Methods and reagents for preparing and using immunological...

Details Methods and reagents for preparing and using immunological... Methods and reagents for preparing and using immunological...

1999-09-16

2003-10-07

Huff, Sheela (Department: 1642)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S069300, C435S070100, C435S070200, C435S440000, C435S449000, C435S452000, C435S451000, C435S325000, C530S387100, C530S387700, C530S387900, C530S388100, C530S388200

Reexamination Certificate

active

06630327

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the use of immunological reagents specific for a human transmembrane efflux pump protein (P-glycoprotein) 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. The invention provides such immunological reagents for immunodiagnostic and therapeutic uses, for isolating lymphocytes and hematopoietic stem cells, and for anticancer drug development.
2. Background of the Invention
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 A 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.
MDR frequently appears to result from decreased intracellular accumulation of drug 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.
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 co-owned U.S. Pat. No. 5,206,352, issued Apr. 27, 1993, having an effective filing date of Mar. 28, 1986), and is generally referred to as MDR 1. The protein product of the MDR 1 gene, most generally known as P-glycoprotein (“Pgp”), is a 170-180 kilodalton (kDa) transmembrane protein having the aforementioned energy-dependent efflux pump activity.
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).
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.
Because Pgp is involved in the resistance of different types of human malignancies to conventional chemotherapy, the expression of Pgp is an important diagnostic and prognostic factor which in many cases helps the physician to choose the most effective combination of chemotherapeutic drugs and to monitor the efficacy of treatment. One way Pgp expression has been evaluated is by detecting the binding of specific immunological reagents (antibodies) to tumor samples. However, frequently the expression level of Pgp in tumor cells is low and cannot be reproducibly detected by routine immunological methods. In addition, there are few immunological or other reagents specific for functionally-active Pgp (which are the only forms of Pgp that are clinically relevant). Thus, there is a need in the art to increase the sensitivity and specificity of immunological and immunohistochemical methods for detecting functional Pgp expression.
Pgp is also constitutively expressed in many normal cells and tissues (see Cordon-Cardo et al., 1990
, J. Histochem. Cytochem
. 3: 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 Rhl23, 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
8: 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, as disclosed more fully in co-pending U.S. patent application Ser. No. 08/658,583, filed Jun. 7, 1996, incorporated by reference herein in its entirety), the physiological role of Pgp in normal immune cells has remained unclear to date.
Expression of Pgp in hematopoietic cells provides an effective means for identifying and purifying lymphocytes and hematopoietic stem cells. As described more completely in co-owned and/or co-pending U.S. Pat. No. 5,434,075, issued Jul. 18, 1995 and U.S. patent application Ser. No. 08/032,056, filed Mar. 16, 1993, functional Pgp assays (such as fluorescent dye efflux) and immunochemical methods (such as fluorescence activated cell sorting (FACS) analysis) can in theory be used to purify lymphocytes and hematopoietic stem cells.
However, the levels of expression of Pgp on stem cells are low, and consequently the amount of an immunological reagent such as a monoclonal antibody (mAb) bound to a hematopoietic stem cell membrane using conventional procedures is generally not high enough to efficiently separate Pgp-positive cells by any conventional immunological technique (such as FACS, immunomagnetic particle separation, cell panning, or other methods known in the art). Thus, there remains a need in this art to improve the efficiency of methods for using Pgp expression to specifically purify lymphocytes and hematopoietic stem cells from biological sources.
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 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 (Akiyama et al., 1988
, Molec. Pharm.
3: 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 c

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