| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Methods and reagents for preparing and using immunological agents specific for P-glycoprotein |
| PATENT 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. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 21, 1999 |
| PATENT REFERENCES CITED | This data is not available for free |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. A method for screening a compound for P-glycoprotein binding comprising the steps of: (a) incubating a mammalian cell expressing P-glycoprotein in the presence or absence of the compound; (b) reacting the mammalian cell with an antibody or antigen-binding fragment thereof specific for P-glycoprotein in a biochemical conformation adopted in the presence of a P-glycoprotein substrate, and (c) comparing binding of the antibody or antigen-binding fragment thereof to the cell in the presence of said compound with binding in the absence of the compound. 2. The method of claim 1 wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody specific for P-glycoprotein in a biochemical conformation adopted in the presence of said P-glycoprotein substrate. 3. The method of claim 1 wherein binding of the antibody or antigen-binding fragment thereof is increased in the presence of the P-glycoprotein substrate. 4. The method of claim 1 wherein the antibody or antigen-binding fragment thereof is detectably-labeled. 5. The method of claim 4 wherein the detectable label is a fluorescent label. 6. The method of claim 5 wherein binding of the antibody or antigen-binding fragment thereof is increased to a detectable level in the presence of the P-glycoprotein substrate and wherein enhanced binding of the fluorescently-labeled antibody or antigen-binding fragment thereof is detected by fluorescence-activated cell sorting. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention 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. 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 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 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. 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 proteinproduct 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. 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 MDR 1 gene expression correlates with resistance to chemotherapy in different types of cancer. 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. 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). 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. Immunological reagents also provide a means for specifically inhibiting drug efflux mediated by Pgp. Monoclonal antibodies specific for Pgp are known in the art. 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. Meyers et al., 1987, Cancer Res. 49: 3209 disclose mAbs HYB-241 and HYB-612, which recognize an external epitope of Pgp. 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. 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. Hamada et al., 1990, Cancer Res. 50: 3167 disclosed a recombinant chimeric antibody that combines the variable region of MRK-16 with the Fc portion of a human antibody, and showed this chimeric antibody to be more effective than MRK-16 mAb in increasing cytotoxicity in vitro. 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. 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. 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. 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. 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. 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. 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) 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 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. 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. 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. 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 FIGS. 1A through 1H depicts 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 highlighted. 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. FIG. 2B illustrates flow cytometric analysis of K562/1-S9 leukemia cells incubated with PE-conjugated UIC2 mAb in the presence or absence of vinblastine at 4.degree. C. FIGS. 3A through 3D 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. 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). FIGS. 5A through 5D illustrates 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). FIGS. 6A through 6D illustrates 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. 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. 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. 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. FIGS. 10A through 10D 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. 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. 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-3 8 as described in Example 5. 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 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. 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 therof. 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. 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). 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 F.sub.v 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). 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 USSN 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). 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.). 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. 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. 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). 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. 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. 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 Fc portion of antibodies, resulting in undesirable non-specific binding of the complete antibody. 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 |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
|
Want more information ? Interested in the hidden information ? Click here and do your request. |