the altered gene are injected into a
blastocyst of
an animal, such as a mouse, to form aggregation chimeras as described for
transgenic
animals. Chimeric embryos are implanted as described above. Transmission of
the
altered gene into the germline of a resultant animal can be confirmed using
standard
techniques and the animal can be used to breed animals having an altered MRP
gene in every cell. Accordingly, a knockout animal can be made which cannot
express a functional MRP protein. Such a knockout animal can be used, for
example, to test the effectiveness of a chemotherapeutic agent in the absence
of an
MRP multidrug resistance protein.
VIII. Uses of the Invention
The isolated nucleic acids of the invention are useful as molecular probes for
use diagnostically to determine multidrug resistance of a tumor. As
demonstrated
in Example 1, multidrug resistance of certain tumor cell lines is associated
with
increased expression of cellular mRNA corresponding to the nucleotide sequence
of
SEQ ID NO: 1. Accordingly, the nucleic acids of the invention can be labelled
with
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a detectable marker, such as a radioactive, fluorescent or bioti~rlated
marker, and
used in cornentional dot blot, Northern hybridization or in situ hybridization
procedures to probe mRNA molecules of total cellular or poly(A)+ RNAs from a .
biological sample, for instance cells of a tumor biopsy.
The nucleic acid probes can be used to detect genes, preferably in human
cells, that encode proteins related to or analogous to the MRP of the
invention.
Preferably, nucleic acid comprising the nucleotide sequence of the invention,
or a
segment thereof, can be used as a probe to identify DNA fragments comprising
genes or parts of genes that are co-ampliiied with the gene of the invention
and
which reside within the same amplification unit, or amplicon, at the
chromosomal
location 16p13.1. More specifically a nucleic acid of the invention can be
used as
a probe to screen human genomic DNA libraries constructed in cosmid or yeast
artifiaal chromosome vectors, using procedures standard in the art, to define
a
contiguous segment of DNA that comprises the amplification unit detected in a
multidrug resistant cell line such as H69AR. In this manner additional genes
can
be identified which also confer or contribute to the multidrug resistance
phenotype
of Hb9AR and other human cell lines yet to be examined but which are known to
include the HeLa cell line J2c and HT1080 DR4 cell line.
The antisense nucleic acids of the invention are useful for inhibiting
expression of nucleic acids (e.g. mRNAs) encoding proteins having MRP
activity,
thereby decreasing expression of proteins having MRP activity. Since increased
expression of proteins having MRP activity is associated with and can confer
multidrug resistance an a cell, decreasing expression of such proteins can
inhibit or
reverse multidrug resistance of a cell into which the antisense nucleic acid
has been
introduced. Antisense nucleic acids can be introduced into a multidrug
resistant cell
in culture to inhibit MRP expression. One or more antisense nucleic acids,
such as
oligonucleoddes, can be added to cells in culture media, typically at 200
~cg/ml. A
cultured multidrug resistant cell in which MRP expression is inhibited is
useful for
testing the efficacy of potential therapeutic agents. For example, MRP
expression
could be inhibited in a tumor cell line which expresses both MRP and P-
glycoprotein
to determine the contribution of MRP to an observed resistance or sensitivity
of the
cell to a particular therapeutic agent.
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The antisense nucleic acids of the invention, or oligonucleotides thereof, can
also be used in gene therapy to correct or prevent multidrug resistance in a
subject.
For example, antisense sequences can be used to render multidrug resistant
malignant cells sensitive to chemotherapeutic agents. Administration of
antisense
nucleic acids to a subject may be most effective when the antisense nucleic
acid is
contained in a recombinant expression vector which allows for continuous
production
of antisense RNA. Recombinant molecules comprising an antisense nucleic acid
or
oligonucleotides thereof, can be directly introduced into tissues, including
lung tissue
in vivo, using delivery vehicles such as liposomes, retroviral vectors,
adenoviral
vectors and DNA virus vectors. A delivery vehicle can be chosen which can be
targeted to a cell of interest in the subject (e.g. a multidrug resistant
tumor cell).
Antisense nucleic ands can also be introduced into isolated cells, such as
those of
the hematopoietic system, ex vivo using viral vectors or physical techniques
such as
microinjection and electroporation or chemical methods such as coprecipitation
and
incorporation of DNA into liposomes and such cells can be returned to the
donor.
Recombinant molecules can also be delivered in the farm of an aerosol or by
lavage.
In the treatment of lung malignancies, antisense sequences can be directly
delivered
to lung tissue by an aerosol or by lavage.
Accordingly, the invention provides a method for inhibiting mulddrug
resistance of a multidrug resistant cell by introducing into the multidrug
resistant cell
a nucleic acid which is antisense to a nucleic acid which encodes the protein
shown
in SEQ ID NO: 2.
The isolated nucleic aads and antisense nucleic acids of the invention can be
used to construct recombinant expression vectors as described previously.
These
recombinant expression vectors are then useful for making transformant host
cells
containing the recombinant expression vectors, for expressing proteins ended
by
the nucleic acids of the invention, and for isolating proteins of the
invention as
described previously. The isolated nucleic acids and antisense nucleic acids
of the
invention can also be used to construct transgenic and knockout animals as
described
previously.
As demonstrated in Examples 5 and 6, a recombinant expression vector
containing a nucleic acid of the invention can be used to transfect a drug
sensitive
cell line to produce a protein in the cell which can confer multidrug
resistance on
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the transfected cell line. Thus, the recombinant expression vectors of the
invention
are useful for conferring multidrug resistance on a drug sensitive cell.
Accordingly,
the invention provides a method for protecting a drug sensitive cell from
cytotoxicity
due to exposure to a drug by transfecting the cell with nucleic acid
comprising a
nucleotide sequence shown in SEQ ID NO: 1 to confer drug resistance on the
cell.
In preferred embodiments, the drug sensitive cell is a cardiac muscle cell or
a
hematopoietic stem cell. The ability to confer drug resistance on a cell has
important clinical applications. A major dose-limiting factor for
chemotherapeutic
agents is their cytotoxicity for normal cells in a patient as well as tumor
cells. In
patients with mufti-drug resistant tumors, increasing the dosage of
chemotherapeutic
agents is prohibited by the toxicity of these agents for normal cells. In the
case of
anthracyclines, cardiotoxicity of the drugs can be a major clinical
limitation. For
chemotherapeutic drugs which target rapidly dividing cells, toxicity to
hematopoietic
cells can be a major clinical limitation. Additionally, neurotoxicity can
occur.
Protecting nonresistant nontumor cells from the effects of chemotherapeutic
agents,
by conferring on the cell multidrug resistance, thus has major clinical
importance.
The transformant host cells of the invention, and recombinant expression
vectors used to make them, are useful for testing potential therapeutic agents
for
their effectiveness against multidrug resistant cells. These agents include
agents
which are themselves cytotoxic for multidrug resistant cells or which are
chemosensitizers of other therapeutic agents. As used herein, the term
"chemosensitizer" refers to a substance which can increase the efficacy of a
therapeutic agent against a multidrug resistant cell and/or decrease the
resistance
of a multidrug resistant cell for a therapeutic agent.
A method is provided for identifying a chemosensitizer of a therapeutic agent.
The method involves incubating the therapeutic agent with a cell transfected
with
a nucleic acid which confers resistance to the therapeutic agent on the cell,
both with
and without a substance to be tested, determining resistance of the cell to
the
therapeutic agent when incubated with and without the substance to be tested
and
identifying a substance which is a chemosensitizer of the therapeutic agent by
the
ability of the substance to decrease the resistance of the cell to the
therapeutic agent
when incubated with the substance as compared to the resistance of the cell to
the
therapeutic agent when incubated without the substance. In a preferred
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embodiment, the nucleic acid is a recombinant expression vector containing
nucleic
acid comprising a nucleotide sequence shown in SEQ ID NO:1. Preferably, the
cell
into which the nucleic acid is transfected is drug sensitive prior to
transfection so
that the effects of a potential chernosensitizer are assessed in the presence
of a
single, isolated multidrug resistance-conferring protein. The cell used to
test
potential chemosensitizing substances can be a cell in culture, e.g. a
transformant
host cell of the invention, and the therapeutic agent and substance to be
tested are
incubated in culture with the cell. Alternatively, the cell can be a multidrug
resistant
cell in a transgenic animal, transgenic for a nucleic acid of the invention,
and the
therapeutic agent and substance to be tested are administered to the
transgenic
animal. Furthermore, the cell can be a cell in culture isolated from a
multidrug
resistant transgenic animal of the invention. The resistance of the cell for
the
therapeutic agent in the presence and absence of the potential therapeutic
agent is
assessed by determining the concentration of the therapeutic agent which is
cytotoxic
for the cell either in the presence or in the absence of the substance being
tested.
The invention provides a method for identifying a substance which is directly
cytotoxic to a multidrug resistant cell involving incubating a substance to be
tested
with a cell transfected with a nucleic acid which confers multidrug resistance
on the
cell and determining the cytotoxicity of the substance for the cell. In a
preferred
embodiment, the nucleic acid is a recombinant expression vector containing
nucleic
acid comprising a nucleotide sequence shown in SEQ ID NO:1. Preferably, the
cell
into which the nucleic acid is transfected is drug sensitive prior to
transfection so
that the effects of a potential chemosensitizer are assessed in the presence
of a
single, isolated multidrug resistance-conferring protein. The cell used to
test
potential cytotoxic substances can be a cell in culture, e.g. a vansformant
host cell
of the invention, and the substance to be tested is incubated in culture with
the cell.
Alternatively, the cell can be a multidrug resistant cell in a transgenic
animal,
transgenic for a nucleic acid of the invention and the substance to be tested
is
administered to the transgenic animal. Furthermore, the cell can be a cell in
culture
isolated from a multidrug resistant transgenic animal of the invention.
Additionally, a multidrug resistant cell line such as H69AR, or an equivalent
cell line, can be used in the same methods for identifying a chemosensitizer
of a
therapeutic agent or for identifying a substance which is directly cytotoxic
to a
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mulddrug resistant cell.
The isolated proteins of the invention are useful for making antibodies
reactive against proteins having MRP activity, as described previously.
Alternatively,
the antibodies of the invention can be used to isolate a protein of the
invention by
standard immunoaffinity techiques. Furthermore, the antibodies of the
invention,
including bispecific antibodies and tetrameric antibody complexes, are useful
for
diagnostic purposes and for therapeutic purposes.
In one embodiment of the invention, antibodies labelled with a detectable
substance, such as a fluorescent marker, an enzyme or a radioactive marker,
can be
used to identify multidrug resistant tumor cells in a tumor sample or in vivo.
Tumor
tissue removed from a patient can be used as the tumor sample. In order to
prevent
tumor samples from being degraded, the samples can be stored at temperatures
below -20'C. A tissue section, for example, a freeze-dried or fresh frozen
section,
of tumor tissue removed from a patient, can also be used as the tumor sample.
The
samples can be fixed and the appropriate method of fixation is chosen
depending
upon the type of labelling used for the antibodies. Alternatively, a cell
membrane
fraction can be separated from the tumor tissue removed from a patient and can
be
used as the tumor sample. Conventional methods such as differential or density
gradient centrifugation can be used to separate out a membrane fraction.
A multidrug resistant tumor cell is identified by incubating an antibody of
the
invention, for example a monoclonal antibody, with a tumor cell to be tested
for
multidrug resistance. Binding of the antibody to the tumor cell is indicative
of the
presence on the tumor cell of a protein having MRP activity. The level of
antibody
binding to the tumor cell can be compared to the level of antibody binding to
a
normal control cell, and increased binding of the antibody to the tumor cell
as
compared to the normal cell can be used as an indicator of multidrug
resistance.
Binding of the antibody to a cell (e.g. a tumor cell to be tested or a normal
control
cell) can be determined by detecting a detectable substance with which the
antibody
is labelled. The detectable substance may be directly coupled to the antibody,
or
alternatively, the detectable substance may be coupled to another molecule
which
can bind the antibody. For example, an antibody of the invention which has a
rabbit
Fc region (e.g. which was prepared by immunization of a rabbit) can be
detected
using a second antibody directed against the rabbit Fc region, wherein the
second
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antibody is coupled to a detectable substance.
A multidrug resistant tumor cell can be detected as described above in vitro
in a tumor sample prepared as described above. For example, a tumor section on
a microsrnpe slide can be reacted with antibodies using standard
immunohistochemistry techniques. Additionally, if a single cell suspension of
tumor
cells can be achieved, tumor cells can be reacted with antibody and analyzed
by flow
cytometry. Alternatively, a multidrug resistant tumor cell can be detected in
vivo in
a subject bearing a tumor. Labelled antibodies can be introduced into the
subject
and antibodies bound to the tumor can be detected. For example, the antibody
can
be labelled with a radioactive marker whose presence and location in a subject
can
be detected by standard imaging techniques.
The antibodies of the invention, and compositions thereof can also be used
to inhibit the multidrug resistance of a mulddrug resistant cell. The
invention
provides a method for inhibiting the multidrug resistance of a multidrug
resistant cell
comprising inhibiting activity of a protein comprising an amino aad shown in
SEQ
ID NO: 2 expressed by the multidrug resistant cell. Preferably, the multidrug
resistant cell is a tumor cell. In preferred embodiments, the molecule which
binds
to a protein comprising an amino acid sequence shown in SEQ ID NO: 2 is a
monoclonal antibody, bispecific antibody or tetrameric immunological complex
of the
invention. Multidrug resistance can be inhibited by interfering with the MRP
activity
of the protein to which the molecule binds. For example, the ability of an MRP
protein to transport drugs may be impaired. Accordingly, any molecule which
binds
to a protein having MRP activity and whose binding inhibits the MRP activity
of the
protein are encompassed by invention. Isolated proteins of the invention,
comprising
the amino acid sequence shown in SEQ ID NO: 2, can be used to identify
molecules,
including and in addition to the antibodies of the invention, which can bind
to a
protein having MRP activity in a standard binding assay. A multidrug resistant
cell
in which multidrug resistance is inhibited, by inhibiting the activity of an
MRP
protein, can further be treated with a therapeutic agent to which the cell is
no longer
resistant or less resistant due to inhibition of MRP activity in order to kill
the cell.
Molecules which bind to a protein comprising an amino acid sequence shown
in SEQ ID NO: 2 can also be used in a method for killing a multidrug resistant
cell
which expresses the protein. Preferably, the multidrug resistant cell is a
tumor cell.
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Destruction of a multidrug resistant cells can be accomplished by labelling
the
molecule with a substance having toxic or therapeutic activity. The term
"substance
having toxic or therapeutic acitivit~" as used herein is intended to include
molecules
whose action can destroy a cell, such as a radioactive isotope, a toxin (e.g.
diptheria
toxin or ricin), or a chemotherapeutic drug, as well as cells whose action can
destroy
a cell, such as a cytotoxic cell. The molecule binding to multidrug resistant
cells can
be directly coupled to a substance having toxic or therapeutic activity (e.g.
a
ricin-linked monoclonal antibody), or may be indirectly linked to the
substance. For
example, a bispecific antibody which is capable of crosslinking a tumor cell
and a
cytotoxic cell can be used, thereby facilitating lysis of the tumor cell. A
bispeci~c
antibody can crosslink a tumor cell and the cytotoxic cell by binding to the
Fc
receptors of cytotoxic cells.
The compositions and methods of the invention can be used to treat patients
with tumors displaying mulddrug resistance particularly those displaying
resistance
to anthracyclines, epipodophyllotoxins, vinca alkaloids, and hydrophobic
drugs. The
methods of the invention for inhibiting the multidrug resistance of a tumor
cell and
for killing a multidrug resistant tumor cell can be applied to patients having
a
multidrug resistant tumor. The compositions and methods can be particularly
useful
in treating breast cancer, leukemias, fibrosarcomas, cervical cancer, and lung
cancer
in particular, small cell lung cancers and non small cell lung cancers.
The invention also provides a diagnostic kit for identifying multidrug
resistant
tumor cells comprising a molecule which binds to a protein comprising an amino
cad sequence shown in SEQ ID NO: 2 for incubation with a sample of tumor
cells;
means for detecting the molecule bound to the protein, unreacted protein or
unbound molecule; means for determining the amount of protein in the sample;
and
means for comparing the amount of protein in the sample with a standard.
Preferably, the molecule is a monoclonal antibody. Other molecules which can
bind
a protein having MRP activity can be used, including the bispeci8c antibodies
and
tetrameric antibody complexes of the invention. The diagnostic kit can also
contain
an instruction manual for use of the kit.
The invention further provides a diagnostic kit for identifying multidrug
resistant tumor cells comprising a nucleotide probe complementary to the
sequence,
or an oligonucleotide fragment thereof, shown in SEQ ID NO: 1 for
hybridization
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with mRNA from a sample of tumor cells; means for detecting the nucleotide
probe
bound to mRNA; means for determining the amount of mRNA in the sample; and
means for comparing the amount of mRNA in the sample with a standard. The
diagnostic kit can also contain an instruction manual for use of the kit.
The invention is further illustrated by the following examples. However, the
examples are merely intended to illustrate embodiments of the invention and
are not
to be construed to limit the scope of the invention. The rnntents of all
references
and published patents and patent applications cited throughout this
application are
hereby incorporated by reference.
Example 1: ISOLATION OF cDNA SEQUENCES DERIVED FROM
mRNAS OVEREXPRESSED IN H69AR CELLS
As part of a search of proteins responsible for the multidrug resistance
displayed by H69AR cells, a randomly primed cDNA library constructed from
H69AR mRNA was screened using differential hybridization with total cDNA
prepared from H69 and H69AR mRNA. One of the clones isolated contained a 2.8
kb cDNA insert and gave a particularly strong differential signal when
analyzed on
northern blots (Figure 1A). The analysis of lug of poly(A+)RNA from each cell
line
was carried out using standard procedures. Poly(A+)RNA was obtained using a
Fast
Track' mRNA isolation kit (Invitrogen) and 1 ug was electrophoresed on a
denaturing formaldehyde agarose gel. The RNA was transferred to nitrocellulose
membrane and prehybridized in 50% formamide, SX SSPE(1X = 150 mM NaCI,10
mM NaHiPO,,, 1 mM EDTA, pH 7.4), 2.5X Denhardt's solution (50X = 1% bovine
serum albumin, 1% polyvinylpyrrolidone, 1% ficoll) and sheared, denatured
herring
testes DNA (100 ~cg/ml) for 4-16 hours at 42°C. The blot was probed
with a 1.8 kb
EcoRI fragment of MRP, labelled to a specific activity of > 5 x 108 cmp/~eg
DNA
with a-[32P}-dCTP (3000 Ci/mmol; Dupont/NEN) by the random priming method
[A.P. Feinberg, B. Vogelstein, Analyt. Biochem. 132, 6 (1983)]. Hybridization
was
carried out for 16-20 hours at 42°C. Blots were washed three times in
0.1% SDS
and O.1X SSC(pH 7.0) for 30 minutes each at 52°C and then exposed to
film. To
estimate variation in RNA loading of the gel, the blot was reprobed with a
~P-labelled &actin cDNA (201pBv2.2)[H. Ueyama, H. Hamada, N. Battula, T.
Kakunaga, Mol. Cell. Biol. 4,1073 (1984)}. The autoradiograph shown in Figure
1A
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is a 5 hour exposure with intensifying screens at -70°C. The size of
the
overexpressed mRNA in H69AR cells was estimated to be approximately 7 kb.
Prolonged exposure of the film revealed low levels of this mRNA in H69 and
H69PR cells. The concentration of this mRNA was increased 100 fold in H69AR
. cells relative to H69 cells. The level of this mRNA in H69PR, a drug
sensitive
revenant of H69AR, had decreased approximately 20-fold relative to that found
in
H69AR, further substantiating the correlation of overexpression of this
particular
mRNA with a multidrug resistance phenotype.
Southern blot analyses of H69, H69AR and H69PR DNA indicated that the
major mechanism underlying overexpression was gene amplification. Ten ~cg of
each
DNA was digested with EcoRI, electrophoresed through a 0.7% agarose gel and
blotted onto a nitrocellulose membrane. The DNA was hybridized with a 1.8 kb
EcoRI cDNA fragment of MRP, labelled by random pruning with a-[~P]-dG"fP.
The autoradiograph shown in Figure 1B is a 6 hour exposure at -70°G
Based on
the examination of several restriction digests and normalization of the
amounts of
DNA loaded, the MRP gene was amplified 40-50 fold in resistant H69AR cells and
no differences in the copy number of the gene in H69 and H69PR cells were
detected.
The mRNA was also overexpressed 10-15 fold in a doxorubicin-selected
multidrug resistant HeLa cell line that does not overexpress P-glycoprotein
(Figure
1C). S3 and J2c are drug sensitive and resistant HeLa cell lines obtained from
the
laboratory of Dr. R.M. Baker (Roswell Park Memorial Institute). Two ~g of
poly(A+)RNA from each cell line was electrophoresed, blotted and probed with
MRP cDNA as described for Figure 1A. The MRP and B-actin autoradiographs
shown in Figure 1C are 18 hour and 1 hour exposures, respectively, at -
70°C.
Southern blotting of DNA from S3 and J2c cells indicated that the MRP gene was
amplified 10-15 fold in the resistant cell line. These findings provide
further
evidence of the association of elevated levels of this mRNA with multidrug
resistance.
The initial 2.8 kb cDNA clone was sequenced, allowing the isolation of
overlapping clones by rescreening the H69AR cDNA library with synthetic
oligonucleotides. A single, extended open reading frame of 1531 amino acids
was
defined encoding a protein designated as multidrug resistance associated
protein
CA 02448557 2003-11-25
WO 94110303 PCT/CA93/OOA39
(MRP). The translated GenBank and SwissProt databases wer a searched for
similarities to MRP using the FASTA program. The search revealed that MRP is
a novel member of the ATP-binding cassette (ABC) superfamily of transport
systems. Members of this superfamily are involved in the energy dependent
transport of a wide variety of molecules across cell membranes in both
eukaryotes
and prokaryotes. Included in this superfamily are the human multidrug
transporter
P-glycoprotein (MDR1) and the cystic fibrosis transmembrane conductance
regulator
(CFTR).
Example 2: RELATIONSHIP OF MRP TO OTHER MEMBERS OF
THE ABC TRANSPORTER SUPERFAMILY
The relationship of MRP to the various members of the ABC transporter
superfamily was examined using the PILEUP program from the Genetics Computer.
Group package (version 7) using a modified version of the progressive
alignment
method of Feng and Doolittle [J. Mol. Evol. 25, 351 (198'n]. A representative
selection of a phylogenetically broad range of ABC proteins that are comprised
of
hydrophobic transmembrane regions followed by nucleotide binding regions, and
whose sequences could be retrieved from GenBank and SwissProt databases, were
included in this analysis. The analysis divides this family of proteins into
two major
subgroups (Figure 2). One of the major subgroups consists of the cluster
containing
MRP {Hum/MRP), the leishmania P-glycoprotein-related molecule (Lei/PgpA) and
the CFTRs (Hum/CFTR, Bov/CFTR, Mus/CFTR and Squ/CFTR). The other
subgroup consists of the P-glycoproteins, the MHC class II-linked peptide
transporters (Hum JTap2, Mus/Tap 1), the bacterial exporters (Eco/HIyB,
Pas/LktB),
the heterocyst differentiation protein (Ana/HetA), the malarial parasite
transporter
(Pfa/Mdrl) and the yeast mating factor exporter (Ysc/Sted).
The dendrogram in Figure 2 indicates that MRP is only distantly related to
previously identified members of the ABC transporter superfamily. Although the
analysis suggests that it is most closely related to Lei/PgpA, the similarity
between
MRP and Lei/PgpA resides predominantly in two regions, both containing
signatures ,
of nucleotide binding folds (NBFs) (Figure 3A). The alignment was generated
using
PILEUP as described in Figure 2. The MRP sequence shown was compiled from
four overlapping lambda gtll cDNA clones. The alignment begins at a methionine
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residue in MRP that aligns with the initiator methionine of Lei/PgpA. The
predicted initiator methionine of MRP itself is located 66 amino acids
upstream.
Identical and conserved amino acids are identified in Figure 3A by double and
single
dots, respectively. The Walker A and B motifs and the 'active transport'
family
signature that are characteristic of nucleotide binding folds (NBFs) of ABC
transporters are indicated by single lines and denoted A, B, and C,
respectively. The
predicted transmembrane regions of each protein are indicated by double lines.
The
region in Lei/PgpA indicated by a dashed line has a mean hydrophobicity value
approaching that of a transmembrane region.
It has been proposed that the bipartite structure of P-glycoproteins reflects
duplication of an ancestral gene that occurred prior to the evolutionary
separation
of animals and plants. However, rnmparison of the NH2- and COOH-terminal
NBFs of MRP and Lei/PgpA revealed less similarity than typically found between
the two corresponding regions of P-glycoproteins. To determine whether this
was
a common structural feature of MRP, Lei/PgpA and Hum/CFTR, their NH2- and
COOH-terminal NBFs were aligned with each other and those of several
P-glycoproteins. One such comparison using human P-glycoprotein (Hum/Mdr1) as
an example is shown in Figure 3B. Shown in Figure 3B are the NHZ-terminal (N)
and COOH-terminal (C) halves of the deduced amino acid sequence of MRP
corresponding to ltpgpA (Lei/PgpA) (amino acids 650-799 and 1303-1463), human
CFTR (Hum/CFTR) (amino acids 441-590 and 1227-1385), and MDR1
(Hum/Mdrl)(amino acids 410-573 and 1053-1215). The sequences are presented as
aligned by PILEUP. Reverse type indicates that 3 of 4 amino acids at that
position
are identical or conserved. The conserved motifs A, B, and C described in
Figure
3A are underscored by a single line. The NHZ-terminal NBFs of MRP, Hum/CFTR
and Lei/PgpA share structural features that clearly distinguish them from the
NHZ-terminal NBF of Hum/Mdrl, particularly in the spacing of conserved motifs.
This difference in spacing also contributes to the relatively low similarity
between
NH2 and COOH-terminal NBFs in each of the three proteins. In addition, the
. COOH-terminal NBFs of MRP, Lei/PgpA and Hum/CFTR are more similar to
each other than to either the COOH or NH-terminal NBFs of Hum/Mdrl.
Similarity scores for the NH-terminal NBFs relative to MRP are: Lei/PgpA
(0.93),
Hum/CFTR (0.85) and Hum/Mdrl (0.60). Comparable COOH-terminal scores are
47
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Lei/PgpA (0.87), Hum/CFTR (0.84) and Hum/Mdrl (0.73). Similarity scores for
NH- and COOH-terminal NBFs within the same protein are: MRP (0.61),
Lei/PgpA (0.60), Hum/CFTR (0.62) and Hum/Mdrl (1.10). These observations,
combined with the overall analysis shown in Figure 2, suggest that MRP,
Lei/PgpA
and CF'1"R evolved from a common ancestor containing both NHz- and ,
COOH-terminal NBFs, which was distinct, or diverged from the ancestral gene of
the P-glycoproteins prior to the animal/plant separation. .
Example 3: EXPRESSION OF MRP IN NORMAL TISSUES
Despite knowledge of its structure and its ability to act as a drug efflux
pump,
the normal physiological roles) of P-glycoprotein has not been elucidated.
Some
possible clues to its function have been provided by its distribution in
normal tissues.
P-glycoprotein is highly expressed in secretory organs and tissues, such as
the
adrenals, kidneys, lumenal epithelium of the colon and the marine gravid
uterus.
It has also been detected in the lung although this finding is variable. Based
on the
cell types in which it is expressed, it has been postulated that P-
glycoprotein may be
involved in steroid transport and/or protection against xenobiotics. Northern
blot
analyses of total RNA preparations from a range of human tissues shown that
MRP
is expressed at relatively high levels in lung, testis and peripheral blood
mononuclear
cells (PBMCs)(Figure 4). Lung and testis RNAs were obtained from Clontech
Laboratories (Palo Alto, CA). PBMC RNA was prepared from cells isolated by
centrifugation over Ficoll-Isopaque (specific gravity 1.078 g/ml; Pharmacia)
of
peripheral blood from healthy volunteers. Total RNAs from lung, testis and
PBMCs
(30 fig) and H69AR cells (10 fig) were analyzed as for Figure 1A. The
autoradiograph shown in Figure 4 is from a blot probed with a 0.9 kb EcoRI
cDNA
fragment of MRP and exposed for 38 hours for the normal tissue RNAs and for 24
hours for the H69AR RNA The blot was stripped and reprobed with ~P-labelled
B-actin cDNA. The actin autoradiograph is a 24 hour exposure. MRP transcripts
were below the level of detection in placenta, brain, kidney, salivary gland,
uterus,
liver and spleen.
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Example 4: MAPPING OF THE HUMAN MRP GENE
The human CFTR and MDR1 genes have been mapped to chromosome 7 at
bands q31 and q21, respectively. The possible evolutionary relationship of MRP
to
these proteins prompted examination of the possibility that the MRP gene may
be
linked to one of these previously identified loci. In situ hybridization of a
1.8 kb
EcoRI fragment of MRP cDNA was performed using the method of Harper and
Saunders [Chromosoma 8, 431 (1981)]. Metaphase chromosomes on slides were
denatured for 2 minutes at 70°C in 70% deionized formamide, 2X SSC and
then
dehydrated with ethanol. The hybridization mixture consisted of SO% deionized
formamide,10% dextran sulfate, 2X SSC (pH 6), 20 ~g/ml sonicated salmon sperm
DNA and 0.2 ~cg/ml 3H-labelled MRP cDNA. The cDNA probe was labelled to a
specific activity of 8.5 x 10g cpm/ug DNA with [3H]-dTTP and [3H]-dATP (New
England Nuclear) using a Multiprime DNA Labelling System (Amersham) and
denatured in the hybridization solution at 70°C for 5 minutes. Fifty
~cl of the probe
solution was placed on each slide and incubated at 37°C overnight.
After
hybridization, the slides were washed in 50% deionized formamide, 2X SSC
followed
by 2X SSC (pH 7} and then dehydrated sequentially in ethanol. The slides were
coated with Kodak NTB/2 emulsion and developed after exposure for 5 weeks at
4°C. Chromosomes were stained with a modified fluorescence, 0.25%
Wright's stain
procedure (C.C. Lin, P.N. Daper, M. Braekeleer, C~ytogenet. Cell Genet. 39,
269
(1985)]. The positions of 200 silver grains directly over or touching well-
banded
metaphase chromosomes were recorded on the ISCN-derived idiogram of the human
karyotype. A significant clustering of grains (40) was observed in the 16p
region
(p<0.0001) and the peak of the distribution was at 16p13.1, confirming that
MRP
was not linked to either CFTR or MDR genes. Approximately 160 metaphases were
examined. These results are summarized in Figure 5.
Example 5: EXPRESSION OF MRP IN A DRUG SENSITIVE CELL
CONFERS DOXORUBICIN RESISTANCE ON THE CELL
While increased concentrations of MRP and mRNA have been detected in
multidrug resistant cell lines derived from a variety of tissues and several
of these
cell lines have also been shown to contain multiple copies of the MRP gene as
a
result of amplification and translocation of a region of chromosome 16
spanning the
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MRP gene at band p13.1, it remained possible, in view of the multistep
selection
procedures used to derive the cell lines, that overexpression of the MRP gene
is only
one component of a set of alterations required to confer multidrug resistance.
The
ability of MRP alone to confer drug resistance on a drug sensitive cell line
was
determined by constructing an MRP expression vector, transfecting the
expression
vector into drug sensitive cells and assessing the relative drug resistance of
the
transfected cell populations.
A DNA fragment corresponding to the complete coding region of MRP
mRNA plus 86 nucleotides of 5' and 32 nucleotides of 3' untranslated sequence
was
assembled and transferred into the expression vector pRc/CMV under the control
of the human cytomegalovirus promoter. A DNA fragment containing the complete
coding region of MRP mRNA was assembled in the vector, pBluescript 11 KS+
(Stratagene), using overlapping cDNA clones or PCR products generated from
these
clones. The fidelity of the MRP sequence was con firmed by DNA sequence
analysis
before moving the intact MRP fragment to the eukaryotic expression vector,
pRc/CMV (Invitrogen). The integrity of the MRP fragment in the expression
vector
was assessed by detailed restriction mapping and DNA sequence analysis of the
cloning sites. In the pRc/CMV vector, MRP expression is under the control of
the
enhancer/promoter sequence from the immediate early gene of human
cytomegalovirus. The MRP transcript also contains part of the 3' untranslated
region and the polyadenylation signal from bovine growth hormone mRNA which
is provided by the vector. Thus, the pRc/CMV-MRP construct generates a
transcript of 5.2 to 5.3 kb that includes the entire coding sequence (86
nucleotides
of which are derived from MRP mRNA sequence), and approximately 250
nucleotides of 3' untranslated sequence (32 nucleotides of which are derived
from
MRP mRNA sequence). This vector also contains the bacterial aminoglycoside 3'
phosphotransferase gene which confers resistance to geneticin (G418).
HeLa cells were transfected with either the parental vector, or the vector
containing the MRP coding region, using supercoiled DNA and a standard calcium
phosphate transfecdon procedure. HeLa cells were uansfected with the pRc/CMV
vector or the vector containing the MRP coding sequence using a standard
calcium
phosphate transfection procedure [J. Sambrook, E.F. Firtsch, T. Maniatis,
Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
Harbor, NY (1989)]. Approximately, 50,00(? cells in each well of a 6-well
tissue
culture plate were exposed for 16 hours to 10 beg of supercoiled DNA in a
calcium
phosphate precipitate. After forty-eight hours, the growth medium was changed
to
include 6418 at 200 ~g/ml which selected for cells that expressed the neomycin
resistance gene encoded by the pRc/CMV vector. Three weeks later, six
independently transfected populations of cells were tested for resistance to
. doxorubicin using a tetrazolium salt microtiter plate assay (S.P.C. Cole,
Cancer
' Chemother. Pharmacol. 26, 250 (1990)). Those populations demonstrating
increased
relative resistance to the drug were expanded for testing for cross-resistance
to other
cytotoxic drugs, and analysis of MRP mRNA and protein levels.
Poly(A)+ RNA was isolated using the Micro-FastTrack RNA isolation kit
(Invitrogen). The RNA was subjected to electrophoresis on a
formaldehydeagarose
gel and transferred to Zetaprobe membrane (Bio-Rad). The blots were hybridized
with 3zP-labeled cDNA fragment probes complementary to the mRNas for MRP,
MDRl (A.M. Van der Bliek, F. Baas, T. Ten Houte de Lange, P.M. Kooiman,
T.Van der Velde Koerts, P. Borst, EMBO J. 6, 3325 (1987)], topoisomerase 11 a
[T.D.Y. Chung, F.H. Drake, K.B. Tan, S.R. Per, S.T. Crooke, C.K. Mirabelli,
Proc.
Natl. Acad. Sci. U.S.A. 86 9431 (1989)], topoisomerase 11 B [ibid.], annexin
ll (S. P.
C. Cole, M. J. Pinkoski, G. Bhardwaj, R. G. Deeley, Br. J. Cancer 65, 498
(1992)),
and a region of the pRc/CMV vector encoding part of the 3' untranslated region
and
polyadenylation signal from the bovine growth hormone gene. Hybridization of
the
probes was quantified by densitometry of the autoradiographs on a Molecular
Dynamics Computing Densitometer. Care was taken to compare autoradiographic
exposures that were within the linear range of the film. In addition,
variations in
loading of RNA on the gels were estimated by probing blots with a 3zP-labeled
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAfragment (ATCC/NIH
#57090), and by densitometric scanning of the ethidium bromide-stained
ribosomal
RNA bands on photographic negatives of the RNA gels.
The relative amounts of MRP protein were assessed by immunoblot analysis
of total cell extracts and membrane-enriched fractions. Cell pellets were
. resuspended at 5 X 10' cells/ml in buffer containing lOmM Tris-HCI, pH 7.4,
lOmM
KC1,1.5 mM MgCI,, and protease inhibitors (2mM phenylmethylsulfonyfluoride, 50
S1
CA 02448557 2003-11-25
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ug/ml antipain, 2 ~g/ml aprotinin, 200 ~g/ml EDTA, 200 ~g/ml benzamidine, 1
ug/ml pepstatin). After 10 min on ice, cells were homogenized with
approximately
80 strokes of a Tenbroeck homogenizer. The homogenate was adjusted to 250 mM
in sucrose before remaining intact cells and nuclei were removed by
centrifugation
at 800 X g at 4°C for 20 min. To prepare a membrane-enriched fraction,
the ,
supernatant was centrifuged at 100,000 X g at 4°C for 20 min in a
Beckman TIr100
ultracentrifuge and the pellet resuspended in 10 mM Tris-HCI, pH 7.4, 125 mM
sucrose, and the protease inhibitors listed above. For sodium dodecyl sulphate
(SDS) polyacrylamide gel electrophoresis and immunoblotting, appropriate
amounts
of protein were mixed 1:1 with solubilizing buffer (final concentration 4 M
urea,
0.5% SDS, 50 mM dithiothreitol). Samples were loaded without heating onto a 7%
resolving gel with a 4% stacking gel. Proteins were transferred to Immobilon-P
PVDF membranes (Millipore) using 50 mM 3-(cyclohexylamino)-1-propanesulfonate,
pH 11Ø For detection of MRP, blots were incubated with an affinity-purified,
rabbit polyclonal antibody raised against a synthetic peptide, the sequence of
which
was predicted from that of the cloned MRP cDNA and which is not found in any
other known protein. Antibody binding was visualized with horseradish
peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence
detection (Amersham). The affinity-purified anti-MRP antibody recognizes a
glycosylated, integral membrane protein with an apparent molecular weight of
190
kilodaltons. In its deglycosylated form, the molecular weight of the protein
decreases to 165- to 170 kilodaltons which is in agreement with the molecular
weight
of 171 kilodaltons predicted from the primary amino acid sequence of MRP.
At this time, the level of 6418 in the growth medium was increased to 400
or 800 ug/ml without any noticeable effect on the growth rate of cells
transfected
with either the parental vector or the vector containing the MRP coding
sequence.
Transfected populations have been grown continuously for up to four months in
6418-containing medium without any change in the level of resistance to
doxorubicin. Integration of these vectors into genomic DNA has the potential
to
alter the expression of endogenous genes that might adventitiously increase
drug
resistance. Consequently, chemotherapeutic drugs were not used as selecting
agents.
Populations of transfected cells were selected solely by their ability to grow
in the
presence of 6418. Since cells overexpressing MRP do not display increased
52
CA 02448557 2003-11-25
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resistance to this antibiotic, variable levels of expression of MRP are to be
expected
in the transfected cell populations.
The relative resistan~s to doxorubian are shown for two examples of 6418
resistant cell populations transfected with the MRP expression vector (T2 and
TS),
as well as untransfected Hela cells and a population transfected with the
parental
vector (C1) (Fig. 6). Key: HeLa cells (~); HeLa cells transfected with the
expression vector pRc/CMV (C1, °); HeLa cells transfected with the
vector
containing the MRP coding sequence (1'2, O; T5, ~); and a clone isolated from
the
doxorubicin-resistant uansfected TS cells shown (eT5-5). Each point represents
the
mean of triplicate determinations in a single experiment and standard
deviations
were < 5%. Similar results were obtained in three additional experiments. The
ICS
is indicated on the figure and is defined as the concentration of doxorubicin
required
to decrease by 50% the values obtained with untreated cells. In the examples
shown, one of the populations transfected with the MRP expression vector (T2)
displayed little change in doxorubian resistance while resistance of the other
(TS)
was increased 15-fold. In addition, several clones from the resistant
population were
grown in the presence of 6418 and their degree of doxorubicin resistance
determined. Dose response curves for two of the transfectants (T2, TS) and for
one
of the clones (T5-5) were then compared to determine whether their resistance
to
doxorubicin correlated with the concentrations of MRP mRNA.
The MRP mRNA produced from the expression vector has a predicted length
of 5.2 to 5.3 kb including a poly(A) tail, thus allowing it to be
distinguished from the
longer, endogenous MRP mRNA by Northern analysis. A blot of poly(A)+ RNA
from the cell populations shown in Fig. 6 that was hybridized with a cloned
cDNA
probe corresponding to part of the MRP coding sequence, revealed a relatively
abundant mRNA of approximately 5.3 Kb in the resistant transfectants and low
levels of the endogenous MRP mRNA (Fig. 7A). The relative concentration of the
5.3 kb mRNA is 70- to 80-fold and 20- to 30- fold higher in the resistant cell
population (TS) and clone (TS-5), respectively, than- that of endogenous MRP
mRNA present in the control population (C1). Relative levels of mRNAs were
determined by densitometry and normalization to the levels of GAPDH mRNA.
Expression of the 5.3 kb MRP mRNA in the transfected cell population which
showed little change in resistance (T2) was only approximately half that of
53
CA 02448557 2003-11-25
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endogenous MRP mRNA. Similar RNA blots were also probed with a DNA
fragment from the pRc/CMV plasmid that forms part of the 3' untranslated
region
of the vector encoded MRP mRNA. This probe hybridized only with the 5.3 kb
MRP mRNA, confirming that it was transcribed from the vector and did not
result
from the increased expression of an endogenous MRP-related gene (Fig. 7B).
Thus
in cells transfected with the MRP expression vector the relative level of drug
resistance increases with the concentration of MRP mRNA.
The concentration of endogenous MRP mRNA in the multidrug resistant
H69AR cells (labeled AR in the figures) is approximately 100-fold higher than
in
the H69 parental cells (labeled H69 in the figures) and the relative
resistances of the
two cell lines to doxorubicin also differ by 50- to 100-fold. Vector encoded
MRP
mRNA levels in the TS Hel,a cell population are 70- to 80-fold higher than
endogenous MRP mRNA levels in the parental cells. However, drug resistance is
increased only 15-fold. To investigate why the relative increase in drug
resistance
was lower in the transfectants than in H69AR cells, we compared the levels of
MRP
mRNA and protein in the two different cell types. Northern analysis revealed
that
the levels of endogenous MRP mRNA in H69 cells and HeLA cells transfected with
the pRc/CMV parental vector were similar. The relative abundance of vector
encoded MRP mRNA in the drug resistant transfectant cell population (TS) was
also
comparable to that of endogenous MRP mRNA in H69AR cells (Fig. 7C).
However, a protein blot with affinity purified anti-MRP antibody indicated
that the
level of protein in the TS HeLa cell transfectants was 5- to 8-fold lower than
in
H69AR cells (Fig. 7C). These findings are consistent with the 15-fold increase
in
resistance observed in the transfected TS cells compared to the 50- to 100-
fold
increase in H69AR cells. The lower level of protein in the transfected cells
is most
likely attributable to a difference in translational efficiency between the
vector
encoded and endogenous MRP mRNAs, although a difference in rates of
degradation of the protein between the two cell types cannot be excluded.
Since H69AR cells were obtained by multistep selection, it is possible that
additional alterations have occurred which may, either independently or in
concert
with MRP, influence their degree of resistance to some drugs. H69AR cells have
been shown to have decreased levels of topoisomerase 11 a and B mRNA and
protein
which could enhance their resistance to anthracyclines and
epipodophyllotoxins.
54
CA 02448557 2003-11-25
WO 94!10303 PCT/CA93/00439
They have also been shown to overexpress anne~n 11 which may affect the
trafficking
of membraae proteins. C.D. Evans, S. E. L Mirski, M. K Danks, W. T. Beck, S.
P.
C. Cole, Proc. Amer. Assoc. Cancer Res. 33, 2694 (1992). Annexin a has been
shown to be involved in formation of fusogenic vesicles and in exocytosis. S.
P. C.
Cole, M. J. Pinkoski, G. Bhardwaj, R. G. Deeley, Br. J. Cancer 65, 498 (1992).
It
is unknown to what extent these additional changes influence the degree of
' resistance of H69AR cells or whether they are linked in any way to
overexpression
of MRP. However, overexpression of MRP in the transfected cells does not alter
the levels of mRNAs specifying either topoisomerase ll isoform (Fig. 8A) or
annexin
ll (Fig. 8B), nor do the transfected HeLa cells display any alterations in the
level of
Mdrl mRNA. These observations strongly support the conclusion that increased
resistance to doxorubicin in the transfected cells is directly attributable to
overexpression of MRP.
Example 6: EXPRESSION OF MRP IN A DRUG SENSITIVE CELL
CONFERS MULTIDRUG RESISTANCE ON THE CELL
To determine whether the increased doxorubicin resistance of transfeeted
cells was accompanied by increased resistance to other classes of
chemotherapeutic
drugs, the cells were tested for cross-resistance to vincristine (a Vinca
alkaloid),
VP-16 (an epipodophyllotoxin) and cisplatin (Fig. 9). Cytotoxiaty assays were
performed on untransfected HeLa cells (~), HeLa cells transfected with the
expression vector pRc/CMV (C!, °), HeLa cells transfected with the
expression
vector pRc/CMV-MRP and maintained in 6418 at 400 ug/ml for 4 months (T5, ~),
and TS cells maintained at 800 ug/ml 6418 for 1 month (TS-800/1, o) and 3
months
(TS-800/3, C~. Each point represents the mean of triplicate determinations in
a
single experiment and standard deviations were < 5%. Similar results with
vincristine and VP-16 were obtained in two to three additional experiments.
The
IC~,~ of the various cell lines are indicated on the figure. Dose response
curves for
several independently propagated cultures of MRP transfectants indicate that
they
~ are approximately 25-fold and 5- to 10-fold resistant to vincristine and VP-
I6,
respectively, relative to untransfected HeLa cells or cells transfected with
parental
vector (C!). The transfectants showed no increase in cisplatin resistance
which is
consistent with the pharmacological phenotype of H69AR cells and which is also
CA 02448557 2003-11-25
WO 94!10303 PCTlCA93l00439
characteristic of cells that overexpress P-glycoprotein. These results
demonstrate for
the first time that this phenotype can be conferred by a member of the ABC
superfamily of transporters that is structurally very different from the
P-glycoproteins.
Example 7: PREPARATION OF ANTI-MRP ANTZBOD1ES
AND USE THEREOF IN IMMUNOPRECIPITATION
AND IMMUNOBLOTTING EXPERIMENTS
MRP is encoded by a mRNA of approximately 6.5 kb with an extended open
reading frame of 1531 amino acids. The protein is predicted to contain two
nucleotide binding folds (NBFs) and 12 transmembrane regions, divided 8 and 4
between the NH2 and COOH-proximal halves of the molecule, respectively. To
confirnn that a protein of the predicted size and sequence is overexpressed in
resistant H69AR cells, polyclonal antibodies were prepared against synthetic
peptides
based on the deduced amino acid sequence of MRP and used in immunoanalyses.
One peptide of sequence AELQKAEAI~EE was selected from the highly
divergent cytoplasmic linker domain of MRP (MRP-L, position 932-943) while the
second peptide (GENLSVGQRQLVCLA) was chosen from the second nucleotide
binding domain of MRP (MRP-2, position 1427-1441). Both peptides were
synthesized on Ultrasyn D resin for direct immunization by the Biotechnology
Service Centre at the Hospital for Sick Children (Toronto, Ont.).
Approximately 400
ug of bound peptide was resuspended in distilled water and sonicated. The
resulting
suspension was emulsified in an equal volume of complete Freund's adjuvant
(Difco)
and injected s.c. at four sites in 3-month old female New Zealand White
rabbits. At
2- to 3-week intervals, the same amount of immunogen emulsified in inrnmplete
Freund's adjuvant was injected s.c. Rabbits were bled by arterial puncture
beginning
2 weeks following the third immunization and their sera were tested for the
presence
of antibodies by an enzyme-linked immunosorbent assay (ELISA) and by
immunoblotting.
Rabbit antisera obtained after immunization with peptide MRP-L that were
positive by ELISA or western blotting were concentrated by ammonium sulfate
preapitation and purified by affinity chromatography. Affinity columns were
constructed by coupling the MRP-L peptide to CNBr-activated Sepharose (5 umole
56
CA 02448557 2003-11-25
WO 94!10303 PCT/CA93/00439
peptide/ml gel) according to the instructions of the supplier (Pharmaaa LKB
Biotechnology Inc.) followed by extensive washing with 10 mM Tris, pH ?.5. The
ammonium sulfate precipitate was dissolved in phosphate-buffered saline,
dialyzed
extensively against the same buffer and then applied to the prewashed affinity
column. The loaded column was washed first with 10 mM Tris pH 7.5 followed by
mM Tris, pH 7S, OS M NaCI before eluting the antibody with 0.1 M giycine, pH
2S. Fractions were neutralized in collection tubes containing 1 M Tris, pH
8Ø The
desired fractions were pooled, dialyzed extensively against phosphate-buffered
saline
and concentrated by Amicon concentrators/filtration. The final protein
concentration of the purified antibody was adjusted to 0.7-1.5 mg/ml. Rabbit
antisera obtained after immunization with peptide MRP-2 were used without
further
purification.
ELISA positive antisera from these rabbits were used in immunoblot analyses.
Polyacrylamide gel electrophoresis was carried out by the method of Laemmli
with
a 5 % or 7 % separating gel and a 4% stacking gel. Samples were diluted 1:1 in
solubilizing buffer to a final concentration of 4 M urea, OS% SDS, 50 mM DTT
and
loaded on the gels without heating. For immunnblotting, proteins were
transferred
after gel electrophoresis to Immobilon-P PVDF membranes (Millipore,
Mississauga.
Ont.) using SO mM CAPS, pH 11Ø Blots were incubated for 1 h in blocking
solution (5 % normal goat serum/5% HyClone serum/1% BSA) in TBS-T (10 mM
Tris, pH 7.5, 0.05 % Tween 20, 150 mM NaCI). Anti-MRP antibodies were added
directly to the blocking solution and incubated for 2 h. The blot was washed 3
x 5
min in TBS-T and goat anti-rabbit IgG horseradish peroxidase-conjugate
[affinity
purified F(ab')2 fragment (Jackson ImmunoResearch) or whole molecule (ICN
Biomedicals)] diluted in blocking buffer added. After a 1 h incubation, the
blot was
washed 5 x 5 min in TBS-T, and antibody binding detected by ECL (Amersham,
UK) and exposure on Kodak XOMAT film. The antisera detected a 190 kD protein
in resistant H69AR cells which was not detectable in sensitive H69 and
revenant
H69PR cells.
The antisera were also used in immunoprecipitation experiments using cell
membrane preparations of cells metabolically labelled with ~S-methionine.
Cells
were cultured in 50 uCi/ml ~Smethionine (Tran ~S-label; cell labelling grade;
specific activity, 710 Ci/mmol) (Dupont NEN) overnight in methionine-deficient
57
CA 02448557 2003-11-25
WO 94110303 PGT/CA93100439
RPMI 1640 medium (Sigma) or with 500 ~Ci/mt ~P-orthophosphoric aad (Carrier
free, 500 mCi/ml) (Dupont NF.N) in phosphate-deficient RPMI 1640 medium (ICN)
for 4 h. Crude radiolabelled 100,000 x g membrane-enriched fractions were
prepared
and immunoprecipitated as follows. Frozen or fresh cells (50 x lOb/ml) were
suspended in 10 mM Tris-HCI, pH 7.4 containing 10 mM KC1,1.5 mM MgCl2 with
protease inhibitors (2 mM phenylmethylsulfonylfluoride, 50 ~g/ml andpain,
2 ug/ml aprotinin, 200 ug/ml EDTA, 200 ~g/ml benzamidine, OS ~ug/ml leupeptin,
1 ~g/ml pepstatin) and 0.025 mg/ml RNase A and 0.05 mg/ml DNase 1. After 10
min., the suspension was homogenized in a chilled Tenbroeck homogenizer with
80
strokes of the pestle. The homogenate was then centrifuged at 800 x g at
4°C for 15
min. to remove nuclei and remaining intaM cells. A membrane-enriched fraction
was prepared by ultraoentrifugation of the supernatant at 100,000 x g at
4°C for 20
min. The pellets were resuspended in 10 mM Tris HCI, pH 7.6 with 125 mM
sucrose and protease inhibitors as above. Protein concentrations were
determined
by the Peterson modification of the Lowry assay and aliquots were stored at -
80°C.
Proteins were solubilized in 1% CHAPS,100 mM KCI, 50 mM Tris-HCI, pH
7.5, at a detergent to protein ratio of 20:1 for 1 h at 4°C with
frequent vortexing
followed by centrifugation at 100,000 x g for 20 min using a T1003 rotor in a
Beckman Ultracentrifuge. The supernatant (whatever percentage of protein is
solubilized from an initial 40 ug of membrane protein) was incubated with
affinity
purified MRP-L antisera (25 mg solubilized in 1% CHAPS, 100 mM KCI, 50 mM
Tris-HCi, pH 7.5) overnight at 4°C. The samples were made up 700 u1
with 1 %
CHAPS buffer then incubated with 59 u1 (10% w/v) Protein A Sepharose Cl-4B
(Pharmacia) for 3 h at 4°C with gentle rocking. The samples were
centrifuged for
sec at 14,000 x g and sequentially washed for 5 min with 1 ml each of Buffer 1
(10 mM Tris-HCI, pH 8.0, 0.5 mM NaCI, 0.5% Nonidet P-40, 0.05% SDS), Buffer
2 (10 mM Tris-HCI, pH 8.0, 0.15 M NaCI, 0.5% Nonidet P-40, 0.05% SDS, 0.5%
deoxycholate) and Buffer 3 (10 mM Tris-HCI, pH 8.0, 0.05% SDS). The washed
beads were incubated with 100 gel of 4 M urea, OS% SDS, 50 mM DTT for 1 h at
room temperature with frequent vortexing. The samples were centrifuged and the
supernatants analyzed on 7% polyacrylamide gels. The gels were fixed in
isopropanol:water:acetic acid (25:65:10) for 30 min followed by the addition
of the
fluorographic reagent Amplify (Amersham). The gels were dried and then exposed
58
CA 02448557 2003-11-25
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to film overnight at -80°C. A 190 kD protein was detectable by
immunoprecipitation of membrane-associated proteins from ~S-methionine
labelled
H69AR cells with the immunoreactive antisera.
The apparent molecular weight of the immunodetectable 190 kD protein in
the H69AR cell membranes is approximately 20 kD greater than the predicted 171
kD molecular weight of MItP based upon the deduced primary amino acid
sequence.
However, analysis of the MItP sequence indicates the presence of three
potential N-
glycosyladon sites in regions predicted to be asymmetrically distributed about
a
membrane bilayer. To determine whether or not the 190 kD protein was N-
glycosylated, two sets of experiments were carried out. First, resistant H69AR
cells
were grown in the presence of tunicamycin, a potent inhibitor of N-linked
glycosylation. N-linked glycosylation was inhibited in H69AR cells by
culturing in
15 I~g/ml tunicamycin (Sigma) for 24 h. Treated cells were washed twice with.
phosphate-buffered saline and then whole cell lysates were prepared by
homogenization in lysis buffer (20 mM Tris HCl, pH 7.5, 20 mM KCI, 3 mM MgCl2.
0.5 mg/ml DNase 1, 0.25 mg/ml RNase A) with protease inhibitors as described
above. Polyacrylamide gel eletrophoresis and immunoblotting of the whole cell
lysates were carried out as before. In the second approach, HG9AR 100,000 x g
membranes were incubated with the deglycosylase PNGase F. Membrane-enriched
fractions (200 ~g protein) were diluted to a Foal concentration of 1 ug/ml in
50 mM
Na phosphate buffer, pH 7.5, containing 25,000 NEB units PNGase F (New England
Biolabs). After 8 h at 37°C, an additional 25,000 NEB units PNGase F
was added
followed by incubation overnight at 37°C. Sample buffer was added
directly and
SDS-PAGE and immunoblotting carried out as before. In both cases, a 170 kD
protein was detected by immunoblot analyses which correlates well with the 171
kD
predicted molecular weight of M1ZP.
To confirm that MRP is an ATP-binding protein, as suggested by the
presence of ATP-binding signature motifs, membranes from resistant H69AR and
sensitive H69 cells were photolabelled with ~P-8-azido ATP. Crude membrane-
- enriched fractions were resuspended at 1 ug/~d protein in 10 mM Tris-HCI, pH
7.6,
buffer containing 1 mM MgCl2 and protease inhibitors as described above. After
the
' addition of 3-4 ~eCi 32P-8-azido-ATP (specific activity 2-10 Ci/mmol; ICN
Biomedical, Mississauga, Ont.), incubation on ice was continued for 1-5 min.
The
59
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
azido-ATP was cross-linked to the protein on ice by irradiation at 366 nm
about 10
cm from the light source for 8 min. using a Stratalinker set at 1100 ~W. The
labelled proteins were stored at -80°C until polyacrylamide gel
electrophoresis or
immunoprecipitations were carried out. Specificity of the labelling was
confirmed by
competition with cold excess ATP (Boehringer Mannheim, Laval, Que.) which was
added to the membrane preparations prior to the addition of ~P-8-azido-ATP.
These studies reveaied strong, specific labelling of a 190 kD protein in
membranes
from the H69AR cells that was not detected in drug sensitive H69 cells. Our
results
indicate that in H69AR cells, the MRP gene encodes an N-glycosylated ATP-
binding
protein of 194 kD.
Forming part of the present disclosure is the appended Sequence Listing for
the multidrug resistance protein of the present invention.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than routine experimentation, many equivalents of the specific embodiments of
the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Cole, Susan P.C.
Deeley, Roger G.
(ii) TITLE OF INVENTION: MULTIDRUG RESISTANCE PROTEIN
(iii)NUMBER
OF
SEQUENCES:
2
(iv)CORRESPONDENCE
ADDRESS:
(A) ADDRESSEE: LAHIYE & COCKFIELD
(B) STREET: 60 STATE STREET, SUTTE
510
(C) CITY: BOSTON
(D) STATE: MASSACHUSETTS
(E) COUNTRY: USA
(F) ZIP: 02109
(v) COMPUTER
READABLE
FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release h1.0,
Version #1.25
(vi)CURRENT
APPLICATION
DATA:
(A) APPLICATION NUMBER: US 000000
(B) FILTNG DATE: -OC'T'-1993
(C) CLASSIFICATION:
(viii)ATTORNEY/AGENT
INFORMATION:
(A) NAME:
(B) REGISTRATION NUMBER: 000000
(C) REFERENCE/DOCKET NUMBER: PQI-002
(ix)TELECOMMUNICATION
INFORMATION:
(A) TELEPHONE: (617) 227-7400
($) TELEFAX: (617) 227-5149
(2) INFORMATION FOR SEQ ID NO:1:
(ij SEQUENCE CHARACTERTSTICS:
(A) LENGTH: 5011 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATTON: 196..4788
61
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
(xi.) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CAGGCGGCGT TGCGGCCCCG GCCCCGGCTC CCTGCGCCGC CGCCGCCGCC GCCGCCGCCG 60
CCGCCGCCGC CGCCGCCAGC GCTAGCGCCA GCAGCCGGGC CCGATCACCC GCCGCCCGGT 120
GCCCGCCGCC GCCCGCGCCA GCAACCGGGC CCGATCACCC GCCGCCCGGT GCCCGCCGCC 180
GCCCGCGCCA CCGGC ATG GCG CTC CGG GGC TTC TGC AGC GCC GAT GGC TCC 231
Met Ala Leu Arg Gly Phe Cys Ser Ala Asp Gly Ser
1 S 10
GAC CCG CTC TGG GAC TGG AAT GTC ACG TGG AAT ACC AGC AAC CCC GAC 279
Asp Pro Leu Trp Asp Trp Asn Val Thr Trp Asn Thr Ser Asn Pro Asp
15 20 25
TTC ACC AAG TGC TTT CAG AAC ACG GTC CTC GTG TGG GTG CCT TGT TTT 327
Phe Thr Lys Cys Phe Gln Asn Thr Val Leu Val Trp Val Pro Cys Phe
30 35 ~ 40
TAC CTC TGG GCC TGT TTC CCC TTC TAC TTC CTC TAT CTC TCC CGA CAT 375
Tyr Leu Trp Ala Cys Phe Pro Phe Tyr Phe Leu Tyr Leu Ser Arg His
45 50 55 60
GAC CGA GGC TAC ATT CAG ATG ACA CCT CTC AAC AAA ACC AAA ACT GCC 423
Asp Arg Gly Tyr Ile Gln Met Thr Pro Leu Asn Lys Thr Lys Thr Ala
65 70 75
TTG GGA TTT TTG CTG TGG ATC GTC TGC TGG GCA GAC CTC TTC TAC TCT 471
Leu Gly Phe Leu Leu Trp Ile Val Cys Trp Ala Asp Leu Phe Tyr Ser
80 85 90
TTC TGG GAA AGA AGT CGG GGC ATA TTC CTG GCC CCA GTG TTT CTG GTC 519
Phe Trp Glu Arg Ser Arg Gly Ile Phe Leu Ala Pro Val Phe Leu Val
95 100 105
AGC CCA ACT CTC TTG GGC ATC ACC ACG CTG CTT GCT ACC TTT TTA ATT 567
Ser Pro Thr Leu Leu Gly Ile Thr Thr Leu Leu Ala Thr Phe Leu Ile
110 115 120
CAG CTG GAG AGG AGG AAG GGA GTT CAG TCT TCA GGG ATC ATG CTC ACT 615
Gln Leu Glu Arg Arg Lys Gly Val Gln Ser Ser Gly Ile Met Leu Thr
125 130 135 .140
TTC TGG CTG GTA GCC CTA GTG TGT GCC CTA GCC ATC CTG AGA TCC AAA 663
Phe Trp Leu Val Ala Leu Val Cys Ala Leu Ala Ile Leu Arg Ser Lys
145 150 155
ATT ATG ACA GCC TTA AAA GAG GAT GCC CAG GTG.GAC CTG TTT CGT GAC 711
Ile Met Thr Ala Leu Lys Glu Asp Ala Gln Val Asp Leu Phe Arg Asp ,
160 165 170
ATC ACT TTC TAC GTC TAC TTT TCC CTC TTA CTC ATT CAG CTC GTC TTG 759 _
Ile Thr Phe Tyr Val Tyr Phe Ser Leu Leu Leu Ile Gln Leu Val Leu
175 180 185
62
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
TCC TGT TTC TCA GAT CGC TCA CCC CTG TTC TCG GAA ACC ATC CAC GAC 807
Ser Cys Phe Ser Asp Arg Ser Pro Leu Phe Ser Glu Thr Ile His Asp
190 195 200
CCT AAT CCC TGC CCA GAG TCC AGC GCT TCC TTC CTG TCG AGG ATC ACC 855
Pro Asn Pro Cys Pro Glu Ser Ser Ala Ser Phe Leu Ser Arg Ile Thr
205 210 215 220
TTC TGG TGG ATC ACA GGG TTG ATT GTC CGG GGC TAC CGC CAG CCC CTG 903
Phe Trp Trp Iie Thr Gly Leu Ile Val Arg Gly Tyr Arg Gln Pro Leu
225 230 235
GAG GGC AGT GAC CTC TGG TCC TTA AAC AAG GAG GAC ACG TCG GAA CAA 951
Glu Gly Ser Asp Leu Tzp Ser Leu Asn Lys Glu Asp Thr Ser Glu Gln
240 245 250
GTC GTG CCT GTT TTG GTA AAG AAC TGG AAG AAG GAA TGC GCC AAG ACT 999
Val Val Pro Val Leu Val Lys Asn Trp Lys Lys Glu Cys Ala Lys Thr
255 260 265
AGG AAG CAG CCG GTG AAG GTT GTG TAC TCC TCC AAG GAT CCT GCC CAG 1047
Arg Lys Gln Pro Val Lys Val Val Tyr Ser Ser Lys Asp Pro Ala Gln
270 275 280
CCG AAA GAG AGT TCC AAG GTG GAT GCG AAT GAG GAG GTG GAG GCT TTG 1095
Pro Lys Glu Ser Ser Lys Val Asp Ala Asn Glu Glu Val Glu Ala Leu
285 290 295 300
ATC GTC AAG TCC CCA CAG AAG GAG TGG AAC CCC TCT CTG TTT AAG GTG 1143
Ile Val Lys Ser Pro Gln Lys Glu Trp Asn Pro Ser Leu Phe Lys Val
305 310 315
TTA TAC AAG ACC TTT GGG CCC TAC TTC CTC ATG AGC TTC TTC TTC AAG 1191
Leu Tyr Lys Thr Phe Gly Pro Tyr Phe Leu Met Ser Phe Phe Phe Lys
320 325 330
GCC ATC CAC GAC CTG ATG ATG TTT TCC GGG CCG CAG ATC TTA AAG TTG 1239
Ala Ile His Asp Leu Met Met Phe Ser Gly Pro Gln Ile Leu Lys Leu
335 340 345
CTC ATC AAG TTC GTG AAT GAC ACG AAG GCC CCA GAC TGG CAG GGC TAC 1287
Leu Ile Lys Phe Val Asn Asp Thr Lys Ala Pro Asp Trp Gln Gly Tyr
350 355 360
TTC TAC ACC GTG CTG CTG TTT GTC ACT GCC TGC CTG CAG ACC CTC GTG 1335
Phe Tyr Thr Val Leu Leu Phe Val Thr Ala Cys Leu Gln Thr Leu Val
365 370 375 380
CTG CAC CAG TAC TTC CAC ATC TGC TTC GTC AGT GGC l.ITG AGG ATC AAG 1383
Leu His Gln Tyr Phe His Ile Cys Phe Val Ser Gly Met Arg Ile Lys
385 390 395
ACC GCT GTC ATT GGG GCT GTC TAT CGG AAG GCC CTG GTG ATC ACC AAT 1431
Thr Ala Val ile Gly Ala Val Tyr Arg Lys Ala Leu Val Ile Thr Asn
400 405 410
TCA GCC AGA AAA TCC TCC ACG GTC GGG GAG ATT GTC AAC CTC ATG TCT 1479
Ser Ala Arg Lys Ser Ser Thr Val Gly Glu Ile Val Aszi Leu Met Ser
415 420 425
63
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
GTG GAC GCT CAG AGG TTC ATG GAC TTG GCC ACG TAC ATT AAC ATG ATC 1527
Val Asp Ala Gln Arg Phe Met Asp Leu Ala Thr Tyr Ile Asn Met Ile
430 435 440
TGG TCA GCC CCC CTG CAA GTC ATC CTT GCT CTC TAC CTC CTG TGG CTG 1575
Trp Ser Ala Pro Leu Gln Val Ile Leu AIa Leu Tyr Leu Leu Trp Leu
445 450 455 460
AAT CTG GGC CCT TCC GTC CTG GCT GGA GTG GCG GTG ATG GTC CTC ATG 1623
Asn Leu Gly Pro Ser Val Leu Ala Gly Val Ala Val Met Val Leu Met
465 470 475
GTG CCC GTC AAT GCT GTG ATG GCG ATG AAG ACC AAG ACG TAT CAG GTG 1671
Val Pro Val Asn Ala Vai Met Ala Met Lys Thr Lys Thr Tyr Gln Val
480 485 490
GCC CAC ATG AAG AGC AAA GAC AAT CGG ATC AAG CTG ATG AAC GAA ATT 1719
Ala His Met Lys Ser Lys Asp Asn Arg Ile Lys Leu Met Asn Glu Ile
495 500 505
CTC AAT GGG ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GCA TTC 1767
Leu Asn Gly Ile Lys Val Leu Lys Leu Tyr Ala Trp Glu Leu Ala Phe
510 515 520
AAG GAC AAG GTG CTG GCC ATC AGG CAG GAG GAG CTG AAG GTG CTG AAG 1815
Lys Asp Lys Val Leu Ala Ile Arg Gln Glu Glu Leu Lys VaI Leu Lys
525 530 535 540
AAG TCT GCC TAC CTG TCA GCC GTG GGC ACC TTC ACC TGG GTC TGC ACG 1863
Lys Ser Ala Tyr Leu Ser Ala Val Gly Thr Phe Thr Trp Val Cys Thr
545 550 555
CCC TTT CTG GTG GCC TTG TGC ACA TTT GCC GTC TAC GTG ACC ATT GAC 1911
Pro Phe Leu Val Ala Leu Cys Thr Phe Ala Val Tyr Val Thr Ile Asp
560 565 570
GAG AAC AAC ATC CTG GAT GCC CAG ACA GCC TTC GTG TCT TTG GCC TTG 1959
Glu Asn Asn Ile Leu Asp Ala Gln Thr Ala Phe Val Ser Leu Ala Leu
575 580 585
TTC AAC ATC CTC CGG TTT CCC CTG AAC ATT CTC CCC ATG GTC ATC AGC 2007
Phe Asn Ile Leu Arg Phe Pro Leu Asn Ile Leu Pro Met Val Ile Ser
590 595 600
AGC ATC GTG CAG GCG AGT GTC TCC CTC AAA CGC CTG RGG ATC TTT CTC 2055
Ser Ile Val Gln Ala Ser Val Ser Leu Lys Arg Leu Arg Ile Phe Leu
605 610 615 620
TCC CAT GAG GAG CTG GAA CCT GAC AGC ATC GAG CGA CGG CCT GTC AAA 2103
Ser His Glu Glu Leu Glu Pro Asp Ser Ile Glu Arg Arg Pro Val Lys
625 630 635
GAC GGC GGG GGC ACG AAC AGC ATC ACC GTG AGG AAT GCC ACA TTC ACC 2151
Asp Gly Gly Gly Thr Asn Ser Ile Thr Val Arg Asn Ala Thr Phe Thr
640 695 650
TGG GCC AGG AGC GAC CCT CCC ACA CTG AAT GGC ATC ACC TTC TCC ATC 2199
Trp Ala Arg Ser Asp Pro Pro Thr Leu Asn Gly Ile Thr Phe Ser Ile
64
SUBS'~iTUTE S~iEET
CA 02448557 2003-11-25
WO 94110303 PCT/CA93/00439
655 660 665
CCC GAA GGT GCT TTG GTG GCC GTG GTG GGC CAG GTG GGC TGC GGA AAG 2247
Pro Glu Gly Ala Leu Val Ala Val Val Gly Gln Val Gly Cys Gly Lys
670 675 680
TTG TCC CTG CTC TCA GCC CTC TTG GCT GAG ATG GAC AAA GTG GAG GGG 2295
Leu Ser Leu Leu Ser Ala Leu Leu Ala Glu Met Asp Lys Val Glu Gly
685 690 695 700
CAC GTG GCT ATC AAG GGC TCC GTG GCC TAT GTG CCA CAG CAG GCC TGG 2343
His Val Ala Ile Lys Gly Ser Val Ala Tyr Val Pro Gln Gln Ala Trp
705 710 715
ATT CAG AAT GAT TCT CTC CGA GAA AAC ATC CTT TTT GGA TGT CAG CTG 2391
Ile Gln Asn Asp Ser Leu Arg Glu Asn Ile Leu Phe Gly Cys Gln Leu
720 725 730
GAG GAA CCA TAT TAC AGG TCC GTG ATA CAG GCC TGT GCC CTC CTC CCA 2439
Glu Glu Pro Tyr Tyr Arg Ser Val Ile Gln Ala Cys Ala Leu Leu Pro
735 740 745
GAC CTG GAA ATC CTG CCC AGT GGG GAT CGG ACA GAG ATT GGC GAG AAG 2487
Asp Leu Glu Ile Leu Pro Ser Gly Asp Arg Thr Glu Ile GIy Glu Lys
750 755 760
GGC GTG AAC CTG TCT GGG GGA CAG AAG CAG CGC GTG AGC CTG GCC CGG 2535
Gly Val Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Ser Leu Ala Arg
765 770 775 780
GCC GTG TAC TCC AAC GCT GAC ATT TAC CTC TTC GAT GAT CCC CTC TCA 2583
Ala Val Tyr Ser Asn Ala Asp Ile Tyr Leu Phe Asp Asp Pro Leu Ser
785 790 795
GCA GTG GAT GCC CAT GTG GGA AAA CAC ATC TTT GAA AAT GTG ATT GGC 2631
Ala Val Asp Ala His Val Gly Lys His Ile Phe Glu Asn Val Ile Gly
800 805 810
CCC AAG GGG ATG CTG AAG AAC AAG ACG CGG ATC TTG GTC ACG CAC AGC 2679
Pro Lys Gly Met Leu Lys Asn Lys Thr Arg Ile Leu Val Thr His Ser
815 820 825
ATG AGC TAC TTG CCG CAG GTG GAC GTC ATC ATC GTC ATG AGT GGC GGC 2727
Met Ser Tyr Leu Pro Gln Val Asp Val Ile Ile Val Met Ser Gly Gly
830 835 840
AAG ATC TCT GAG ATG GGC TCC TAC CAG GAG CTG CTG GCT CGA GAC GGC 2775
Lys Ile Ser Glu Met Gly Ser Tyr Gln Glu Leu Leu_Ala Arg Asp Gly
845 850 855 860
GCC TTC GCT GAG TTC CTG CGT ACC TAT GCC AGC ACA GAG CAG GAG CAG 2823
Ala Phe Ala Glu Phe Leu Arg Thr Tyr Ala Ser Thr Glu Gln Glu Gln
865 870 875
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
GAT GCA GAG GAG AAC GGG GTC ACG GGC GTC AGC GGT CCA GGG AAG GAA 2871
Asp Ala Glu Glu Asn Gly Val Thr Gly Val Ser Gly Pro Gly Lys Glu
880 885 890
GCA AAG CAA ATG GAG AAT GGC ATG CTG GTG ACG GAC AGT GCA GGG AAG 2919
AIa Lys Gln Met Glu Asn Gly Met Leu Val Thr Asp Ser Ala Gly Lys
895 900 905
CAA CTG CAG AGA CAG CTC AGC AGC TCC TCC TCC TAT AGT GGG GAC ATC 2967
Gln Leu Gln Arg Gln Leu Ser Ser Ser Ser Ser Tyr Ser Gly Asp Ile
910 915 920
AGC AGG CAC CAC AAC AGC ACC GCA GAA CTG CAG AAA GCT GAG GCC AAG 3015
Ser Arg His His Asn Ser Thr Ala GIu Leu Gln Lys Ala Glu A1a Lys
925 930 935 940
AAG GAG GAG ACC TGG AAG CTG ATG GAG GCT GAC AAG GCG CAG ACA GGG 3063
Lys GIu Glu Thr Trp Lys Leu Met Glu Ala Asp Lys Ala Gln Thr Gly
945 950 955
CAG GTC AAG CTT TCC GTG TAC TGG GAC TAC ATG AAG GCC ATC GGA CTC 3111
Gln Val Lys Leu Ser Val Tyr Trp Asp Tyr Met Lys Ala Ile Gly Leu
960 965 970
TTC ATC TCC TTC CTC AGC ATC TTC CTT TTC ATG TGT AAC CRT GTG TCC 3159
Phe Ile Ser Phe Leu Ser Ile Phe Leu Phe Met Cys Asn His Val Ser
975 980 985
GCG CTG GCT TCC AAC TAT TGG CTC AGC CTC TGG ACT GAT GAC CCC ATC 3207
Aia Leu Ala Ser Asn Tyr Trp Leu Ser Leu Trp Thr Asp Asp Pro Ile
990 995 1000
GTC AAC GGG ACT CAG GAG CAC ACG AAA GTC CGG CTG AGC GTC TAT GGA 3255
Val Asn Gly Thr Gln Glu His Thr Lys Val Arg Leu Ser Val Tyr Gly
1005 1010 1015 1020
GCC CTG GGC ATT TCA CAA GGG ATC GCC GTG TTT GGC TAC TCC ATG GCC 3303
Ala Leu Gly Ile Ser Gln GIy Ile Ala Val Phe GIy Tyr Ser Met Ala
1025 1030 1035
GTG TCC ATC GGG GGG ATC TTG GCT TCC CGC TGT CTG CAC GTG GAC CTG 3351
Val Ser Ile Gly Gly Ile Leu Ala Ser Arg Cys Leu His Val Asp Leu
1040 1045 1050
CTG CAC AGC ATC CTG CGG TCA CCC ATG AGC TTC TTT GAG CGG ACC CCC 3399
Leu His Ser Ile Leu Arg Ser Pro Met Ser Phe Phe Glu Arg Thr Pro
1055 1060 1065
AGT GGG AAC CTG GTG AAC CGC TTC TCC AAG GAG CTG GAC ACA GTG GAC 3447
Ser Gly Asn Leu Val Asn Arg Phe Ser Lys Glu Leu Asp Thr Val Asp
1070 1075 1080
TCC ATG ATC CCG GAG GTC ATC AAG ATG TTC ATG GGC TCC CTG TTC AAC 3495
Ser Met Ile Pro Glu Val Ile Lys Met Phe Met Gly Ser Leu Phe Asn
1085 1090 1095 1100
66
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 ' PCT/CA93/00439
GTC ATT GGT GCC TGC ATC GTT ATC CTG CTG GCC ACG CCC ATC GCC GCC 3543
Val Ile Gly Ala Cys Ile Val ile Leu Leu Ala Thr Pro Ile Ala Ala
1105 1110 1115
ATC ATC ATC CCG CCC CTT GGC CTC ATC TAC TTC TTC GTC CAG AGG TTC 3591
Ile Ile Ile Pro Pro Leu Gly Leu Ile Tyr Phe Phe Val Gln Arg Phe
1120 1125 1130
TAC GTG GCT TCC TCC CGG CAG CTG AAG CGC CTC GAG TCG GTC AGC CGC 3639
Tyr Val Ala Ser Ser Azg Gln Leu Lys Arg Leu Glu Ser Val Ser Arg
1135 1140 1145
TCC CCG GTC TAT TCC CAT TTC AAC GAG ACC TTG CTG GGG GTC AGC GTC 3687
Ser Pro Val Tyr Ser His Phe Asn Glu Thr Leu Leu GIy Val Ser Val
1150 1155 1160
ATT CGA GCC TTC GAG GAG CAG GAG CGC TTC ATC CAC CAG AGT GAC CTG 3735
Ile Arg Ala Phe Giu Glu Gln Glu Arg Phe Ile His Gln Ser Asp Leu
1165 1170 1175 1180
AAG GTG GAC GAG AAC CAG AAG GCC TAT TAC CCC AGC ATC GTG GCC AAC 3783
Lys Val Asp Glu Asn Gln Lys Ala Tyr Tyr Pro Ser Ile Val Ala Asn
1185 1190 1195
AGG TGG CTG GCC GTG CGG CTG GAG TGT GTG GGC AAC TGC ATC GTT CTG 3831
Arg Trp Leu Ala Val Arg Leu Glu Cys Val Gly Asn Cys Ile Val Leu
1200 1205 1210
TTT GCT GCC CTG TTT GCG GTG ATC TCC AGG CAC AGC CTC AGT GCT GGC 3879
Phe Ala Ala Leu Phe Ala Val Ile Ser Arg His Ser Leu Ser Ala Gly
1215 1220 1225
TTG GTG GGC CTC TCA GTG TCT TAC TCA TTG CAG GTC ACC ACG TAC TTG 3927
Leu Val Gly Leu Ser Val Ser Tyr Ser Leu Gln Val Thr Thr Tyr Leu
1230 1235 1240
AAC TGG CTG GTT CGG ATG TCA TCT GAA ATG GAA ACC AAC ATC GTG GCC 3975
Asn Trp Leu Val Arg Met Ser Ser Glu Met Glu Thr Asn Ile Val Ala
1245 1250 1255 1260
GTG GAG AGG CTC AAG GAG TAT TCA GAG ACT GAG AAG GAG GCG CCC TGG 4023
Val Glu Arg Leu Lys Glu Tyr Ser Glu Thr G1u Lys Glu Ala Pro Trp
1265 1270 1275
CAA ATC CAG GAG ACA CGT CCG CCC AGC AGC TGG CCC CAG GTG GGC CGA 4071
Gln Ile Gln Glu Thr Arg Pro Pro Ser Ser Trp Pro Gln Val Gly Arg
1280 1285 1290
GTG GAA TTC CGG AAC TAC TGC CTG CGC TAC CGA GAG GAC CTG GAC TTC 4119
Val Glu Phe Arg Asn Tyr Cys Leu Arg Tyr Arg Glu Asp Leu Asp Phe
1295 1300 1305
GTT CTC AGG CAC ATC AAT GTC ACG ATC AAT GGG GGA GAA AAG GTC GGC 4167
Val Leu Arg His Ile Asn Val Thr Ile Asn Gly Gly Glu Lys Val Gly
1310 1315 1320
67
SUBSTITUTE S~3EET
CA 02448557 2003-11-25
WO 94/10303 PGT/CA93/00439
ATC GTG GGG CGG ACG GGA GCT GGG AAG TCG TCC CTG ACC CTG GGC TTA 4215
Ile Val Gly Arg Thr Gly Ala Gly Lys Ser Ser Leu Thr Leu Gly Leu
1325 1330 1335 1340
TTT CGG ATC AAC GAG TCT GCC GAA GGA GAG ATC ATC ATC GAT GGC ATC 4263
Phe Arg Ile Asn Glu Ser Ala Glu Gly Glu Ile Ile Ile Asp Gly Ile
1345 1350 1355
AAC ATC GCC AAG ATC GGC CTG CAC GAC CTC CGC TTC AAG ATC ACC ATC 4311
Asn Ile Ala Lys Ile Gly Leu His Asp Leu Arg Phe Lys Ile Thr Ile
1360 1365 1370
ATC CCC CAG GAC CCT GTT TTG TTT TCG GGT TCC CTC CGA ATG AAC CTG 4359
Ile Pro Gln Asp Pro Val Leu Phe Ser Gly Ser Leu Arg Met Asn Leu
1375 1380 1385
GAC CCA TTC AGC CAG TAC TCG GAT GAA GAA GTC TGG ACG TCC CTG GAG 4407
Asp Pro Phe Ser Gln Tyr Ser Asp Glu Glu Val Trp Thr Ser Leu G1u
1390 1395 1400
CTG GCC CAC CTG AAG GAC TTC GTG TCA GCC CTT CCT GAC AAG CTA GAC 4455
Leu Ala His Leu Lys Asp Phe Val Ser Ala Leu Pro Asp Lys Leu Asp
1405 1410 1415 1420
CAT GAA TGT GCA GAA GGC GGG GAG AAC CTC AGT GTC GGG CAG CGC CAG 4503
His Glu Cys Ala Glu Gly Gly Glu Asn Leu Ser Val Gly Gln Arg Gln
1425 1430 1435
CTT GTG TGC CTA GCC CGG GCC CTG C't~G AGG AAG ACG AAG ATC CTT GTG 4551
Leu Val Cys Leu Ala Arg Ala Leu Leu Arg Lys Thr Lys Ile Leu Val
1440 1445 1450
TTG GAT GAG GCC ACG GCA GCC GTG GAC CTG GAA ACG GAC GAC CTC ATC 4599
Leu Asp Glu Ala Thr Ala Ala Val Asp Leu Glu Thr Asp Asp Leu Ile
1455 1460 1465
CAG TCC ACC ATC CGG ACA CAG TTC GAG GAC TGC ACC GTC CTC ACC ATC 4647
Gln Ser Thr Ile Arg Thr Gln Phe Glu Asp Cys Thr Val Leu Thz Ile
1470 1475 1480
GCC CAC CGG CTC AAC ACC ATC ATG GAC TAC ACA AGG GTG ATC GTC TTG 4695
Ala His Arg Leu Asn Thr Ile Met Asp Tyr Thr Arg Val Ile Val Leu
1485 1490 1495 1500
GAC AAA GGA GAA ATC CAG GAG TAC GGC GCC CCA TCG GAC CTC CTG CAG 4743
Asp Lys Gly Glu Ile Gln Glu Tyr Gly Ala Pro Ser Asp Leu Leu Gln
1505 1510 1515
CAG AGA GGT CTT TTC TAC AGC ATG GCC AAA GAC GCC GGC TTG GTG 4788
Gla Arg Gly Leu Phe Tyr Ser Met Ala Lys Asp Ala Gly Leu Val
1520 1525 1530
TGAGCCCCAG AGCTGGCATA TCTGGTCAGA ACTGCAGGGC CTATATGCCA GCGCCCCAGG 4848
GAGGAGTCAG TACCCCTGGT AAACCAAGCC TCCCACACTG AAACCAAAAC ATAAAAACCA 4908
AACCCAGACA ACCAAAACAT ATTCAAAGCA GCAGCCACCG CCATCCGGTC CCCTGCCTGG 4968
AACTGGCTGT GAAGACCCAG GAGAGACAGA GATGCGAACC ACC 5011
68
SUBSTETUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
(2) INFORMATIOLd FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1531 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ri) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ptet Ala Leu Arg Gly Phe Cys Ser Ala Asp Gly Ser Asp Pro Leu Trp
1 5 10 15
Asp Trp Asn Val Thr Trp Asn Thr Ser Asn Pro Asp Phe Thr Lys Cys
20 25 30
Phe Gln Asn Thr Val Leu Val Trp Val Pro Cys Phe Tyr Leu Trp Ala
35 40 45
Cys Phe Pro Phe Tyz Phe Leu Tyr Leu Ser Arg His Asp Arg Gly Tyr
50 55 60
Ile Gln Met Thr Pro Leu Asn Lys Thr Lys Thr Ala Leu Gly Phe Leu
65 70 75 80
Leu Trp Ile Val Cys Trp Ala Asp Leu Phe Tyr Ser Phe Trp Glu Arg
85 90 95
Ser Arg Gly Ile Phe Leu Ala Pro Val Phe Leu Val Ser Pro Thr Leu
100 105 110
Leu Gly Ile Thr Thr Leu Leu Ala Thr Phe Leu Ile Gln Leu Glu Arg
115 120 125
Arg Lys Gly Val Gln Ser Ser Gly Ile Met Leu Thr Phe Trp Leu Val
130 135 140
Ala Leu Val Cys Ala Leu Ala Ile Leu Arg Ser Lys Ile Met Thr Ala
145 150 155 160
Leu Lys Glu Asp Ala Gln Val Asp Leu Phe Arg Asp Ile Thr Phe Tyr
165 170 175
Val Tyr Phe Ser Leu Leu Leu Ile Gln Leu Val Leu Ser Cys Phe Ser
180 185 190
Asp Arg Ser Pro Leu Phe Ser Glu Thr Ile His Asp Pro Asn Pro Cys
195 200 ~ 205
Pro Glu Ser Ser Ala Ser Phe Leu Ser Arg Ile Thr Phe Trp Trp Ile
210 215 220
Thr Gly Leu Ile Val Arg Gly Tyr Arg Gln Pro Leu Glu Gly Ser Asp
225 230 235 240
Leu Trp Ser Leu Asn Lys Glu Asp Thr Ser Glu Gln Val Val Pro Val
245 250 255
69
SUBST~TUT~ S~~E~T
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
Leu VaI Lys Asn Trp Lys Lys Glu Cys Ala Lys Thr Arg Lys Gln Pro
260 265 270
Val Lys Val Val Tyr Ser Ser Lys Asp Pro Ala Gln Pro Lys Glu Ser
275 280 285
Ser Lys Val Asp Ala Asn Glu Glu Val Glu Ala Leu Ile Val Lys Ser
290 295 300
Pro Gln Lys Glu Trp Asn Pro Ser Leu Phe Lys Val Leu Tyr Lys Thr
305 310 315 320
Phe Gly Pro Tyr Phe Leu Met Ser Phe Phe Phe Lys Ala Ile His Asp
325 330 335
Leu Met Met Phe Ser Gly Pro Gln Ile Leu Lys Leu Leu Ile Lys Phe
340 345 350
Val Asn Asp Thr Lys Ala Pro Asp Trp Gln Gly Tyr Phe Tyr Thr Val
355 360 365
Leu Leu Phe Val Thr Ala Cys Leu Gln Thr Leu Val Leu His Gln Tyr
370 375 380
Phe His Ile Cys Phe Val Ser Gly Met Arg Ile Lys Thr Ala Val Ile
3B5 390 395 400
Gly Ala Val Tyr Arg Lys Ala Leu Val Ile Thr Asn Ser Ala Arg Lys
405 410 415
Ser Ser Thr Val Gly Glu Ile Val Asa Leu Met Ser Val Asp Ala Gln
420 425 430
Arg Phe Met Asp Leu Ala Thr Tyr Ile Asn Met Ile Trp Ser Ala Pro
435 440 445
Leu Gln Val Ile Leu Ala Leu Tyr Leu Leu Trp Leu Asn Leu Gly Pro
450 455 460
Ser Val Leu Ala Gly Val Ala Val Met Val Leu Met Val Pro Val Asn
465 470 475 480
Ala Val Met Aia Met Lys Thr Lys Thr Tyr Gln Val Ala His Met Lys
485 490 495
Ser Lys Asp Asn Arg Ile Lys Leu Met Asn Glu Ile Leu Asn Gly Ile
500 SOS 510
Lys Val Leu Lys Leu Tyr Ala Trp Glu Leu Ala Phe Lys Asp Lys Val
515 520 525
Leu Ala Ile Arg Gln Glu Glu Leu Lys Val Leu Lys Lys Ser Ala Tyr
530 535 540
SUBSTITUTE S~IEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
Leu Ser Ala Val Gly Thr Phe Thr Trp Val Cys Thr Pro Phe Leu Val
545 550 555 560
Ala Leu Cys Thr Phe Ala Val Tyr Val Thr Ile Asp Glu Asn Asn Ile
565 570 575
' Leu Asp Ala Gln Thr Ala Phe Val Ser Leu Ala Leu Phe Asn Ile Leu
580 585 590
Arg Phe Pro Leu Asn Ile Leu Pro Met Val Ile Ser Ser Ile Val Gln
595 600 605
Ala Ser Val Ser Leu Lys Arg Leu Arg Ile Phe Leu Ser His Glu Glu
610 615 620
Leu Glu Pro Asp Ser Ile Glu Arg Arg Pro Val Lys Asp Gly Gly Gly
625 630 635 640
Thr Asn Ser Ile Thr Val Arg Asn Ala Thr Phe Thr Trp Ala Arg Ser
645 650 655
Asp Pro Pro Thr Leu Asn Gly Ile Thr Phe Ser Ile Pro Glu Gly Ala
660 665 670
Leu Val Ala Val Val Gly Gln Val Gly Cys Gly Lys Leu Ser Leu Leu
675 680 685
Ser Ala Leu Leu Ala Glu Met Asp Lys Val Glu Gly His Val Ala Ile
690 695 700
Lys Gly Ser Val Ala Tyr Val Pro Gln Gln Ala Trp Ile Gln Asn Asp
705 710 715 720
Ser Leu Arg Glu Asn Ile Leu Phe Gly Cys Gln Leu Glu Glu Pro Tyr
725 730 735
Tyr Arg Ser Val Ile Gln Ala Cys Ala Leu Leu Pro Asp Leu Glu Ile
740 745 750
Leu Pro Ser Gly Asp Arg Thr Glu Ile Gly Glu Lys Gly Val Asn Leu
755 760 765
Ser Gly Gly Gln Lys Gln Arg Val Ser Leu Ala Arg Ala Val Tyr Ser
770 775 780
Asn Ala Asp Ile Tyr Leu Phe Asp Asp Pro Leu Ser Ala Val Asp Ala
785 790 795 800
His Val Gly Lys His Ile Phe Glu Asn Val Ile Gly Pro Lys Gly Met
805 ~ 810 815
Leu Lys Asn Lys Thr Arg Ile Leu Val Thr His Ser Met Ser Tyr Leu
820 825 830
Pro Gln Val Asp Val Ile Ile Val Met Ser Gly Gly Lys Ile Ser Glu
835 840 845
~1
SUBST1TUTF S~IEE T
CA 02448557 2003-11-25
WO 94/10303 _ PCT/CA93/00439
Met Gly Ser Tyr Gln Glu Leu Leu Ala Arg Asp Gly Ala Phe Ala Glu
850 855 B60
Phe Leu Arg Thr Tyr Ala Ser Thr Glu Gln Glu Gln Asp Ala Glu Glu
865 970 875 880
Asn Gly Val Thr Gly Val Ser Gly Pro Gly Lys Glu Ala Lys Gln Met '
885 890 895
Glu Asn Gly Met Leu Val Thr Asp Ser Ala Gly Lys Gln Leu Gln Arg
900 905 910
Gln Leu Ser Ser Ser Ser Ser Tyr Ser Gly Asp Ile Ser Arg His His
915 920 925
Asn Ser Thr Ala Glu Leu Gln Lys Ala Glu Ala Lys Lys Glu Glu Thr
930 935 940
Trp Lys Leu Met Glu Ala Asp Lys Ala Gln Thr Gly Gln Val Lys Leu
945 950 955 960
Ser Val Tyr Trp Asp Tyr Met Lys Ala Ile Gly Leu Phe Ile Ser Phe
965 970 ~ 975
Leu Ser Ile Phe Leu Phe Met Cys Asn His Val Ser Ala Leu Ala Ser
980 985 990
Asn Tyr Trp Leu Ser Leu Trp Thr Asp Asp Pro Ile Val Asn Gly Thr
995 1000 1005
Gln Glu His Thr Lys Val Arg Leu Ser Val Tyr Gly Ala Leu Gly Ile
1010 1015 1020
Ser Gln Gly Ile Ala Val Phe Gly Tyr Ser Met Ala Val Ser Ile Gly
1025 1030 1035 1040
Gly Ile Leu Ala Ser Arg Cys Leu His Val Asp Leu Leu His Ser Ile
1045 1050 1055
Leu Arg Ser Pro Met Ser Phe Phe Glu Arg Thr Pro Sex Gly Asn Leu
1060 1065 1070
Val Asn Arg Phe Ser Lys Glu Leu Asp Thr Val Asp Ser Met Ile Pro
1075 1080 1085
Glu Val Ile Lys Met Phe Met Gly Ser Leu Phe Asn Val Ile Gly Ala
1090 1095 1100
Cys Ile Val Ile Leu Leu Ala Thr Pro Ile Ala Ala Ile Ile Ile Pro
1105 1110 1115 1120
Pro Leu Gly Leu Ile Tyr Phe Phe Val Gln Arg Phe Tyr Val Ala Ser
1125 1130 1135
Ser Arg Gln Leu Lys Arg Leu Glu Ser Val Ser Arg Ser Pro Val Tyr
1140 1145 1150
72
SUBSTITUTE SHEET
CA 02448557 2003-11-25
WO 94/10303 PCT/CA93/00439
Ser His Phe Asn Glu Thr Leu Leu Gly Val Ser Val Ile Arg Ala Phe
1155 1160 1165
Glu Glu Gln Glu Arg Phe Ile His Gln Ser Asp Leu Lys Val Asp Glu
1170 1175 1180
Asn Gln Lys Ala Tyr Tyr Pro Ser Ile Val Ala Asn Arg Trp Leu Ala
1185 1190 1195 1200
Val Arg Leu Glu Cys Val Gly Asn Cys Ile Val Leu Phe Ala Ala Leu
1205 1210 1215
Phe Ala Val Ile Ser Arg His Ser Leu Ser Ala Gly Leu Val Gly Leu
1220 1225 1230
Ser Val Ser Tyr Ser Leu Gln Val Thr Thr Tyr Leu Asn Trp Leu Val
1235 1240 1245
Arg Met Ser Ser Glu Met Glu Thr Asn Ile Val Ala Val Glu Arg Leu
1250 1255 1260
Lys Glu Tyr Ser Glu Thr Glu Lys Glu Ala Pro Trp Gln Ile Gln Glu
1265 1270 1275 1280
Thr Arg Pro Pro Ser Ser Trp Pro Gln Val Gly Arg Val Glu Phe Arg
1285 1290 1295
Asn Tyr Cys Leu Arg Tyr Arg Glu Asp Leu Asp Phe Val Leu Arg His
1300 1305 1310
Ile Asn Val Thr Ile Asn Gly Gly Glu Lys Val Gly Ile Val Gly Arg
1315 1320 1325
Thr Gly Ala Gly Lys Ser Ser Leu Thr Leu Gly Leu Phe Arg Ile Asn
1330 1335 1340
Glu Ser Ala Glu Gly Glu Ile Ile Ile Asp Gly Ile Asn Ile Ala Lys
1345 1350 1355 1360
Ile Gly Leu His Asp Leu Arg Phe Lys Ile Thr Ile Ile Pro Gln Asp
1365 1370 1375
Pro Val Leu Phe Ser Gly Ser Leu Arg Met Asn Leu Asp Pro Phe Ser
1380 1385 1390
Gln Tyr Ser Asp Glu Glu Val Trp Thr Ser Leu Glu Leu Ala His Leu
1395 1400 1405
Lys Asp Phe Val Ser Ala Leu Pro Asp Lys Leu Asp His Glu Cys Ala
1410 1415 1420
Glu Gly Gly Glu Asn Leu Ser Val Gly Gln Arg Gln Leu Val Cys Leu
1425 1430 1435 1440
Ala Arg Ala Leu Leu Arg Lys Thr Lys Ile Leu Val Leu Asp Glu Ala
1445 1450 1455
73
SUBST~1UTE St~EET
CA 02448557 2003-11-25
WO 94/1U303 PCf/CA93/00439
Thr Ala Ala Val Asp Leu Glu Thr Asp Asp Leu Ile Gln Ser Thr Ile
1460 1465 1470
Arg Thr Gln Phe Glu Asp Cys Thr Val Leu Thr Ile Ala His Arg Leu
1475 1480 1485
Asn Thr Ile Met Asp Tyr Thr Arg Val Ile Val Leu Asp Lys Gly Glu
1490 1495 1500
Ile Gln Glu Tyr Gly Ala Pro Ser Asp Leu Leu Gln Gln Arg Gly Leu
1505 1510 1515 1520
Phe Tyr Ser Met Ala Lys Asp Ala Gly Leu Val
1525 1530
74
SUBS'~iTUTE S~iEET
