U.S. patent application number 11/566989 was filed with the patent office on 2009-11-12 for methods and reagents for preparing and using immunological agents specific for p-glycoprotein.
This patent application is currently assigned to ONCOTECH, INC.. Invention is credited to John Fruehauf, Eugene Mechetner.
Application Number | 20090280508 11/566989 |
Document ID | / |
Family ID | 25026377 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280508 |
Kind Code |
A1 |
Mechetner; Eugene ; et
al. |
November 12, 2009 |
Methods and Reagents for Preparing and Using Immunological Agents
Specific for P-Glycoprotein
Abstract
This invention relates to immunological reagents and methods
specific for a mammalian, transmembrane protein termed Pgp, having
a non-specific efflux pump activity established in the art as being
a component of clinically-important multidrug resistance in cancer
patients undergoing chemotherapy. The invention provides methods
for developing and using immunological reagents specific for
certain mutant forms of Pgp and for wild-type Pgp in a conformation
associated with substrate binding or in the presence of ATP
depleting agents. The invention also provides improved methods for
identifying and characterizing anticancer compounds.
Inventors: |
Mechetner; Eugene; (Irvine,
CA) ; Fruehauf; John; (Tustin, CA) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF LLP
300 S. WACKER DRIVE, 32ND FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
ONCOTECH, INC.
Tustin
CA
|
Family ID: |
25026377 |
Appl. No.: |
11/566989 |
Filed: |
December 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10114847 |
Apr 2, 2002 |
7144704 |
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11566989 |
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09316167 |
May 21, 1999 |
6365357 |
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10114847 |
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08752447 |
Nov 15, 1996 |
5994088 |
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09316167 |
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Current U.S.
Class: |
435/7.21 |
Current CPC
Class: |
G01N 33/5014 20130101;
G01N 2333/705 20130101; C12N 2799/027 20130101; G01N 33/5008
20130101; G01N 2333/70596 20130101; A61K 38/00 20130101; G01N
33/5011 20130101; G01N 33/5094 20130101; C07K 16/28 20130101; G01N
33/56972 20130101; C07K 2319/00 20130101 |
Class at
Publication: |
435/7.21 |
International
Class: |
G01N 33/567 20060101
G01N033/567 |
Claims
1. A method for characterizing binding of a test compound to
P-glycoprotein expression in a mammalian cell, the method
comprising the steps of: (a) incubating a first mammalian cell
expressing P-glycoprotein in the presence of a first P-glycoprotein
binding compound having a first binding affinity for
P-glycoprotein; (b) incubating a second mammalian cell expressing
P-glycoprotein in the presence of the test compound; (c) reacting
the first mammalian cell and the second mammalian cell with an
immunological reagent specific for P-glycoprotein in a biochemical
conformation adopted in the presence of a P-glycoprotein substrate;
and (d) comparing binding of the immunological reagent to the first
mammalian cell with binding of the immunological reagent to the
second mammalian cell.
2. The method of claim 1 wherein the immunological reagent is a
monoclonal antibody specific for P-glycoprotein in a biochemical
conformation adopted in the presence of an ATP-depleting agent.
3. The monoclonal antibody of claim 2 that is UIC2 (A.T.C.C.
Accession No. HB11027).
4. The method of claim 1, wherein binding of the immunological
reagent is increased in the presence of the P-glycoprotein
substrate.
5. The method of claim 1 wherein the immunological reagent is
detectably-labeled.
6. The method of claim 5 wherein the detectable label is a
fluorescent label.
7. The method of claim 1 wherein binding of the immunological
reagent is increased in the presence of the P-glycoprotein
substrate and wherein enhanced binding of the fluorescently-labeled
immunological reagent is detected by fluorescence-activated cell
sorting.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/114,847, filed Apr. 2, 2002, now U.S. Pat.
No. 7,144,704, granted Dec. 5, 2006, which is a divisional of U.S.
patent application Ser. No. 09/316,167, filed May 21, 1999, now
U.S. Pat. No. 6,365,357, granted Apr. 2, 2002, which is a
continuation-in-part of U.S. patent application Ser. No.
08/752,447, filed Nov. 15, 1996, now U.S. Pat. No. 5,994,088,
granted Nov. 30, 1999, the disclosure of each of which is
incorporated by reference in its entirety herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to uses of immunological
reagents specific for a human transmembrane efflux pump protein
(P-glycoprotein). Specifically, the invention relates to uses of
such immunological reagents that specifically recognize
P-glycoprotein that is in a biochemical conformation adopted in the
presence of certain cytotoxic, lipophilic drugs that are substrates
for P-glycoprotein, in the presence of cellular ATP depleting
agents, and by certain mutant embodiments of Pgp. In particular,
the invention provides methods of using such immunological reagents
for anticancer drug screening and development.
[0004] 2. Background of the Invention
[0005] Many human cancers express intrinsically or develop
spontaneously resistance to several classes of anticancer drugs,
each with a different structure and different mechanism of action.
This phenomenon, which can be mimicked in cultured mammalian cells
selected for resistance to certain plant alkaloids or antitumor
antibiotics such as colchicine, vinblastine and doxorubicin
(formerly known as Adriamycin), is generally referred to as
multidrug resistance ("MDR"; see Roninson (ed)., 1991, Molecular
and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum
Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for
Multidrug Resistance in Cancer, Academic Press, N.Y., Chapter 11
for reviews). The MDR phenotype presents a major obstacle to
successful cancer chemotherapy in human patients.
[0006] MDR frequently appears to result from decreased
intracellular accumulation of anticancer drugs as a consequence of
increased drug efflux related to alterations at the cellular plasma
membrane. When mutant cell lines having the MDR phenotype are
isolated, they are found to express an ATP-dependent non-specific
molecular "pump" protein (generally known as P-glycoprotein) that
is located in the plasma membrane and keeps the intracellular
accumulation of an anti-cancer drug low enough to evoke the
drug-resistance phenotype. This protein (which has been determined
to be the gene product of the MDR1 gene in humans) facilitates
active (i.e., energy-dependent) drug efflux from the cell, against
a concentration gradient of (generally) lipophilic compounds,
including many cytotoxic drugs.
[0007] The gene encoding P-glycoprotein (which is also known as
gp170-180 and the multidrug transporter) has been cloned from
cultured human cells by Roninson et al. (see U.S. Pat. No.
5,206,352, issued Apr. 27, 1993), and is generally referred to as
MDR1. The protein product of the MDR1 gene, most generally known as
P-glycoprotein ("Pgp"), is a 170-180 kilodalton (kDa) transmembrane
protein having the aforementioned energy-dependent efflux pump
activity.
[0008] Molecular analysis of the MDR1 gene indicates that Pgp
consists of 1280 amino acids distributed between two homologous
halves (having 43% sequence identity of amino acid residues), each
half of the molecule comprising six hydrophobic transmembrane
domains and an ATP binding site within a cytoplasmic loop. Only
about 8% of the molecule is extracellular, and carbohydrate
moieties (approximately 30 kDa) are bound to sites in this region
(Chen et al., 1986, Cell 47: 381-387).
[0009] Expression of Pgp on the cell surface is sufficient to
render cells resistant to many (but not all) cytotoxic drugs,
including many anti-cancer agents. Pgp-mediated MDR appears to be
an important clinical component of drug resistance in tumors of
different types, and MDR1 gene expression correlates with
resistance to chemotherapy in different types of cancer.
[0010] Pgp is also constitutively expressed in many normal cells
and tissues (see Cordon-Cardo et al., 1990, J. Histochem. Cytochem.
38: 1277; and Thiebaut et al., 1987, Proc. Natl. Acad. Sci. USA 84:
7735 for reviews). In hematopoietic cells, Neyfakh et al. (1989,
Exp. Cancer Res. 185: 496) have shown that certain subsets of human
and murine lymphocytes efflux Rh123, a fluorescent dye that is a
Pgp substrate, and this process can be blocked by small molecule
inhibitors of Pgp. It has been demonstrated more recently that Pgp
is expressed on the cell-surface membranes of pluripotent stem
cells, NK cells, CD4- and CD8-positive T lymphocytes, and B
lymphocytes (Chaudhary et al., 1992, Blood 80: 2735; Drach et al.,
1992, Blood 80: 2729; Kimecki et al., 1994, Blood 83: 2451;
Chaudhary et al., 1991, Cell 66: 85). Pgp expression on the cell
surface membranes of different subsets of human lymphocytes has
been extensively documented (Coon et al., 1991, Human Immunol. 32:
134; Tiirikainen et al., 1992, Ann. Hematol. 65: 124; Schluesener
et al., 1992, Immunopharmacology 23: 37; Gupta et al., 1993, J.
Clin. Immunol. 13: 289). Although recent studies suggest that Pgp
plays a role in normal physiological functions of immune cells
(Witkowski et al., 1994, J. Immunol. 153: 658; Kobayashi et al.,
1994, Biochem. Pharmacol. 48: 1641; Raghu et al., 1996, Exp.
Hematol. 24: 1030-1036), the physiological role of Pgp in normal
immune cells has remained unclear to date.
[0011] Once the central role in MDR played by Pgp was uncovered,
agents with a potential for reversing MDR phenotypes were developed
that target Pgp. Several classes of drugs, including calcium
channel blockers (e.g., verapamil), immunosuppresants (such as
cyclosporines and steroid hormones), calmodulin inhibitors, and
other compounds, were found (often fortuitously) to enhance the
intracellular accumulation and cytotoxic action of Pgp-transported
drugs (Ford et al., 1990, Pharm. Rev. 42: 155). Many of these
agents were found to inhibit either drug binding or drug transport
by Pgp (Akiyamaetal., 1988, Molec. Pharm. 33: 144; Horio et al.,
1988, Proc. Natl. Acad. Sci. USA 84: 3580). Some of these agents
themselves were found to bind to and be effluxed by Pgp, suggesting
that their enhancing effects on the cytotoxicity of Pgp substrates
are due, at least in part, to competition for drug binding sites on
this protein (Cornwell et al., 1986, J. Bio. Chem. 261: 7921;
Tamai, 1990, J. Biochem. Molec Biol. 265: 16509).
[0012] Many of these agents, however, also have strong, deleterious
side effects at physiologically-achievable concentrations. These
systemic side effects severely limit the clinical use of these
agents as specific inhibitors of Pgp or for negative selection
against Pgp-expressing tumor cells. Most of the known MDR-reversing
drugs used in clinical trials have major side effects unrelated to
inhibition of Pgp, such as calcium channel blockage (verapamil) or
immunosuppression (cyclosporines and steroids). Similarly,
targeting of cytotoxic drugs to Pgp-expressing cells is capable of
compromising normal tissue function in normal cells (such as
kidney, liver, colonic epithelium, etc.) that normally express Pgp.
These drawbacks restrict the clinically-achievable dose of such
agents and ultimately, their usefulness.
[0013] Immunological reagents also provide a means for specifically
inhibiting drug efflux mediated by Pgp. Monoclonal antibodies
specific for Pgp are known in the art.
[0014] Hamada et al., 1986, Proc. Natl. Acad. Sci. USA 83: 7785
disclose the mAbs MRK-16 and MRK-17, produced by immunizing mice
with doxorubicin-resistant K-562 human leukemia cells. MRK-16 mAb
was also reported to modulate vincristine and actinomycin D
transport in resistant cells, and MRK-17 was shown to specifically
inhibit growth of resistant cells with these drugs.
[0015] Meyers et al., 1987, Cancer Res. 49: 3209 disclose mAbs
HYB-241 and HYB-612, which recognize an external epitope of
Pgp.
[0016] O'Brien et al., 1989, Proc. Amer. Assoc. Cancer Res. 30:Abs
2114 disclose that mAbs HYB-241 and HYB-612 increased the
accumulation of vincristine and actinomycin D in tumor cells and
increased the cytotoxicity of combinations of these drugs with
verapamil.
[0017] Tsuruo et al., 1989, Jpn. J. Cancer Res. 80: 627 reported
that treatment of athymic mice that had been previously inoculated
with drug resistant human ovarian cancer cells with the mAb MRK16
caused regression of established subcutaneous tumors.
[0018] Hamada et al., 1990, Cancer Res. 50: 3167 disclosed a
recombinant chimeric antibody that combines the variable region of
MRK-16 with the F.sub.c portion of a human antibody, and showed
this chimeric antibody to be more effective than MRK-16 mAb in
increasing cytotoxicity in vitro.
[0019] Pearson et al., 1991, J. Natl. Cancer Inst. 88: 1386
disclosed that MRK-16 mAb increased the in vivo toxicity of
vincristine to a human MDR colon cancer cell line grown as a
xenograft in nude mice. The in vitro potentiation of drug
cytotoxicity by MRK-16 mAb was, however, weak relative to known
chemical inhibitors of Pgp action, and was apparently limited to
only two Pgp substrates (vincristine and actinomycin D), having no
effect on cytotoxicity by doxorubicin.
[0020] Cinciarelli et al., 1991, Int. J. Cancer 47: 533 disclosed a
mouse IgG.sub.2a mAb, termed MAb657, having cross reactivity to
Pgp-expressing human MDR cells. This mAb was shown to increase the
susceptibility of MDR cells to human peripheral blood
lymphocyte-mediated cytotoxicity, but was not shown to have an
inhibitory effect on the drug efflux activity of Pgp.
[0021] Arcesi et al., 1993, Cancer Res. 53:310-317 disclosed mAb
4E3 that binds to extracellular epitopes of Pgp but does not
disrupt drug efflux or potentiate MDR drug-induced
cytotoxicity.
[0022] Mechetner and Roninson, U.S. Pat. No. 5,434,075, issued Jul.
18, 1995, disclose mAb UIC2, having specificity for extracellular
Pgp epitopes. This antibody was also shown to effectively inhibit
Pgp-mediated drug efflux in MDR cells, and to reverse the MDR
phenotype in vitro thereby, for a number of structurally and
functional different cytotoxic compounds and all tested
chemotherapeutic drugs known to be substrates for Pgp-mediated drug
efflux.
[0023] There is a need in the art to develop new Pgp inhibitors for
preventing or overcoming multidrug resistance in human cancer. In
developing new pharmaceuticals, it is essential to determine
whether a drug candidate is a Pgp substrate and is effluxed by Pgp
expressed in normal or tumor cells. This is important because, on
the one hand, such drugs are expected to inhibit Pgp expression in
normal cells (in the gastrointestinal tract, excretory organs like
kidney, certain hematopoietic cells and the blood-brain and
testicular barriers), as well as tumor cells, and to compromise
normal function in such organs thereby. On the other hand, tumors
derived from such Pgp-expressing tissues are frequently
intrinsically multidrug resistant and therefore unaffected by
chemotherapeutic intervention. Finally, in all multidrug resistant
tumor cells, anti-cancer drugs transported by Pgp decrease
intracellular drug concentration, reduce the drug's "therapeutic
window" and ultimately reduce the effectiveness of chemotherapeutic
treatment. Thus, there is a great need in the art for reagents and
assays that permit the rapid, efficient and economical screening
and development of effective Pgp inhibitors.
[0024] It has been shown that small molecules that are transported
by Pgp can be used to competitively inhibit Pgp-mediated efflux of
chemotherapeutic drugs that are Pgp substrates. Inhibition of
cytotoxic drug efflux from tumor cells in the presence of small
molecule Pgp inhibitors has been shown to increase intracellular
concentrations of drug and thereby increase its cytotoxic
effectiveness. Such small molecules are considered promising drug
candidates for selective potentiation of the antitumor effects of
several anticancer drugs, including doxorubicin, taxol, vinblastine
and VP-16. For example, recent clinical trials of a (relatively)
non-toxic cyclosporin analog (PSC833, Novartis Corp.) demonstrated
the feasibility of using small molecule Pgp inhibitors for
reversing multidrug resistance in patients with hematological
malignancies. These results are being actively pursued by a variety
of pharmaceutical and biotechnology companies and academic
researchers. Thus, development of inexpensive and reliable tests
for high throughput screening and identification of new Pgp
substrates is important for the development of potent Pgp reversing
agents.
[0025] At present there are two techniques available for
identifying Pgp transport substrates. The first is a dye-efflux
assay performed using flow cytometry and is based on competitive
inhibition of Pgp-mediated efflux of fluorescent dyes such as
rhodamine 123. The second is an in vitro cytotoxicity assay that
uses the ability of Pgp substrates to competitively inhibit
Pgp-mediated efflux of cytotoxic drugs in Pgp-expressing multidrug
resistant cells. In this assay, competitive inhibition of Pgp in
cells cultured in the presence of cytotoxic concentrations of
Pgp-effluxed cytotoxic drugs results in increased intracellular
concentration of such drugs and decreased cell growth. Both assays
suffer from the disadvantage that they are laborious and
time-consuming and are not suitable for high throughput screening
or clinical laboratory testing. In addition, these assays are not
specific for Pgp because fluorescent dyes and cytotoxic drugs are
also transport substrates for related multidrug resistance
transporters (such as MRP; Grant et al., 1994, Cancer Res. 54:
357-361)
[0026] There remains a need in the art for a rapid, reliable,
efficient and inexpensive method for high throughput screening of
compounds for Pgp inhibiting activity in order to develop more
effective chemotherapeutic treatment of human cancer patients.
SUMMARY OF THE INVENTION
[0027] The invention also provides methods for evaluating novel
cytotoxic, chemotherapeutic drugs and Pgp inhibitors. The methods
of the invention are based on the development of novel
immunological reagents specific for Pgp in a biochemical
conformation adopted in the presence of Pgp-mediated transport
substrates or ATP depleting agents. The capacity to discriminate
between compounds that induce this conformation in Pgp and those
that do not provides a way to identify Pgp inhibitors that can be
used in high throughput screening assays. These methods can be used
as screening assays based on enhanced binding of certain
immunological reagents such as UIC2 mAb (A.T.C.C. Accession No. HB
11027) or its derivatives in the presence of Pgp substrates and
enable rapid, reliable and cost-effective characterization of
potential new Pgp-targeted drugs.
[0028] The methods of the invention comprise the steps of
contacting a mammalian cell expressing Pgp with an immunological
reagent such as UIC2 mAb in the presence and absence of a putative
Pgp binding substrate and comparing binding of the immunological
reagent in the presence of the test compound with immunological
reagent binding in the absence of the test compound. In preferred
embodiments, the immunological binding agent is detectably labeled.
More preferably, the immunological reagent is detectably labeled
with a fluorescent label, and binding affinity is detected by
fluorescence activated cell sorting (FACS), immunohistochemistry
and similar staining methods. In one aspect of the methods of the
invention, Pgp expression levels are determined, providing the
capacity to quantitatively compare results between assays. In a
second aspect, enhanced binding activity of the immunological
reagents provide a way of determining Pgp binding capacity of the
test compound. The assays of the invention thus advantageously
provide information on both Pgp expression and function
simultaneously.
[0029] An additional advantage of the methods of the invention is
that the use of immunological reagents specific for Pgp reduces the
possibility that the assay results contain contributions from
related species involved in multidrug resistance, such as MRP.
[0030] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A through 1H depict the predicted nucleic acid
sequence of human Pgp (Seq. I.D. No. 1), wherein the initiation
(ATG) and termination (TGA) codons, as well as codons encoding
mutations at amino acid positions 433 and 1076, are underlined.
[0032] FIG. 2A illustrates flow cytometric analysis of K562/I-S9
leukemia cells incubated with phycoerythrin (PE)-conjugated mAb in
the presence or absence of vinblastine.
[0033] FIG. 2B illustrates flow cytometric analysis of K562/I-S9
leukemia cells incubated with PE-conjugated UIC2 mAb in the
presence or absence of vinblastine at 4.degree. C.
[0034] FIGS. 3A and 3B illustrate flow cytometric analysis of
K562/i-S9 leukemia cells incubated with PE-conjugated UIC2 mAb
(FIG. 3A) or MRK16 mAb (FIG. 3B) in the presence or absence of
different cytotoxic drugs.
[0035] FIG. 4 illustrates flow cytometric analysis of K562/i-S9
leukemia cells incubated with PE-conjugated UIC2 mAb in the
presence of increasing concentrations of vinblastine (1-625 .mu.M),
taxol (0.96-600 .mu.M), verapamil (1.8-1125 .mu.M), colchicine
(2-1250 .mu.M), etoposide (1.36-850 .mu.M) and puromycin (1.72-1075
.mu.M).
[0036] FIGS. 5A through 5D illustrate flow cytometric analysis of
mouse L cell transfectants expressing wildtype (KK-L) double mutant
(MM) or single mutant (MK-H or KM-H) human Pgp incubated with
PE-conjugated UIC2 (FIGS. 5A and 5C) or MRK16 (FIGS. 5B and
5D).
[0037] FIGS. 6A through 6D illustrate flow cytometric analysis of
mouse L cell transfectants expressing wildtype (KK-L; FIGS. 6A and
6B), or double mutant (MM; FIGS. 6C and 6D) human Pgp incubated
with PE-conjugated UIC2 (FIGS. 6A and 6C) or MRK16 (FIGS. 6B and
6D) in the presence of absence of taxol, vinblastine or
etoposide.
[0038] FIGS. 7A through 7F illustrate flow cytometric analysis of
mouse L cell transfectants expressing wildtype (KK-H) or single
mutant (KM-H and MK-H) human Pgp incubated with PE-UIC2 (FIGS. 7A,
7C and 7E) or PE-MRK16 (FIGS. 7B, 7D and 7F) in the presence or
absence of vinblastine, taxol or etoposide.
[0039] FIGS. 8A through 8E illustrate flow cytometric analysis of
mouse L cell transfectants expressing wildtype (KK-L; FIG. 8A or
KK-H; FIG. 8B), single mutant (MK-H; FIG. 8C; or KM; FIG. 8D) or
double mutant (MM; FIG. 8E) human Pgp incubated with PE-conjugated
UIC2 in the presence or absence of vinblastine and the ATP
depletion agents oligomycin, azide and cyanide.
[0040] FIGS. 9A through 9C illustrate flow cytometric analysis of
K562/i-S9 leukemia cells incubated with PE-conjugated UIC2 in the
presence or absence of vinblastine and varying concentrations of
the ATP depletion agents oligomycin, azide and cyanide.
[0041] FIGS. 10A and 10B illustrate flow cytometric analysis of
KK-L cells incubated with PE-conjugated UIC2 (FIG. 10A) or MRK16
(FIG. 10B) in the presence or absence of vinblastine and varying
concentrations of the ATP depletion agents oligomycin, azide and
cyanide.
[0042] FIG. 11 illustrates flow cytometric analysis of K562/i-S9
leukemia cells incubated with PE-conjugated UIC2 in the presence or
absence of cyclosporine, BSO or SN-38 as described in Example
5.
[0043] FIG. 12 illustrates flow cytometric analysis of MCF7-40F P4
breast cancer cells incubated with PE-conjugated UIC2 in the
presence or absence of cyclosporine, cisplatin or SN-38 as
described in Example 5.
[0044] FIG. 13 illustrates flow cytometric analysis of human KB-8-5
tumor cells incubated with PE-conjugated UIC2 in the presence or
absence of taxol or taxotere as described in Example 6.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0045] The present invention provides a variety of methods related
to P-glycoprotein mediated multidrug resistance in mammalian, most
preferably human, cells. For the purposes of the present invention,
"multidrug resistance" is defined as cross-resistance to at least
the following cytotoxic drugs: vinblastine, vincristine,
doxorubicin, colchicine, actinomycinD, etoposide, taxol, puromycin,
and gramicidin D; it will be recognized that cross-resistance to
other cytotoxic drugs also falls within the meaning of multidrug
resistance as it is understood by those with skill in the art. Such
drugs are generally referred to herein as MDR drugs.
[0046] The methods of the invention ire based in significant part
on the discovery by the present inventors that the mAb UIC2, which
is capable of inhibiting drug efflux from Pgp-expressing cells,
specifically binds to Pgp in a particular biochemical conformation.
For the purposes of this invention this biochemical conformation is
functionally defined as the conformation adopted by human Pgp in
the presence of Pgp substrates or ATP depleting agents, and results
in enhanced binding of the mAb UIC2. Also within this definition
are certain mutant forms of Pgp having disabling mutations in the
nucleotide binding sites, wherein ATPase activity is disabled, as
described below, in Loo and Clarke (1995, J. Biol. Chem. 270:
21449-21452) and in Muller et al. (1996, J. Biol. Chem 271:
1877-1883). For the purposes of this invention, exemplary Pgp
transport substrates include a variety of lipophilic, cytotoxic
natural product drugs used in cancer chemotherapy, including but
not limited to Vinca alkaloids, epipodophyllotoxins,
anthracyclines, etoposide, colchicine, colcemid and taxol, as well
as the antibiotics monensin and actinomycin D and the interleukin
cytokines. For the purposes of this invention, the term
"ATP-depleting agent" is intended to include, but is not limited
to, 2-deoxyglucose, cyanine, oligomycin, valinomycin and azide, as
well as salts and derivatives thereof.
[0047] The invention provides methods for detecting functional
P-glycoprotein expression in a mammalian cell, particularly a
malignant mammalian cell and most particularly a multidrug
resistant malignant mammalian cell. For the purposes of this
invention, the term "functional Pgp expression" is intended to
encompass the production of Pgp protein in a cell membrane, most
preferably the plasma membrane, wherein the Pgp is capable of
transporting an MDR drug across said membrane and against a
concentration or solubility gradient. "Functional Pgp expression"
is also intended to encompass Pgp protein molecules having an
ATPase activity.
[0048] In the methods of the invention provided to detect
functional Pgp expression in a mammalian cell, the immunological
reagent is preferably provided wherein the extent and amount of
specific binding of the reagent to Pgp expressed by the mammalian
cell is increased in the presence of a Pgp substrate or
ATP-depleting agent. For the purposes of this invention, it will be
understood that the invention thus provides methods and reagents
wherein specific binding of the immunological reagents is enhanced
in the presence of a Pgp substrate or ATP-depleting agent, as
compared with specific binding of the immunological reagent to the
mammalian cell in the absence of a Pgp substrate or ATP-depleting
agent. Such enhanced binding is detected using any method known to
the skilled artisan, including but not limited to detection of
binding of detectably-labeled embodiments of the immunological
reagents of the invention, and detection of specific binding of the
immunological reagents of the invention using a detectably-labeled
immunological reagent that is specific for the immunological
reagents of the invention (e.g., in a "sandwich-type" immunoassay).
Alternatively, and preferably, the methods of the invention include
conventional cell separation methods and techniques, including but
not limited to fluorescence activated cell sorting techniques. In
other embodiments, the methods of the invention are provided
wherein the immunological reagents of the invention are recognized
by detectably-labeled second immunological reagents which
specifically recognize the immunological reagents of the invention
(for example, based on isotypic, allotypic or species-specific
antibodies or antisera).
[0049] For the purposes of this invention, the term "immunological
reagents" is intended to encompass antisera and antibodies,
particularly monoclonal antibodies, as well as fragments thereof
(including F(ab), F(ab).sub.2, F(ab)' and Fv fragments). Also
included in the definition of immunological reagent are chimeric
antibodies, humanized antibodies, and recombinantly-produced
antibodies and fragments thereof. Immunological methods used in
conjunction with the reagents of the invention include direct and
indirect (for example, sandwich-type) labeling techniques,
immunoaffinity columns, immunomagnetic beads, fluorescence
activated cell sorting (FACS), enzyme-linked immunosorbent assays
(ELISA), and radioimmune assay (RIA). For use in these assays, the
Pgp-specific immunological reagents can be labeled, using
fluorescence, antigenic, radioisotopic orbiotin labels, among
others, or a labeled secondary immunological detection reagent can
be used to detect binding of the Pgp-specific immunological
reagents (i.e., in secondary antibody (sandwich) assays).
[0050] The UIC2 mAb is one example of the immunological reagents of
the invention. This mAb is directed to an epitope in an
extracellular domain of human Pgp, and was made by immunizing mice
with mouse cells that have been made MDR by transfection with an
isolated human MDR1-encoding cDNA (see U.S. Ser. No. 07/626,836,
incorporated by reference). Briefly, immunogenic cells (preferably
transfected syngeneic mouse fibroblasts) were used to immunize
BALB/c mice (e.g., transfected BALB/c mouse 3T3 fibroblasts). MDR
derivatives of mouse BALB/c 3T3 fibroblasts were generated with
human MDR1-encoding DNA, and cells selected and grown in cytotoxic
concentrations of an MDR drug. Once produced, MDR fibroblasts were
selected in which the transfected MDR1 gene had been amplified, by
consecutive steps of selection in progressively higher
concentrations of an MDR drug. This produced highly multidrug
resistant cells that expressed large amounts of Pgp inserted into
the cellular plasma membrane resulting in high levels of MDR (e.g.,
BALB/c 3T3-1000 cells are resistant to vinblastine at a
concentration of 1000 ng/mL).
[0051] Such cells were used to immunize syngeneic mice. Appropriate
numbers of cells were injected subcutaneously (s.c.) or
intraperitoneally (i.p.) by art-recognized immunization protocols
(see U.S. Pat. No. 5,434,075, issued Jul. 18, 1995, incorporated in
their entirety herein). Typically, 10.sup.5 to 10.sup.8 transfected
cells were injected 5 or 6 times at two week intervals, and a final
boosting was done with, for example, 10.sup.6 cells subcutaneously
and/or intravenously. At an appropriate time after the booster
injection, typically 3 to 5 days thereafter, the spleen was
harvested from a hyperimmune mouse, and hybridomas generated by
standard procedures (see, e.g., Kearney et al., 1979, J. Immunol.
123: 1548) using human myeloma cells, for example, P3-X63-Ag 8.653
(A.T.C.C., Rockville, Md.).
[0052] Extracellular fluids from individual hybridoma cultures were
screened for specific mAb production by conventional methods, such
as by indirect immunofluorescence using non-Pgp expressing control
cells (e.g. non-transfected fibroblasts) and human Pgp-expressing
(e.g. BALB/c 3T3-1000) cells affixed to glass slides, and
FITC-labeled goat anti-mouse polyvalent immunoglobulins (Sigma
Chemical Co., St. Louis, Mo.) as the secondary, reporter antibody.
The particular screening method used was not critical provided that
it was capable of detecting anti-human MDR1 Pgp mAb. It is
important, however, that cells are not permeabilized and fixed
during screening (i.e., they are living cells), so that only
antibodies reactive with extracellular protein domains are
detected.
[0053] A stable hybridoma producing the UIC2 mAb was established by
conventional methods, such as by consecutive rounds of subcloning
by, e.g., end-point dilution, and screening the culture medium for
monoclonal antibodies. The hybridoma was propagated by, for
example, growth in ascites fluid in vivo in syngeneic animals, and
the secreted antibody isolated and purified from ascites fluid by
affinity chromatography with a Sepharose-Protein A matrix specific
for an IgG isotype. It will be understood that other procedures for
immunoglobulin purification well known in the art are also useful
for producing hybridomas that express Pgp-specific antibodies.
[0054] Alternative methods for producing mAbs are known in the art
(as described in U.S. Pat. No. 5,434,075, issued Jul. 18, 1995,
incorporated in its entirety herein).
[0055] mAbs produced by the UIC2 hybridoma, as well as fragments
and recombinant derivatives thereof, were characterized as to
immunoglobulin isotype, reactivity with different Pgp-expressing
cell lines and binding to Pgp in MDR cells using art-recognized
techniques (see U.S. Pat. No. 5,434,075, issued Jul. 18, 1995,
incorporated by reference). As provided herein, preferred mAbs of
the invention specifically bind to Pgp in a biochemical
conformation adopted in the presence of Pgp-mediated transport
substrates or ATP depleting agents, or in certain Pgp mutants as
described herein.
[0056] The effect of anti-Pgp mAbs, fragments or recombinant
derivatives thereof on Pgp function was assessed by studying the
efflux of fluorescent or radioactively labeled drugs from MDR cells
in the presence of absence of mAb. The effects of antibody
preparations on drug cytotoxicity were assessed by incubating
suspensions of MDR and control cells with the antibody preparation,
then testing for cell growth inhibition in the absence and presence
of an anti-cancer drug such as one of the Vinca alkaloids. Such
assays are by definition preferred, as the mAbs of the invention
are intentionally provided to be specific for substrate-bound
Pgp.
[0057] Fragments of the UIC2 mAb that maintain the antigenic
specificity of the complete antibody are derived by enzymatic,
chemical or genetic engineering techniques (for example, partial
digestion with proteolytic enzymes such as papain, pepsin or
trypsin; papain digestion produces two Fab fragments and one
F.sub.c fragment, while pepsin cleavage releases F(ab).sub.2 (two
antigen-binding domains bound together) fragments). mAb fragments
lacking the constant (F.sub.c) portion are advantageous over the
complete antibody for in vivo applications, as such fragments are
likely to possess improved tissue permeability. Furthermore, many
cells and tissues in the body express receptors capable of binding
to the F.sub.c portion of antibodies, resulting in undesirable
non-specific binding of the complete antibody.
[0058] The methods of the invention are not intended to be limited
in scope to immunological reagents comprising the UIC2 mAb and
hybridomas producing this mAb. The invention provides a variety of
methods, all related to specific binding of mAbs to Pgp in a
biochemical conformation adopted in the presence of Pgp-mediated
transport substrates or ATP depleting agents. The UIC2 mAb is
provided solely as one illustrative example of an mAb that
specifically binds to Pgp and mutants thereof having such a
biochemical conformation.
[0059] The Examples which follow are illustrative of specific
embodiments of the invention, and various uses thereof. They set
forth for explanatory purposes only, and are not to be taken as
limiting the invention.
Example 1
1. Cell Lines, Monoclonal Antibodies, and Reagents
[0060] MRK-16 mAb (IgG.sub.2a) was obtained from Dr. T. Tsuruo,
University of Tokyo, Japan. UIC2 was produced from UIC2 and UIC2/A
hybridomas as described in U.S. Pat. No. 5,434,075, issued Jul. 18,
1995.
[0061] All mAb samples were at least 95% pure according to
SDS-PAGE. Concentrations of the mAb were determined by the
quantitative mouse Ig radial immunodiffusion kit (ICN, Costa Mesa,
Calif.). When necessary, mAb's were further concentrated and
dialyzed against phosphate-buffered saline (PBS) or Dulbecco
modified Eagle's medium (DMEM). mAbs were conjugated with
R-phycoerythrin (PE) or fluorescein isothiocyanate (FITC) at 1:1
(PE) and 1:4 (FITC) mAb:label and purified using standard
techniques (Maino et al., 1995, Cytometry 20: 127-133).
IgG.sub.2a-PE conjugates were purchased from Becton-Dickinson
Immunocytometry Systems (BDIS, San Jose, Calif.) and used as a
negative isotype control for nonspecific staining.
[0062] The K562/Inf cell line was derived by infection of human
K562 leukemia cells with a recombinant retrovirus pLMDR1L6 carrying
human MDR1 cDNA (Choi et al., 1991, Proc. Natl. Acad. Sci. USA 88:
7386-7390), and subsequently subcloned without cytotoxic selection
(e.g., by FACS sorting based on Pgp-specific immunostaining or
Pgp-mediated efflux of fluorescent dyes). Clones expressing
relatively high levels of Pgp were selected by repeated selection
of Pgp-positive clones by FACS after clonal expansion. Clone
K562/I-S9 is one such FACS-selected clone (produced as described in
Weisberg et al., 1996, J. Exp. Med. 183: 2699-2704).
[0063] LMtk.sup.- cells transformed with wildtype and mutant forms
of P-glycoprotein were prepared according to Morse (1996, Doctoral
Dissertation, Department of Genetics, University of Illinois at
Chicago, incorporated by reference herein). MDR1 cDNA-comprising
constructs encoding wildtype (KK), single mutant (KM, MK) and
double mutant (MM) forms of P-glycoprotein were prepared as
described in Morse, wherein the mutant forms have a
lysine.fwdarw.to.fwdarw.methionine mutation within either (single
mutant) or both (double mutant) of the consensus ATP binding sites
in the amino- and carboxyl-terminal halves of P-glycoprotein,
introduced at amino acid positions 433 and 1076 by site-directed
mutagenesis techniques (see Kramer et al., 1984, Nucleic Acids Res.
12: 9441-9456; Carter et al., 1985, Nucleic Acids Res. 13:
4431-4443). Each of these constructs further comprises the
bacterial neomycin-resistance gene (neo), fused to the MDR1 gene
via an overlapping translation termination/initiation codon (ATGA).
As a consequence, MDR1 and neo are expressed in mammalian cells in
a bicistronic messenger RNA. The MDR1-encoding portions of these
constructs are shown in Seq. I.D. No. 1. These sequences, cloned
into the mammalian expression vector expression vector pUCFVX were
introduced into LMtk cells by calcium phosphate coprecipitation or
electroporation (see Sambrook et al., ibid.) and transfectants
selected in G418 (Grand Island Biological Co. (GIBCO), Long Island,
N.Y.)-containing media. Clonal populations of Pgp wildtype or
mutant-expressing cells expressing approximately equal amounts of
Pgp at the cell surface were selected by FACS using fluorescently
labeled mAb MRK16 and were then expanded under G418 selection.
[0064] All chemotherapeutic drugs were purchased from Sigma
Chemical Co. (St. Louis, Mo.), diluted in water, DMSO or alcohol,
aliquoted and stored at +4.degree. C. for 10-14 days or at
20.degree. C. until use.
2. Preparation of Anti-Pgp Monoclonal Antibodies
[0065] Monoclonal antibodies specific for human P-glycoprotein were
prepared as disclosed in co-owned and co-pending U.S. patent
application Ser. No. 07/854,881, filed Mar. 20, 1992, now U.S. Pat.
No. 5,434,075, issued Jul. 18, 1995, and Ser. No. 08/032,056, filed
Mar. 16, 1993, each of which are incorporated in its entirety
herein.
[0066] Briefly, mouse fibroblast BALB/c 3T3 cells expressing the
MDR1 gene encoding P-glycoprotein (Pgp) were derived by
transfecting fibroblasts with isolated human MDR1 cDNA in a
eukaryotic expression vector pUCFVXMDR1 (Choi et al., 1988, Cell
53: 519-529), isolating multidrug-resistant cells after cytotoxic
selection in 20 ng/mL of vinblastine, and subsequently amplifying
the transfected gene by consecutive steps of selection in 250
ng/mL, 500 ng/mL and 1000 ng/mL of vinblastine. The resultant
multidrug-resistant fibroblasts were termed BALB/c 3T3-250, BALB/c
3T3-500 and BALB/c 3T3-1000, respectively.
[0067] BALB/c mice were immunized with 1-2.times.10.sup.7 of BALB/c
3T3-1000 cells, injected subcutaneously (s.c.) and/or
intraperitoneally (i.p.) six times at two-week intervals. The final
immunogenic boost was done with 2.times.10.sup.7 cells i.p., and
5.times.10.sup.6 cells administered intravenously (i.v.). Four days
after the last administration of fibroblasts, the spleen from one
animal was removed, and hybridomas generated by art-recognized
techniques using the human myeloma cell line P3-X63-Ag8.653
(A.T.C.C. Accession No. CRL-1580).
[0068] Tissue culture supernatant fluids from individual hybridomas
were screened for monoclonal antibody (mAb) production by indirect
immunofluorescence labeling of live BALB/c 3T3 and BALB/c 3T3-1000
cells attached to glass slides. Fluorescein isothiocyanate
(FITC)-labeled goat anti-mouse polyvalent immunoglobulins (obtained
from Sigma Chemical Co., St. Louis, Mo.) were used as a secondary
antibody reagent at 1:100 dilution. Of 556 tested hybridomas, mAb
produced by only two hybridomas reacted with BALB/c 3T3-1000 cells,
and of these two only one hybridoma (termed UIC2) produced an
antibody reactive with BALB/c 3T3-1000 cells, but not with control
BALB/c 3T3 cells.
[0069] A stable hybridoma line secreting UIC2 mAb was established
by three consecutive rounds of subcloning by end-point dilution and
screening of the supernatant fluids, as described in co-owned and
co-pending U.S. patent application Ser. No. 07/854,881, filed Mar.
20, 1992, now U.S. Pat. No. 5,434,075, issued Jul. 18, 1995, and
Ser. No. 08/032,056, filed Mar. 16, 1993, each of which are
incorporated in its entirety herein.
[0070] The UIC2 hybridoma was propagated as ascites in syngeneic
BALB/c mice, and the immunoglobulin was purified from ascites fluid
by Sepharose-Protein A (Bio-Rad, Richmond, Calif.) affinity
chromatography. UIC2 mAb, tested by SDS-PAGE, was at least 95% pure
IgG. The UIC2 hybridoma is on deposit in the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. U.S.A.
20110-2209 (Accession No. HB11027) and is available to the
public.
[0071] Application of Ouchterlony and immunoblotting tests using a
standard set of anti-mouse Ig antibodies revealed that the UIC2 mAb
belongs to the IgG.sub.2a subclass.
[0072] UIC2 mAb was shown to induce complement-mediated
cytotoxicity using Low-Tox-M rabbit complement (Cedarland Labs,
Homby, Ontario) on BALB/c, BALB/c 3T3-1000, CEM/VLB.sub.100, K562
and K562/Inf cell lines.
3. Fluorescence Activated Cell Sorting/Flow Cytometry Analysis
[0073] Cells were trypsinized, when necessary, and washed twice
with PBS at room temperature or 4.degree. C. and distributed in 2
mL conical plastic tubes at a concentration of 10.sup.6 cells/tube
in 1 mL of pre-warmed (37.degree. C.) Ca.sup.++, Mg.sup.++-free PBS
and incubated for 37.degree. C. for 10 min. Thereafter, aliquots of
20 .mu.L of drug stock solutions at 1 mg/mL (or at different
concentrations, when necessary) were added. The cells were
incubated with drugs at 37.degree. C. for 10 min. Aliquots of 50
.mu.L mAb stock solutions (UIC2 conjugated with R-phycoerythrin
(UIC2-PE), MRK16-PE, and IgG2a-PE conjugates, or UIC2 conjugates
with fluorescein isothiocyanate (UIC2-FITC) and IgG2a-FITC
conjugates), prepared at 1:10 dilution, were added and the tubes
mixed thoroughly. The amount of mAb added per 10.sup.6 cells/mL was
determined by preliminary titration. mAb stock solutions were used
at a concentration of 0.08 mg/mL in all experiments with
chemotherapeutic drugs. After incubation with mAb for 20-30
minutes, cells were washed twice with ice-cold PBS, transferred
into plastic tubes containing 0.5 mL ice-cold PBS and 1 .mu.g/mL
propidium iodide, and kept on ice until FACS analysis. For indirect
staining experiments, cell samples were washed twice, stirred,
incubated with secondary antibody reagents in 100 .mu.L PBS for 20
min. and prepared as above for FACS analysis. For ATP depletion
experiments, washed cells were incubated with 20 .mu.L aliquots of
stock solutions of azide, oligomycin or cyanide for 15 min. at
37.degree. C. and then immediately treated with chemotherapeutic
drugs, antibodies and propidium iodide as described above.
[0074] Cells were analyzed by FACSort (BDIS) equipped with an argon
laser (Cyonics) tuned to 488 nm, using 4 parameters (forward
scatter, side scatter, FL1 for FITC, FL2 for PE conjugates and FL3
for propidium iodide); dead cells were excluded on the basis of
forward and side scatter and PI (FL3) staining. The FACS data were
analyzed by the Lysis II or CellQuest computer programs.
4. ATP Depletion Experiments
[0075] Cells were depleted of intracellular ATP by incubation with
oligomycin, azide or cyanide at various concentrations under
conditions described in Section 3 above. Intracellular ATP was
measured using the Bioluminescent Somatic Cell Assay kit (Sigma,
St. Louis, Mo.), whereby the amount of ATP in cell lysates is
proportional to light emitted by firefly luciferase. Intracellular
ATP was expressed relative to the amount present in cells treated
with PBS instead of ATP depleting agents. After incubation of cell
lysates with the components of the assay kit, 0.1 mL of the
reaction solution was assayed spectrophotometrically over a
wavelength range of 390-622 nm using an AutoLumat LB953 Universal
Luminometer (EG&G Berthold, Vildbad, Germany). All measurements
were performed at 8.degree. C. in 12.times.75 mm polystyrene
cuvettes (Analytical Luminescence Lab, San Diego, Calif.).
Example 2
mAb UIC2 Reactivity is Increased in the Presence of Pgp-Transported
Compounds
[0076] Flow cytometry was used to analyze the reactivity of
phycoerythrin (PE)-conjugated mAbs UIC2 and MRK16 with
Pgp-expressing cells in the presence of different drugs. The range
of optimal drug concentrations for these experiments (1-5 mg/mL)
was determined by a series of preliminary titration
experiments.
[0077] FIG. 2A illustrates the results obtained with K562/I-S9
leukemia cell line, which was selected to express Pgp by infecting
K562 cells with a MDR1-transducing recombinant retrovirus and
subsequent flow cytometric selection based on MRK16 antibody
staining. Cells were treated in the presence or absence of 25 .mu.M
vinblastine and contacted with PE-conjugates mAbs UIC2, MRK16,
IgG2a (a negative isotype control) and anti-CD54 (a positive
control mAb against a cell surface marker of K562 cells). UIC2
reactivity of this cell line was increased in the presence of the
Pgp-transported drug vinblastine, as seen by the rightwards shift
in the flow cytometric profile at increasing drug concentrations.
This profile shift was not seen with either the positive or
negative control mAbs and was not seen with the Pgp-specific mAb
MRK16. A similar pattern of mAb binding was observed with
FITC-conjugates mAbs and in experiments performed with unlabeled
mAbs detected using labeled secondary antibody (sandwich)
techniques. In addition, increased UIC2 reactivity was only
observed when cells were incubated with drugs and antibody at
37.degree. C., but did not appear when incubations were performed
at 4.degree. C. (FIG. 2B), suggesting that enhanced UIC2 binding in
the presence of certain Pgp substrates requires the cells to be
metabolically active
[0078] A variety of MDR drugs and competitive inhibitors of Pgp
were tested to determine whether these compounds could induce the
FACS profile shift observed with UIC2 binding in the presence of
vinblastine. The tested compounds included vinblastine, taxol,
actinomycin D, gramicidin D, cyclosporine A, reserpine,
5-fluorouracil and methotrexate. The results of these experiments
are shown in FIG. 3A for binding of PE-UIC2 mAb and in FIG. 3B for
binding of MRK16 mAb. In these experiments, a rightwards shift in
the flow cytometry profile of cells contacted with PE-UIC2 mAb was
observed for cells treated with vinblastine, taxol, actinomycin D,
gramicidin D cyclosporine A and reserpine. No FACS profile shift
was observed in cells treated with 5-fluorouracil or methotrexate,
supporting the conclusion that shifting was Pgp specific and was
specifically induced with Pgp substrates (since neither
5-fluorouracil or methotrexate is (typically) a Pgp substrate). In
contrast, and consistent with the earlier results disclosed above,
no change in the flow cytometry profile of cells contacted with
MRK16 mAb was observed in cells treated with any of the tested
drugs. Stimulation of UIC2 reactivity by these compounds was
dose-dependent, for some compounds, while for others no shift was
observed at any concentration tested (as illustrated in FIG.
4).
[0079] Increased UIC2 reactivity in the presence of Pgp substrates
was also observed with other Pgp-expressing cells and cell lines,
including PA3 17 cells expressing Pgp via an MDR1-encoding
retrovirus (Choi et al., ibid.), NIH 3T3 cells, KB-3-1, VSV1 and
GSV1 cells transfected with MDR1 cDNA (Choi et al., 1988, Cell 53:
519-529), Pgp-positive leukemia/lymphoma and tumor samples and
normal B- and T-lymphocyte subpopulations and hematopoietic stem
cells expressing Pgp (Chaudhary et al., 1992, Blood ibid.;
Chaudhary et al., 1992, Cell ibid.). The concentrations of Pgp
substrates producing maximal stimulation of UIC2 reactivity
differed slightly for different cell lines and appeared to
correlate with the levels of Pgp expressed on the corresponding
cell lines.
[0080] A summary of these results are shown in Table I.
TABLE-US-00001 TABLE I UIC2 MRK 16 MDR Drugs taxol + - vinblastine
+ - reserpine + - verapamil + - gramicidin + - cyclosporine + -
vincristine + - actinomycin D + - colchicine* - - etoposide* - -
puromycin - - Non-MDR Drugs 5-fluorouracil - - cisplatin - -
carboplatin - - methotrexate - - azidothymidine - -
cyclophosphamide - - *weak Pgp substrate
Example 3
Mutations at Pgp Nucleotide-Binding Sites Alter UIC2 Reactivity
[0081] The ability of Pgp transport substrates to increase UIC2
reactivity as described in Example 2 suggested that mAb UIC2 reacts
more strongly with Pgp having a conformation associated with
functioning (i.e., drug-transporting) Pgp. To investigate the
relationship between Pgp function and UIC2 reactivity,
nucleotide-binding site mutants of Pgp were used. As described in
Example 1, Pgp was mutagenized at highly conserved lysine residues
(positions 433 and 1076) in the N-terminal and C-terminal
nucleotide-binding sites of the human Pgp. These lysine residues
were substituted with methionine residues (i.e.,
lysine-to-methionine (K.fwdarw.M) substitutions), and the resulting
proteins were designated KK (wild-type Pgp), MM (double mutant), KM
and MK (C-terminal and N-terminal single mutants, respectively).
Analysis of immunoprecipitated Pgps showed that nucleotide binding,
as measured by specific photolabeling with .sup.32P-8-azido-ATP,
was decreased in the single mutants (KM and MK) and undetectable in
MM (as disclosed in Muller et al., 1996, J. Biol. Chem. 271:
1877-1883). In addition, all three mutants (MM, KM and MK) lost
detectable ATPase activity (see Muller et al., ibid.). The double
mutant, MM, also lost the ability to confer drug resistance to all
tested MDR drugs (including vinblastine and vincristine). KM and MK
mutant expressing cells, however, showed a 2-3 fold greater
resistance to vinblastine than control cells not expressing Pgp,
and accumulated 3-4 times more vinblastine than wildtype
(KK)-expressing cells with the same level of vinblastine
resistance. Vinblastine resistance conferred by KK, KM and MK Pgps
was equally sensitive to inhibition with mAb UIC2.
[0082] For UIC2 shift experiments, two sets of murine Lmtk.sup.-
transfectants were used, matched to express very similar levels of
the wild-type or mutant human MDR1 Pgps. The first set includes
cell lines designated KK-L (wild-type) and MM (double mutant)
(FIGS. 5A through 5D and 6A through 6D). The second set, expressing
about five times as much Pgp as the first set, includes cell lines
KK-H (wild type), KM-H and MK-H (single mutants) (FIGS. 7A through
7F). The relative levels of Pgp expression were established on the
basis of indirect immunofluorescence with PE-conjugated MRK16 (see
Morse, 1996, ibid.).
[0083] FIGS. 5A and 5B show a comparison between flow cytometric
analysis of KK-L and MM expressing cells contacted with LTIC2 (FIG.
5A) and MRK16 (FIG. 5B). Similarly, FIGS. 5C and 5D show a
comparison between flow cytometric analysis of KK-H, MK-H and KM-H
expressing cells contacted with UIC2 (FIG. 5C) and MRK16 (FIG. 5D).
The flow cytometric pattern of all of these cells was the same when
assayed using the MRK16 mAb (see FIGS. 5B and 5D). In contrast to
the results obtained using mAb MRK16, UIC2 mAb showed a strikingly
different pattern of reactivity with cell lines transfected with
mutant Pgps. UIC2 reacted much more strongly with the MM double
mutant than with the wild-type Pgp on KK-L cells (compare in FIG.
5A). Similarly, UIC2 binding in single mutant KM-H was equivalent
to wildtype binding (KK-H), while the extent of UIC2 binding to the
MK-H single mutant was diminished.
[0084] UIC2 mAb binding was compared to MRK16 binding in these
cells in the presence or absence of different MDR drugs. These
results are shown in FIGS. 6A through 6D. No rightwards shift in
the flow cytometric profile was observed in any of the cell lines
assayed using MRK16 mAb. In contrast, the wildtype KK-L cell line
showed a rightward shift in the profile when cells treated with
vinblastine or taxol were assayed, but not when cells treated with
etoposide was assayed, consistent with the results disclosed above.
The MM double mutant cell line showed no flow cytometric profile
shift in the presence of these drugs, but the profile was shifted
rightward using UIC2 compared with MRK16 (compare FIGS. 6C and 6D).
Vinblastine induced levels of UIC2 mAb reactivity in KK-L cells
were roughly equivalent to binding levels seen with MM cells. MM
cells showed high levels of UIC2 mAb binding in either the presence
or absence of drugs (FIG. 6D); MRK16 binding was unaffected and
observed at a level consistent with binding to KK-L cells,
confirming our earlier observations on the relative Pgp expression
levels of these cell lines.
[0085] In contrast with these results, the single mutant MK-H cells
showed lower UIC2 reactivity than the wild-type KK-H transfectants,
while the reactivity of the other single mutant cell line KM-H, was
similar to KK-H (FIGS. 7A through 7F). The KK-H, MK-H and KM-H
transfectants were all observed to yield increased UIC2 reactivity
by Pgp substrates, with the final levels becoming very similar for
all three cell lines (compare FIGS. 7A, 7C and 7E). MRK16 binding
levels were approximately the same for all three cell lines in the
presence or absence of drug.
[0086] These results demonstrated that enhanced UIC2 mAb binding
was related to the conformation of Pgp expressed in UIC2-reactive
cell lines, and suggested that the MM mutant had adopted a
conformation equivalent to the biochemically active conformation
presumed to be recognized by UIC2 and which accounted for enhanced
UIC2 mAb binding to Pgp in the presence of certain Pgp
substrates.
Example 4
Intracellular ATP Depletion Maximized UIC2 Reactivity
[0087] The results described in Example 3 above indicated that
maximal UIC2 mAb reactivity was associated with the MM mutant,
which carries disabling mutations in both nucleotide-binding sites.
This result suggested that the biochemical conformation of Pgp that
is specifically recognized by UIC2 mAb could reflect a conformation
in which Pgp had no bound ATP. This further suggested that
intracellular ATP depleting agents would increase UIC2 mAb
reactivity for Pgp. Three different agents that induce ATP
depletion, sodium cyanide, sodium azide and oligomycin (all of
which are specific for mitochondrial enzymes and mechanisms which
generate ATP) were used to deplete Pgp-expressing cells of
intracellular ATP. All three agents were found to increase UIC2 mAb
reactivity to wild-type Pgp in KK-L (FIG. 8A) and K562/I-S9 cells
(FIGS. 9A through 9C). The increase in UIC2 reactivity correlated
with the extent of intracellular ATP depletion, as measured by the
luciferase assay described in Example 1.
[0088] The addition of cyanide, azide or oligomycin to the series
of LMtk.sup.- cells transfected with different Pgp mutants had the
same effect on UIC2 mAb reactivity as the addition of Pgp transport
substrates (FIGS. 8A through 8E). These agents increased the
reactivity of KK-L cells to the level of MM (compare FIGS. 8A and
8B), while having no effect on the MM cell reactivity, and
increased the reactivity of KK-H, MK-H and KM-H cell lines to
similar final levels. Similar results were obtained in K562/I-S9
cells expressing human Pgp (FIGS. 9A through 9C), and a comparison
of UIC2 (FIG. 10A) and MRK16 (FIG. 10B) binding of KK-L cells
expressing the wildtype human Pgp is shown in FIGS. 10A and
10B.
[0089] Thus, these results support the conclusion that ATP
depleting agents have the same effect on UIC2 mAb reactivity as
mutagenesis of both nucleotide-binding sites of Pgp.
Example 5
Characterization of Pgp Binding of a Novel Anticancer Compound
[0090] A novel anticancer drug was tested using the UIC2 binding
assay to determine whether it bound to Pgp.
[0091] SN-38 is the active species of CPT-11 (Irinothecan), a
newly-developed drug for treating colon cancer. Several clinical
trials have demonstrated the efficacy of CPT-11 in colon cancer
patients. However, it was unclear if SN-38 is a Pgp-transported
substrate. This is of particular importance in colon cancer because
all colon cancer tumors express Pgp and, therefore, are
intrinsically resistant to Pgp-transported cyctotoxic drugs.
[0092] A modification of the UIC2 binding assay was used to
determine Pgp substrate specificity of SN-38 in vitro. Flow
cytometry was performed on the K562/S9-I cell line, which had been
infected with a retrovirus containing human MDR1 cDNA and selected
for Pgp expression by flow sorting. Washed K562/S9-I cells were
incubated in phosphate-buffered solution (PBS) with 2% FCS for 10
min at 37.degree. C. in the presence of SN-38, cyclosporin-A (a
known Pgp substrate as a positive control) or buthionine
sulfoximine (BSO, a non-Pgp drug as a negative control for UIC2
shift) at the final concentration of 5 mg/mL. The cells were then
incubated with UIC2 labeled with phycoerythrin (UIC2-PE, obtained
from Immunotech, Marseille, France), or IgG2a-PE (an mAb isotype
control) for 20 minutes at 37.degree. C. in the presence of the
above drugs. The treated samples were washed twice with cold PBS,
mixed with propodium iodide (to exclude non-viable cells) and
analyzed using the BDIS FACSVantage cell sorter. A DMSO control was
also used in the experiment to exclude the possibility that this
diluent affects UIC2 staining. As shown in FIG. 11, cyclosporine A
induced UIC2 shift (increased immunoreactivity, as compared to "no
drug" DMSO control), while treatment with BSO, a non-Pgp drug, did
not result in UIC2 shift in K562/S9-I cells. Incubation of
K562/S9-I cells with UIC2-PE in the presence of SN-38 under
physiological conditions induced UIC2 shift, demonstrating that
this drug is a Pgp-transported substrate.
[0093] This result was confirmed in additional experiments
performed using a Pgp-positive breast cancer cell line, MCF7-P4
Pgp-positive breast carcinoma cell line; in these assays, cisplatin
was used as a negative control. These results are shown in FIG. 12,
wherein binding of labeled UIC2 to these cells was enhanced in the
presence of SN-38. There results established that SN-38 is a
Pgp-biding drug. These results have been confirmed by conventional
in vitro cytotoxicity tests.
Example 6
Characterization of Pgp Binding of a Novel Anticancer Compound
[0094] The cytotxic drugs taxol and taxotere are commonly-used
chemotherapeutic agents for treating a variety of human cancers,
including breast, lung and ovarian tumors, as well as Kaposi's
sarcoma. Taxol was initially purified from Pacific yew tree bark,
and currently its semi-synthetic form is manufactured from other
renewable sources. Taxotere is a semi-synthetic compound derived
from the needles of European yew tree. Both drugs are very similar
with respect to their chemical structure and mechanism of cytotoxic
action on microtubules in tumor cells.
[0095] It has been previously shown that both taxol and taxotere
can be effluxed by the Pgp pump in human tumors. However, it had
also been shown that taxotere had higher anticancer efficacy than
taxol in clinical trials on breast and ovarian tumors. One
explanation for the clinical data was that this difference in the
drugs' activity was caused by their differences in their Pgp
substrate specificity. The modification of the UIC2 binding assay
described in Example 5 was used to determine relative Pgp transport
specificities for taxol and taxotere. In these experiments, a human
tumor cell line, KB-8-5, was incubated as described above with
PE-labeled UIC2 in the presence of taxol or taxotere at four
concentrations: 0.08 mM, 0.31 mM, 1.25 mM, and 5 mM, using DMSO
diluent alone as a negative substrate control and mouse IgG2a as a
negative mAb control. Flow cytometric analysis was performed as
described, and the results are shown in FIG. 13. At low
concentrations (0.08 mM and 0.31 mM), taxotere was found to induce
a smaller binding affinity shift than taxol at equivalent
concentrations (compare the two cytometric analysis curves on the
lower left with the two on the lower right in FIG. 13). These
results were not as evidence in the analyses performed at higher
drug concentrations. This observation indicated that the taxotere
is effuxed less efficiently from these cells than taxol, leading to
increased intracellular taxotere accumulation and the higher
therapeutic efficiency found clinically in cancer patients. This
finding was confirmed by conventional in vitro cytotoxicity
experiments demonstrating that intracellular accumulation of
taxotere is higher than that of taxol
[0096] These results demonstrate that the methods of the invention
can be used to characterize and compare novel anticancer drugs for
Pgp binding efficiency, and that these results can be used to
identify anticancer compounds having higher clinical efficacy based
on lower affinity for Pgp.
[0097] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
Sequence CWU 1
1
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