EP1865987A2 - Inhibition of p-glycoprotein by treatment with a class of modulators and the uic2 monoclonal antibody - Google Patents

Abstract

The present invention is related to compositions and methods directed at the use of the UIC2 monoclonal antibody with a modulator to combat multidrug resistance. Particularly, the present invention relates to methods, compositions and kits useful for blocking the action of Pgp receptors as well as for detecting cells that exhibit Pgp activity. The embodiments include a method of detecting Pgp-expressing cells using a UIC2 monoclonal antibody, a modulating compound, and a fluorescent substrate. Another embodiment comprises a method of inhibiting the pumping of substrates out of a cell using the UIC2 monoclonal antibody in concert with a modulating compound such as cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m-amsacrine or ivermectin.

Description

INHIBITION OF P-GLYCOPROTEIN BY TREATMENT WITH A CLASS OF MODULATORS AND THE UIC2 MONOCLONAL ANTIBODY

FIELD OF THE INVENTION

The present invention is related to compositions and methods directed at the use of the UIC2 monoclonal antibody in combination with a modulator, which may be used at a low concentration, to combat multidrug resistance.

BACKGROUND OF THE INVENTION

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

One of the unresolved problems of cancer as well as of AIDS chemotherapy is the resistance of target tissues against a broad range of compounds applied in the treatment protocols, such as steroids, antineoplastic drugs, immuno-suppressive agents or HIV protease inhibitors (Borst, P., & Elferink, R.O. (2002) Annu. Rev. Biochem. 71, 537-592; Glavinas, H., Krajcsi, P., Cserepes, J., & Sarkadi, B. (2004) Current Drug Delivery 1, 27-42; Jones, K., Bray, P.G., Khoo, S.H., Davey, R.A., Meaden, E.R., Ward, S.A. & Back, DJ. {2QQ\)AIDS 15, 1353-1358; Sankatsing, S.U.C., Beijnen, J.H., Schinkel, A.H., Lange, J. M. A. & Prins, J.M (2004) Antimicrob. Agents Chemother. 48, 1073-1081). This phenomenon is frequently associated with and caused by, the overexpression of certain ABC (ATP Binding Cassette) transporters, including P-glycoprotein (ABCBl; Pgp), multidrug resistance protein 1 (ABCCl; MRPl) and breast cancer resistance protein (ABCG2; BCRP), leading to a decreased accumulation of drugs in these tissues (Borst, P., & Elferink, R.O. (2002) Annu. Rev. Biochem. 71, 537-592; Glavinas, H., Krajcsi, P., Cserepes, J., & Sarkadi, B. (2004) Current Drug Delivery 1, 27-42). Multidrug resistance (mdr) mediated by Pgp was the first to be discovered and it appears to be the most widely observed mechanism in clinical cases of mdr (Glavinas, H., Krajcsi, P., Cserepes, J., & Sarkadi, B. (2004) Current Drug Delivery 1, 27-42). Pgp is comprised of two homologous halves, each containing an ATP binding site characterized by an "ABC signature" element, in addition to Walker A and B sequence motives, and 6 transmembrane α-helices. These α-helices form a pore-like structure allowing the passage of a wide range of hydrophobic substrates against their concentration gradient, effected by ATP fueled conformational changes of the protein (Rosenberg, M.F., Kamis, A.B., Callaghan, R., Higgins, C.F., Ford, R.C. (2003) J. Biol. Chem. 278, 8294-8299). Numerous studies suggest that the principal physiological role for Pgp is to protect the organism from toxic substances, since it is expressed mostly in tissues having barrier functions, e.g., in capillary endothelial cells comprising the blood-brain barrier, placental trophoblasts and in polarized endothelial cells in several organs, like the gut, the liver, or the kidneys. Tumors derived from these tissues are intrinsically resistant to chemotherapy, while other malignancies may express Pgp or other ABC transporters during later steps of disease progression or in response to the chemotherapy (Borst, P., & Elferink, R.O. (2002) Annu. Rev. Biochem. 71, 537- 592; Glavinas, H., Krajcsi, P., Cserepes, J., & Sarkadi, B. (2004) Current Drug Delivery 1, 27- 42; Leonard, G.D., Fojo, T.,& Bates, S. (2003) The Oncologist S, 411-424).

Tumors are often resistant to chemotherapy as a result of a decrease in the intracellular concentrations of chemotherapeutic drugs. This phenomenon may be due to the function of a group of membrane protein receptors that extrude cytotoxic molecules, keeping intracellular drug concentration below a cell-killing threshold. These protein receptors, known as "multidrug transporters", belong to the superfamily of ATP Binding Cassette (ABC) proteins. One member of the ABC transporter family, P-glycoprotein (Pgp), is capable of extruding a wide spectrum of cytostatic drugs from the cell and is often responsible for the failure of chemotherapy.

In view of the great medical importance of overcoming mdr in cancer chemotherapy, and also of combating mdr phenomena limiting the intracellular concentration of anti-HIV drugs, search for effective and specific reversal strategies continues. These tools include the coadministration of reversing agents (mdr modulators) with the specific drugs to overcome their efflux mediated by the pumps. Concerning Pgp, its antagonist may hinder drug extrusion competitively (e.g., CsA, FK506, (Saeki, T., Ueda, K., Tanigawara, Y., Hori, R., Kamano, T. (1993) J. Biol. Chem. 268, 6077-6080)) or allosterically (e.g., XR9576, SR33557 or cis-(Z)- flupentixol, (Martin, C, Berridge, G., Mistry, P., Higgins, C. & Callaghan, R. (1997) Br. J. Pharmacol. 122, 765-771; Mistry, P., Stewart, J.A., Dangerfϊeld, W., Okiji, S., Liddle, C, Bootle, D., Plumb, AJ. Templeton, D. & Charlton, P. (2001) Cancer Res. 61, 749-758; Maki, N., Hafkemeyer, P. & Dey, S. (2003) J. Biol. Chem. 278, 18132-18139)).

Several monoclonal antibodies that recognize discontinuous extracellular epitopes of Pgp have been developed. A few of them (e.g., MRKl 6, MRKl 7, MC57, and UIC2 in particular) appear to partially inhibit Pgp mediated drug export in vitro (Mechetner, E.B. & Roninson, LB. (1992) Proc. Natl. Acad. ScL USA, 89, 5824-5828; Jachez, B., Cianfriglia, M. & Loor, F. (1994) Anticancer Drugs 5, 655-665) or potentiate the reversal activity of different cyclosporine analogues in vivo (Naito, M.,Watanabe, T., Tsuge, H., Koyama, T. Ohhara, T. & Tsuruo, T. (1996) Int. J. Cancer 67, 435-440; Watanabe, T., Naito, M., Kokubu, N. & Tsuruo, T. (1997) J. Natl. Cancer Inst. 89, 512-518). Unfortunately, this modulatory effect is weak and often difficult to reproduce, therefore the application of antibodies for the specific inhibition of Pgp function in vivo has not been preferable thus far.

An assay (antibody competition test, ACT; (Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E.B. & Szabό, G. Jr. (2001) Eur. J. Biochem. 268, 2416-2420)) has been developed based on the competition of UIC2 and certain other anti-Pgp mAbs (e.g., MRKl 6, MMl 2.10) for overlapping Pgp epitopes. This test allowed the differentiation of a distinct class of modulators (referred to herein as ACT-positive modulators) eliciting a marked increase in UIC2 binding (Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E.B. & Szabό, G. Jr. (2001) Eur. J. Biochem. 268, 2416-2420; Nagy, H., Goda, K., Fenyvesi, F., Bacsό, Zs., Szilasi, M., Kappelmayer, J., Lustyik, Gy.,. Cianfriglia, M., & Szabό, G. Jr. (2004) Biochem. Biophys. Res Commun. 315, 942-949).

While the mechanisms of substrate binding and export by multidrug transporters have not yet been fully elucidated, it is known that the transporter undergoes a substrate-dependent conformational change that can be detected by monoclonal antibodies. The monoclonal antibody UIC2 has been shown to preferentially bind to one of the Pgp conformations, and its binding is increased in the presence of certain substrates or modulators (reversing agents). Binding of the UIC2 antibody blocks the transport activity of Pgp and therefore inhibits multidrug resistance. This effect, however, is extremely variable and at most partial.

In order to improve the effectiveness of chemotherapeutic drugs and to identify cases where multidrug resistance is an issue, there is a significant need in the art to efficiently inhibit the activity of the Pgp receptor.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with compositions, methods and kits are meant to be exemplary and illustrative, not limiting in scope.

The present invention relates to methods, compositions and kits useful for blocking the action of Pgp receptors as well as for detecting cells that exhibit Pgp activity. The embodiments include a method of detecting Pgp-expressing cells using an antibody capable of recognizing an extracellular epitope of Pgp {e.g., a UIC2 monoclonal antibody), a modulating compound, and a fluorescent substrate, such as calcein. In a particular embodiment, the modulating compound used in the present inventive method is used at a much lower concentration {e.g., about 15 to about 20 times lower) than the concentration of the modulating compound when used alone. Another embodiment comprises a method of inhibiting the pumping of substrates out of a cell using the antibody capable of recognizing an extracellular epitope of Pgp {e.g., a UIC2 monoclonal antibody) in concert with a modulating compound such as cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m-amsacrine, and ivermectin. In a particular embodiment, the modulating compound used in the present inventive method is used at a much lower concentration as compared to a concentration of the modulating compound when used alone.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 depicts the effect of Pgp substrates/modulators on the inhibition of P- glycoprotein by UIC2 mAb and UIC2 binding visualized by indirect immunofluorescence in a parallel experiment in accordance with various embodiments of the present invention. NIH 3T3 MDRl cells were pretreated with Pgp substrates/modulators for 10 min, and incubated further with UIC2 mAb for an additional 30 min. Then the Pgp substrates/modulators were removed by washing one half of each sample with 1 % BSA-PBS and twice with PBS (black bars), while the other half was left unwashed (grey bars). Finally, all the samples were stained with 0.5 μM calcein-AM. Pgp substrates and modulators were applied at the concentrations causing maximal inhibition of Pgp-mediated calcein-AM efflux: (CsA: 10 μM; vinblastine 75 μM; PSC 833: 8 μM; valinomycin 10 μM; verapamil 75 μM; quinine 20 μM; nifedipine 125 μM), with <10% PI- positive cells in the samples. Means of three independent measurements ± SEM, and the result of one representative experiment carried out simultaneously on one of the samples is shown. This figure also depicts UIC2 reactivity of NIH 3T3 MDRl cells in the presence of cyclosporine A (CsA), verapamil and ATP depletion in accordance with various embodiments of the present invention. The cells were pretreated with 75 μM verapamil or 10 μM CsA for 10 min; cellular ATP production was inhibited by 30 min treatment with 5 μM oligomycin, followed by labeling with FITC-conjugated UIC2 niAb for 30 min. The experiment was reproduced several times with similar results.

Figure 2 depicts the effect of cyclosporine A (CsA) and UIC2 mAb on the accumulation of ""¹Tc-MIBI and daunorubicin into NIH3T3 MDRl cells in accordance with an embodiment of the present invention. Samples were preincubated with Pgp substrates for 10 mins and then further incubated with UIC2 for an additional 30 mins. CsA was removed by washing the cells with 1 % BSA-PBS and PBS. All the samples were subsequently incubated with 0.5 μM daunorubicin (B) or 10 μCi/ml 99mTc-MIBI (A). 99mTc-MIBI uptake is expressed as the percentage of the initial radioactivity of the incubating medium (ID %). Means ± SEM of three independent experiments are shown.

Figure 3 depicts the effects of cyclosporine A, added alone and in combination with UIC2 mAb, on calcein accumulation in NIH 3T3 MDRl cells in accordance with various embodiments of the present invention. The insert shows the UIC2 binding of the cells (expressed as % of maximal labeling) visualized by Cy5-GAMIG in a parallel experiment. The cells were preincubated with cyclosporine A for 10 mins and then further incubated in the presence or absence of UIC2 (10 μg/ml) for an additional 30 mins at 37⁰C. Subsequently, all the samples were stained with 0.5 μM calcein-AM for 15 mins. The raw calcein fluorescence intensities were normalized to that of the CsA and UIC2 untreated control. Means ± SEM of three independent experiments are shown. Figure 4 depicts confocal microscopy images of daunorubicin accumulation in cryosections prepared from NIH 3T3 MDRl (panels A, B, D, E) and NIH 3T3 tumors (panels C, F) in accordance with various embodiments of the present invention. Panel A: 10 mg/kg cyclosporine A + 5 mg/kg UIC2; panel B and C: 5 mg/kg UIC2; panel D: 50 mg/kg CsA; panels E and F: 10 mg/kg cyclosporine A. Figure 5 depicts daunorubicin fluorescence intensity distribution histograms of the whole cryosections (shown in Fig. 4) measured by laser scanning cytometry in accordance with an embodiment of the present invention. Histograms A and B represent cryosections from NIH 3T3 MDRl xenotransplants. Histogram C represents an NIH 3T3 tumor. Mice were treated before the intravenous injection of 8 mg/kg daunorubicin as follows. Histogram A: 10 mg/kg cyclosporine A + 5 mg/kg UIC2; histogram B and C: 5 mg/kg UIC2. Quantification was made on the x-y scattergram according to the fluorescence intensity of the phantom contours. Only the evenly fluorescent middle region of the selected sections was evaluated, leaving out the intensively fluorescent edges arising as a result of the folding back tissue elements.

Figure 6 depicts UIC2 mAb binding visualized by indirect immunofluorescence in xenotransplanted NIH 3T3 MDRl Pgp+ tumor tissues in accordance with an embodiment of the present invention.

Figure 7 depicts UIC2 mAb binding visualized by indirect immunofluorescence in xenotransplanted NIH 3T3 MDRl Pgp⁺ (panels A-C) and NIH3T3 Pgp^' (panels D-F) tumors in accordance with various embodiments of the present invention. Panels A and D: 5 mg/kg (i.v.) UIC2 + 10 mg/kg (i.v) cyclosporine A; panels B and E: 5 mg/kg (i.v.) UIC2; panels C and F: 10 mg/kg (i.v.) cyclosporine A.

DETAILED DESCRIPTION OF THE INVENTION All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley and Sons (New York, NY 1994); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., J. Wiley and Sons (New York, NY 1992); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For references on how to prepare these antibodies, see D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor NY, 1988); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al U. S. Patent No. 5,585,089; and Riechmann et al, Nature 332: 323 (1988).

One of the unresolved problems of cancer chemotherapy is the resistance of cancer cells to a broad range of cytotoxic drugs, referred to as multidrug resistance (mdr). This phenomenon is frequently associated with the expression of P-glycoprotein (Pgp) (Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427, Nooter, K. and Sonneveld, P. (1994) Leukemia Res. 18, 233-243, Bosch, I. and Croop, J. (1996) Biochim. Biophys. Acta 1288, F37F54, Gottesman, M.M., et al. (1995) Annu. Rev. Genet. 29, 607-649, Aran, J.M., et al. (1999) Adv. Pharmacology 46, 142.) . Pgp belongs to the ABC (ATP binding cassette) transporter superfamily of transport ATPases characterized by the presence of evolutionary conserved ATP binding motifs. Pgp is able to recognize structurally diverse, hydrophobic and amphiphylic substrates, including cytotoxic drugs, in the size range of 500-1000 Da and to pump them out of the cells at the expense of ATP hydrolysis (Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427, Seelig, A. (1998) Eur. J. Biochem. 251, 252-261). Pgp is expressed in more than 50% of diagnosed human cancer cases, intrinsically, or after chemotherapy (Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427, Nooter, K. and Sonneveld, P. (1994) Leukemia Res. 18, 233-243, Bosch, I. and Croop, J. (1996) Biochim. Biophys. Acta 1288, F37-F54).

An important goal of mdr research is to develop efficacious strategies to decrease drug resistance of cancer cells without serious side-effects. A number of compounds, often referred to as modulators, reversing agents, or chemosensitizers, are capable of decreasing or eliminating mdr by preventing Pgp-mediated substrate export (Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427, Friche, E. and Beck, W.T. (1996) Molecular, Biochemical, Physiological and Biological Aspects. (Gupta, S. and Tsuruo, T., eds), pp. 361-374. John Wiley and Sons, Chichester, UK., Yusa, K., et al (1996) Molecular, Biochemical, Physiological and Biological Aspects. (Gupta, S. and Tsuruo, T., eds.), pp. 331-344. John Wiley and Sons, Chichester, UK). However, the use of these drugs has been associated with toxic side effects (Pennock GD, et al, J Natl Cancer Inst. 1991 Jan 16;83(2): 105-10.)

The Pgp molecule comprises two homologous halves connected by a linker peptide of approx. 75 amino acids (Gottesman, M.M. and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385- 427). Each half spans the plasma membrane six times forming transmembrane helices as well as extra- and intracellular loops, as predicted by hydropathy plots (Gottesman, M.M. and Pastan, I. (1988) J. Biol. Chem. 263, 12163-12166). Several monoclonal antibodies (mAb), including UIC2 are used for detection of Pgp in clinical samples. The UIC2 monoclonal antibody, which has been previously described and patented, has two unique features. First, it is sensitive to the conformation of Pgp — it binds weakly to one conformation, and more strongly to the other. Second, upon binding to the Pgp transporter, it partially inhibits the extrusion of drugs by the P- glycoprotein.

The modulators that bind to Pgp elicit a conformational change in almost all expressed cell surface Pgps (Nagy, H. et al, (2001) Eur. J. of Biochem. 268, 2416-2420). Modulators, which include cyclosporine A, vinblastine, valinomycin, o-amsacrine, m-amsacrine and ivermectine are called "antibody competition test positive modulators", or "ACT⁺ Modulators", because in combination with UIC2, they cause a near-complete suppression of the Pgp receptor and subsequently prevent labeling with other monoclonal antibodies. In order for the UIC2 antibody to bind in a completely inhibitory fashion, ACT⁺ Modulators must be present in a clinical sample either before or simultaneously with introduction of the UIC2 antibody. Once UIC2 antibody is bound in this manner, it will remain affixed to Pgp even after removal of the ACT⁺ modulators.

The invention described herein provides compositions and methods for blocking the pumping activity of the Pgp multidrug resistance receptor, as well as for detecting cells that express Pgp on their surface. Compositions useful in effecting the methods of the present invention include the monoclonal antibody, UIC2, which recognizes and labels a specific conformation of the Pgp multidrug resistance pump, a modulator, and a fluorescent substrate. The UIC2 monoclonal antibody is described in U.S. Patent Nos. 5,434,075, 5,773,280, 6,030,796, and 6,479,639, all of which are incorporated by reference in their entirety as though fully set forth.

The UIC2 monoclonal antibody binds to a fraction of Pgps present on live cell surfaces in the absence of Pgp modulators or substrates, presumably because the UIC2 antibody binds preferentially to only one conformation of the Pgp receptor. Upon addition of certain modulators or substrates (ACT⁺ modulators), the remaining receptors undergo a conformational and/or topological change and assume the conformation recognized by this antibody. For unknown reasons, the fraction of Pgp receptors that are recognized by the UIC2 antibody in the absence of substrates or modulators is extremely variable. The inventors' earlier data suggested that the position of Pgp within the membrane is favorable to UIC2 binding only in the case of this fraction. Goda et a Conformational heterogeneity of P-glycoprotein. Cancer Detection and Prevention, 24(5):415-421 (2000). While not wishing to be bound by any particular theory, the inventors believe that the variability of the size of this fraction of Pgp is related to the variable relative ratio of raft components in the cellular membrane.

A low concentration of an ACT⁺ modulator, when used alone, is ineffective in Pgp inhibition. Because the UIC2 antibody binds preferentially to one fraction of cell surface Pgps, the antibody alone is unable to block all of the receptors on the cell. However, when the UIC2 antibody and a low concentration of an ACT⁺ modulator are added together, almost complete Pgp inhibition is achieved. The concentration of the ACT⁺ modulator required to achieve the almost complete Pgp inhibitory effect in accordance with the embodiments of the present invention is about 15 to about 20 times lower than the concentration of ACT⁺ modulator needed when used alone. These findings may provide a way to abolish Pgp-mediated multidrug resistance.

Two patent applications (PCT Pat. App. Pub. No. WO02/071061 and U.S. Pat. App. No. 10/469,720) describe methods of detecting conformational changes of the Pgp receptor using the UIC antibody as well as modulators. Both of these applications are incorporated herein in their entirety as though fully set forth. Labeling with UIC2 is increased by approximately two to fivefold in the presence of substrates or modulators (Mechetner, E.B., et al., (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913). This enhanced binding of UIC2 has been used to detect functional Pgps. This method is referred to as the 'UIC2-shift' assay. The UIC2-shift phenomenon can be detected via the altered subsequent binding of another mAb (Nagy et al. P-glycoprotein

Conformation Changes Detected by Antibody Competition. Eur. J. Biochem. 268(8):2415-20 (2001)), such as MM12.10, that is specific for the C-terminal part of the molecule (Romagnoli, G., et al, (1999) Biol. Chem. 380, 553-559).

When the cells are incubated with UIC2 mAb in the presence of "ACT-positive" modulators/substrates, an enhanced and inhibitory binding of the antibody is observed, and this inhibited state of Pgp molecules is preserved after removal of the modulators. At the same time, ACT-negative agents do not induce an inhibitory binding of UIC2 (see Fig.l), even if they elicit a mild increase in UIC2 reactivity (Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E.B. & Szabό, G. Jr. (2001) Eur. J. Biochem. 268, 2416-2420). Since a remarkable increment of calcein accumulation was measured only when ±80 % of all cell surface Pgps bind UIC2, as shown in Fig. 3, significant inhibition of drug transport is not expected when UIC2 binds to the usual 20-50 % of all cell surface Pgps, in the absence of modulators or in the presence of ACT- negative drugs. The ensuing data are best interpreted in terms of conformational/topological changes of the transporter elicited by the "ACT-positive" drugs that make all cell surface Pgps UIC2-reactive.

While not wishing to be bound by any particular theory, the inventors believe that the existence of UIC2 reactive Pgps in the absence of externally added substrates/modulators is due to a catalytically active subpopulation of Pgp molecules involved in the transport of endogeneous ACT-positive substrates, perhaps of cholesterol. This notion is supported by the data (not shown) that these UIC2 reactive Pgps mainly reside in the detergent resistant cholesterol-rich raft microdomains and that the basal ATPase activity of Pgp is propotional to the cholesterol content of the cell membrane (Garrigues, A., Escargueil, A.E., & Orlowski, S. (2002) Proc. Natl. Acad. Sci. USA 99, 10347-103452).

Incubation with ACT-positive drugs (modulators; e.g., CsA) leads to the binding of UIC2 to most cell surface Pgps at drug concentrations approximately 20 times lower than what is necessary for complete blocking of transport by the modulator acting merely as a competitive inhibitor. It seems likely that low concentration of the ACT-positive agents (even under the KM of their transport) is sufficient to kick off the catalytic cycle and set all cell surface Pgps gradually into the UIC2 reactive conformation. This state is transient without UIC2 and the role of the mAb may be to trap a Pgp in this conformation.

The effect of several ACT-positive drugs on Pgp conformation was shown to be indistinguishable from that of ATP depletion, suggesting that these agents block the pump in the same, or in a very similar, conformational state that is part of the catalytic cycle (Goda, K., Nagy, H., Mechetner, E.B. Cianfriglia, M., Szabό, Jr. G. (2002) Eur. J. Biochem. 269, 1-6). This conformational state must be present for periods long enough to be recognized by UIC2 mAb moving around by diffusion in the medium. The strength of modulator binding to Pgp may be a dominant factor in eliciting and maintaining the special UIC2 reactive conformation, since ACT-positive agents are rich in hydrogen bond acceptor moieties, giving rise to a slower transport as a result of lower dissociation rates from the protein (Seelig, A. (1997) Eur. J.

Biochem. 251, 252-261; Seelig, A. & Landwojtowicz, E. (2000) Eur. J. Pharmaceutical Sci. 12, 31-40). In the presence of ACT-negative agents, or in the absence of drugs, the UIC2-reactive state may be by-passed, or its duration may be too short to bind the mAb.

Bivalent binding of the antibody is not required for the inhibitory effect of UIC2, as shown by the fact that its Fab fragment was also effective in blocking pumping. Inhibition of drug transport is not specific for a particular substrate or a class of substrates, as shown by the observation that the inventors could reproduce this effect in the case of chemically unrelated compounds, including calcein-AM, daunorubicin and ""¹Tc-MIBI.

The fact that CsA can be used at a much lower concentration to achieve a complete inhibition of the pump when administered together with UIC2, rather than alone, renders this combinative protocol promising for possible in vivo applications in combating mdr. The inventors confirmed this possibility in xenotransplantation experiments, demonstrating that UIC2 can reach Pgp on cells deeply embedded in a solid tumor and inhibit daunorubicin pumping when used in combination with CsA. The CsA dose applied in the ensuing examples was 5 times lower than the minimal dose required to achieve detectable pump inhibition by CsA alone. Success of mdr reversal in clinical practice is hindered by the side-effects of modulators determined by their pharmacokinetic behavior exhibited in the presence of the cytostatic drugs also applied (depending, among other factors, on the level of P450, and on the activity of other ABC transporters involved; e.g., in bile formation (Leonard, G.D., Fojo, T.,& Bates, S. (2003) The Oncologist 8, 411-424)). The low concentration of the modulator used in this invention may significantly diminish most side-effects. UIC2, when bound to the Pgp⁺ cells, stays there long enough to allow an appropriate time window for the disease-specific cytostatics to enter the cells. The inhibited state of Pgp molecules persists even after removal of CsA in in vitro experiments, suggesting that the cell-bound UIC2 molecules exert their pump inhibitory effect after the plasma concentration of CsA has been diminished. Furthermore, since inhibition of Pgp is achieved by the niAb, the effect will be restricted to this transporter.

One embodiment of the instant invention is a diagnostic test for the presence of Pgp in clinical samples that is more sensitive and reliable than the UIC2-shift assay. This test involves using a fluorescent substrate such as calcein in combination with UIC2 and an ACT⁺ modulator. Calcein and other fluorescent substrates (e.g., rhodamine 123, daunorubicin) are normally pumped out of cells by Pgp. In this embodiment, the fluorescent substrate enters the cell, but is unable to exit due to Pgp being bound by the UIC2. As a result, the cells expressing Pgp become fluorescent and can be detected by a number of methods, including flow cytometry and fluorescence microscopy. This diagnostic method is more sensitive than conventional methods because UIC2 binding is detected through dye accumulation. It contrasts with previous methods that use a fluorescently-labeled antibody to measure the difference in intensity between a control and experimental sample, which may not be sensitive enough when Pgp expression levels are low. Further, the present method requires lower concentrations of modulators than used in other studies because once the UIC2 receptors have undergone a conformational shift resulting from binding of modulators, no further addition of modulator is required.

The near complete inhibition of Pgp by the UIC2 antibody and modulators has been observed in the presence of whole (mouse) blood, indicating that an in vivo application of the antibody may be feasible. This observation suggests that humanized UIC2 antibodies may be useful in human therapeutic applications such as in the case where cancer or HIV drug therapies have failed due to multidrug resistance. Therefore, another embodiment of the invention is a therapeutic method to inhibit Pgp function comprising a combination of the UIC2 antibody and a modulator. These may be formulated together in a single composition or delivered separately, as two co-compositions. This type of therapy would enable drugs to remain in their target cells long enough to have their intended effect. In addition, potential unwanted side effects caused by the modulators would be fewer than in therapies that utilize modulators alone to block Pgp function, as significantly lower concentrations of modulator would be required.

The present invention is not limited to UIC2 as the monoclonal antibody. Antibodies with similar capabilities as the UIC2 monoclonal antibody may be used in concert with the modulators as described in the embodiments of the present invention. Methods of preparing monoclonal antibodies are known in the art. For example, monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include Pgp or a fragment thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (see pp. 59-103 in Goding (1986) Monoclonal Antibodies: Principles and Practice Academic Press). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthinβ guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells.

In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen. In one embodiment, one of the binding specificities is for Pgp or a fragment thereof, the other one is for another antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific. Alternatively, tetramer-type technology may create multivalent reagents.

In another embodiment the antibodies to Pgp or a fragment thereof are humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones, et al. (1986) Nature 321:522-525; Riechmann, et al. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596). Humanization can be essentially performed following methods of Winter and co-workers (see, e.g., Jones, et al. (1986) Nature 321:522-525; Riechmann, et al. (1988) Nature 332:323-327; and Verhoeyen, et al (1988) Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies {e.g., U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter (1991) J. MoI. Biol. 227:381-388; Marks, et al. (1991) J. MoI. Biol. 222:581-597) or the preparation of human monoclonal antibodies {e.g., Cole, et al. (1985) Monoclonal Antibodies and Cancer Therapy Liss; and Boeraer, et al. (1991) J. Immunol. 147(l):86-95). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in most respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks, et al. (1992) Bio/Technology 10:779-783; Lonberg, et al. (1994) Nature 368:856-859; Morrison (1994)

Nature 368:812-13; Fishwild, et al. (1996) Nature Biotechnology 14:845-51; Neuberger (1996) Nature Biotechnology 14:826; Lonberg and Huszar (1995) Intern. Rev. Immunol. 13:65-93. In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of an UIC2 antibody and an ACT⁺ modulator. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a phaπnaceutical composition that is generally safe, nontoxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the fonn of a syrup, elixir, emulsion or an aqueous or nonaqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule. The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000). Typical dosages of an effective amount of an UIC2 antibody and an ACT⁺ modulator can also be as indicated to the skilled artisan by the in vitro responses or responses in animal models. For example, in the inventors' xenotransplantation studies using SCID mice, the animals were pre-treated with lOmg/kg CsA (Sandimmun, Novartis) intraperitoneally and 5 mg/kg of UIC2 mAb intravenously 4 hours prior to the administration of 5 mg/kg of daunorubicin, intravenously. The animals were killed 4 hours after the addition of daunorubicin.

Additionally, an effective dose of other ACT⁺ modulators may be even more favorable. For example, ivermectin is almost as effective as cyclosporine, but has no serious side effects as it is an antihelmintic drug used in clinical practice. (Nagy et al. Distinct Groups of Multidrug Resistance Modulating Agents Are Distinguished by Competition of P-gycloprotein-specifϊc Antibodies. Biochemical & Biophysical Research Communications, (2004) 942-50). Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described. The present invention is also directed to a kit to treat multidrug resistance and a kit to detect cells that express Pgp activity. The kits are useful for practicing the inventive method of treating multidrug resistance or to detect cells that express Pgp activity. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in embodiments treating multidrug resistance, the kit contains a composition that comprises both an antibody capable of recognizing an extracellular epitope of Pgp (e.g., an UIC2 antibody) and an ACT⁺ modulator, as described above. Alternatively, the kit contains two compositions - one that comprises an antibody capable of recognizing an extracellular epitope of Pgp (e.g., an UIC2 antibody) and another that comprises an ACT⁺ modulator, as described above. In embodiments for detecting cells that express Pgp activity, the kit contains an UIC2 antibody, an ACT⁺ modulator, and a fluorescent substrate.

The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of treating multidrug resistance. Other embodiments are configured for the purpose of detecting cells that express Pgp activity. In one embodiment, the kit is configured particularly for the purpose of treating mammalian subjects. In another embodiment, the kit is configured particularly for the purpose of treating human subjects; for example, the antibody capable of recognizing an extracellular epitope of Pgp (e.g., an UIC2 antibody) is a humanized antibody or a human antibody. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.

Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to treat multidrug resistance or to detect cells that express Pgp activity. For example, in kits for the treatment of multidrug resistance, instructions for use may include instructions to administer a therapeutically effective amount of he antibody and the ACT⁺ modulator or substrate, either separately or together, to the mammal. In kits for the detection of cells that express Pgp activity, instructions for use may include instructions to place a sample comprising cells in contact with the antibody capable of recognizing the extracellular epitope of Pgp, the ACT+ modulator or substrate and the fluorescent substrate, and instruction to measure the fluorescence of the sample. Optionally, the kit also contains other useful components, such as, test tubes, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1 Cell lines The NIH 3T3 mouse fibroblast cell line and its human mdrl-transfected counterpart (NIH 3T3 MDRl G185) (Brugemann, E.P., Currier, SJ., Gottesman, M.M. & Pastan, I. (1992) J, Biol. Chem. 267, 21020-21026) obtained from Michael Gottesman's lab (NIH, Bethesda) was used in most of the experiments. The cells were grown as monolayer cultures at 37⁰C in an incubator containing 5% CO₂ and maintained by regular passage in Dulbecco's MEM (supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 25 μg/ml gentamycin). The NIH 3T3 MDRl cells were cultured in the presence of 670 nM doxorubicin. Cells were trypsinized 2-3 days prior to the experiments and maintained without doxorubicin until use. In some measurements the KB-3-1 drug sensitive human epidermoid carcinoma cell line and its multidrug resistant variant KB-Vl (Shen, D.W., Cardarelli, C, Hwang, J., Cornwell, M.M., Richert, N., Ishii, S., Pastan, I. & Gottesman, M.M. (1986) J. Biol. Chem. 261 7762- 7770) (cultured in the presence of 180 nM vinblastine) and the A2780 (Pgp-) / 2780AD (Pgp⁺; maintained with 2 μM doxorubicin (Louie, K.G., Hamilton, T.C., Winkler, M.A., Behrens, B.C., Tsuruo, T., Klecker, R.W., Mchoy, W.M., Grotzinger, K.R., Meyers, C.E., Young R.C. & Ozols, R.F. (1986) 35, 467-472)) human ovarian carcinoma cell pair, were used. The cells were occasionally checked for mycoplasma by the mycoplasma T. C. rapid detection system with a 3H-labeled DNA probe from Gen-Probe Inc. and were found to be negative. Example 2 Chemicals

All the Pgp substrates and modulators were from Sigma-Aldrich (Budapest), except for SDZ PSC-833 that was from Novartis (Basel, Switzerland), and MIBI (hexakis-2- methoxybutylisonitrille) purchased from FJ. Curie Radiobiological Research Institute (Budapest, Hungary) and labeled with ^99mTc according to the kit instructions. The working concentrations of the tested Pgp substrates and modulators were determined by measuring the changes in calcein accumulation, as well as in viability by propidium iodide (PI) exclusion, in response to the above agents applied at a wide concentration range. Working concentrations of the tested agents were chosen to bring about maximal inhibition of Pgp mediated calcein- AM efflux, at <10% increase in PI-positivity. Cell culture media and supplements were also from Sigma. Fluorescein-5-isothiocyanate (FITC) and the succinimidyl ester derivative of sulfoindocyanine dye 5 (Cy5) were purchased from Molecular Probes (Eugene, OR, USA) and Amersham Pharmacia Biotech (Vienna, Austria), respectively. The UIC2 (the hybridoma is from the American Type Culture Collections, Manassas, VA, USA), MM6.15 and MM12.10 anti-Pgp mAb preparations (gifts from M. Cianfriglia, Rome) were >97% pure by SDS/PAGE. The FITC, Alexa 488 and Cy5 conjugates of MM6.15, MM12.10, and UIC2 were prepared as described elsewhere (Spack, Jr. E.G., Packard, B., Wier, M.L., Edidin, M. (1986) Anal. Biochem. 158, 233-237). The UIC2 antibody clone was from ATCC, and IgG was produced from the hybridoma according to standard procedures.

Example 3

Drug accumulation studies

Nearly confluent monolayers of cells were harvested by 2-3 min trypsin treatment [0.05 % trypsin and 0.02 % EDTA in PBS (pH=7.4)] and washed twice with PBS before use. Calcein and daunorubicin accumulation was measured as described in Goda, K., Nagy, H.,

Mechetner, E.B. Cianfriglia, M., Szabό, Jr. G. (2002) Eur. J. Biochem. 269 and 1-6; Hollo, Zs., Homolya, L., Davis, CW. & Sarkadi, B. (1994) Biochim. Biophys. Acta 1191, 384-388. Briefly, cells were pre-incubated with Pgp substrates/modulators for 10 min and further incubated with UIC2 mAb (10 μg/ml) at 37 ⁰C for 30 min. The samples were divided into two aliquots; Pgp substrates/modulators were removed from one of the aliquots by washing with 1 % BSA-PBS and twice with PBS, while the other aliquot was kept at room temperature. Finally, samples were stained with 0.5 μM calcein or 1 μM daunorubicin for 15 min or 30 min, respectively. In some experiments cell bound UIC2 molecules were labeled with cy5-GAMIG following the calcein accumulation assay.

Calcein accumulation experiments were also carried out in the presence of whole mouse blood containing 3.8 % trinatrium-citrate. 2.5 X lO⁵ cells were resuspended in 100 μl whole blood and the subsequent modulator and UIC2 treatment and calcein assay was carried out as described above.

In ""¹Tc-MIBI accumulation experiments, 10 μCi/ml ""¹Tc-MIBI was added to the cells after the modulators and/or UIC2 mAb, and the samples were further incubated for 30 min at 36° C. The uptake was terminated by the addition of ice-cold PBS. The cells were then washed three times with ice-cold PBS and resuspended in 1 ml PBS, and the radioactivity was measured in a Canberra Packard gamma-well counter. The intracellular accumulated amount of radiotracer was always less than 5 % of the total radiotracer concentration. The displayed data are the means ± SEM of the results of at least three independent experiments, with the samples in each applied in triplicates.

Example 4 Flow-cytomefiγ

Two- or three-color cytofluorimetric analysis was performed by using the Becton Dickinson FACScan or FACS Calibur flow cytometers (Mountain View, CA, USA), respectively. Dead cells staining with PI were excluded from the analysis. Fluorescence signals were collected in logarithmic mode and the cytofluorimetric data were analyzed by the BDIS CELLQUEST (Becton Dickinson) software.

Example 5 Laboratory animals

Twenty adult (10 to 12 week-old), pathogen-free B-17 SCID (severe combined immunodefficiency) mice were used in this study (Marian, T., Szabό, G., Goda, K., Nagy, H., Szincsak, N., Juhasz, L, Galuska, L., Balkay, L., Mikecz, P., Trόn, L. & Krasznai, L. (2003) Eur. J. Nucl. Med. MoI. Imaging 30, 1147-1154). The "Principles of laboratory animal care" (NIH) were strictly followed and the experimental protocol was approved by the Laboratory Animal Care and Use Committee of the University of Debrecen. The NIH 3T3 and NIH3T3 MDRl cells (4 X 10⁶ cells in 300 μl serum-free DMEM) were injected subcutaneously into opposite flanks of the mice. The tumors were grown for 10-12 days. Animals were pre-treated with 10 mg/kg CsA (Sandimmun, Novartis) intraperitoneally and/or UIC2 mAb (5 mg/kg, added intravenously) 4 hours before the administration of daunorubicin (5 mg/kg, i.v.). The animals were killed 4 hours after the addition of daunorubicin by cervical dislocation and the tumors were dissected and kept in liquid nitrogen until further use. 6 μm thick cryosections were prepared from the tumor samples and their DNR distribution was subsequently measured in a confocal laser scanning microscope. UIC2 binding was visualized by indirect immunofluorescence. The cryosections were blocked with 10 % goat serum for 20 mins and labeled with GAMIG-Alexa 488 (5 μg/ml in PBS containing 2% goat serum) for 1 hour. Then the slides were washed twice with 1% BSA-PBS and twice with PBS; the coverslips were mounted on slides using the Prolong antifade kit (Molecular Probes, Eugene, Oregon, USA) and confocal images were recorded immediately. DNR accumulation and UIC2 binding of successive sections representing identical cells were compared. The morphology of the tissue sections was routinely checked by hematoxylin-eosin staining.

Example 6 Confocal Laser Scanning Microscopy and Laser Scanning Cytometry The daunorubicin accumulation as well as UIC2 binding of the cryosections prepared from the tumors were studied by confocal laser scanning microscopy (LSM 510, Zeiss, Thornwood, NY, USA) and laser scanning cytometery (LSC; CompuCyte, Cambridge, MA). The 488-nm line of an argon-ion laser was used in confocal microscopy experiments. Fluorescence intensities were detected through a 505- to 550-nm bandpass filter (for Alexa 488 dye) and a >580 nm longpass filter (for daunorubicin). Images were collected through a Plan- Apochromat 63X oil-immersion objective (numerical aperture=1.4).

The cryosections prepared from the tumors were also analyzed with a full featured iCys Research Imaging Cytometer and the iCys Cytometric Analysis Software version 2.6 (both from CompuCyte Corp, Cambridge, MA, USA). The 488 nm wavelength of the argon-ion laser was used for excitation and the fluorescence was detected in the green channel (emission: 530±15 nm) in the case of Alexa 488, while daunorubicin fluorescence was detected in a virtual channel generated by adding together the integrated fluorescence signals of the green channel (emission: 530±15 nm) and the orange channel (emission: 575=1=10 nm). For fast setup of scan areas, the "scout" or low resolution-scanning feature of the "Tissue Scan" input module of the iNovator tool kit was applied. The scout scan was used to find the boundaries of tissue samples based on the fluorescence intensity detected in the green channel, and then a high-resolution scan was conducted, to analyze only the defined areas. The 20x objective was used for the high resolution scan and the phantom contouring feature of the iCys software was applied to characterize the fluorescence intensity distribution in large areas of the sections (Megyeri, A., Bacsό, Zs., Shilds, A., & Eliason, J.F. (2005) Cytometry 64, 62-74). Phantom contouring is a stereologic, random sampling technique where circular contours are randomly generated by the iCys software and analyzed in the same way as in the case of contoured cells. It provides fairly accurate measurement of fluorescence over larger areas, or the entire section, but no longer provides information directly relevant to the cellular distribution of fluorescence signals. In these experiments, the highest possible numbers of phantom contours were arranged randomly throughout the scan area, with the radius of each contour set to 10 μm and with no overlapping allowed between phantom contours. The integral fluorescence (the sum of the pixel intensities inside a contour) of each contour was used to characterize the fluorescence intensities and contour maps were created with Sigma Plot 8.0 (SPSS Inc., Chicago, IL, USA) for each section, according to the method described previously (Megyeri, A., Bacsό, Zs., Shilds, A., & Eliason, J.F. (2005) Cytometry 64, 62-74).

Example 7 The UIC2 mAb mediated pump inhibition appears, at best, to be partial, most likely due to the fact that UIC2 binds only to 10-40 % of all Pgps present in the cell membrane (Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Davis, K.A., Tsuruo, T., & Roninson, LB. (1997) Proc. Natl. Acad. Sci. USA, 94, 12908-12913), when added at saturating concentrations (see Fig. 1). It has been previously demonstrated that the incubation of cells with certain Pgp substrates and modulators increases the reactivity of UIC2 to Pgp which is also reflected by the abolished subsequent binding of other monoclonal antibodies sharing epitopes with UIC2 (Antibody Competition Test, ACT). In the presence of such ("ACT-positive") agents (e.g., cyclosporine A (CsA), SDZ PSC 833, vinblastine, valinomycin, o-amsacrine, m-amsacrine, ivermectin) or after ATP depletion, close to 100 % of the cell surface Pgp molecules become labeled with UIC2, as compared to the control cells or to the increment observed in the presence of ACT-negative agents, like verapamil (see Fig. 1). This phenomenon suggested that the augmented binding of UIC2 in the presence of ACT-positive agents, may also potentiate the pump inhibitory effect of the mAb. Figure 1 also demonstrates that UIC2 strongly increases calcein accumulation in NIH3T3 MDRl cells prelabeled with the mAb in the presence of CsA, vinblastine, SDZ PSC 833, or valinomycin. This Pgp inhibitory effect of UIC2 is preserved after removal of the drugs by washing the cells with 1 % BSA-PBS. At the same time, verapamil, quinine and nifedipine did not elicit an inhibitory binding of UIC2 to Pgp molecules, demonstrated by the fact that calcein accumulation decreased to the control level after removal of these agents (see Fig. 1, black bars). Verapamil, quinine and nifedipine did not significantly change the number of UIC2 reactive cell surface Pgps, while CsA and other ACT-positive agents increased UIC2 binding 2-3 times, as shown in Fig. 1. Similar results were obtained in different cell lines exhibiting various Pgp expression levels (high expressor cell lines: KB-Vl and 2780AD; 5 X 10⁵ molecules/cell; low expressor cell line: KB-8-5, 5-10 X 10³ molecules/cell, data not shown).

Figure 2 shows that the UIC2 mAb also strongly increases the ""¹Tc-MIBI and daunorubicin accumulation in NIH 3T3 MDRl cells, when the cells are prelabeled in the presence of CsA, demonstrating that inhibition of Pgp is not restricted to its calcein-AM pumping function.

The Fab fragment of UIC2 also inhibits the pump significantly when the cells are labeled in the presence of the cyclosporine analogue PSC 833, suggesting that bivalent binding of the antibody is not reguired for the inhibitory effect (data not shown).

When Pgp inhibition was measured as a function of the concentration of CsA, applied alone or together with a saturating amount of UIC2, the combined treatment has lead to a >20x increment in intracellular calcein levels at >0.2 μM concentration of the modulator, while approximately 15-2Ox higher CsA concentration was as effective when the modulator was applied alone (see Fig 3.). Thus, an inhibitory binding of UIC2 is brought about at much lower concentrations of CsA than what is necessary for blocking transport by the drug acting as a competitive inhibitor. In parallel samples of these measurements, the UIC2 binding, as detected by Cy5-GAMIG staining, covered about 70-80 % of all cell surface Pgps; at the CsA concentration when calcein accumulation was increased approx. 20 times (see Fig. 3.)

As a prelude to an in vivo attempt of mdr reversal based on the combined application of CsA and UIC2, inhibition of Pgp function by CsA and antibody was demonstrated in the presence of whole (mouse) blood, using modulator concentrations that were not effective when applied alone (data not shown). In vivo experiments were conducted using SCID mice xenotransplanted with NIH3T3 MDRl and NIH3T3 parental cells in their two flanks, respectively. The mice were pretreated with CsA and/or UIC2 mAb 4 hours prior to the addition of the fluorescent Pgp substrate, daunorubicin. The combined application of 10 mg/kg CsA and the antibody increased daunorubicin accumulation of the Pgp⁺ tumor approx. to the level of the Pgp- tumor (see Fig. 4 panels A and C, and Fig. 7. histograms A and C). The mean daunorubicin fluorescence intensity of the Pgp⁺ tumor section treated with 10 mg/kg CsA and the antibody was 271±32 (mean ± SD) and that of the section prepared from the NIH 3T3 tumor was 315±44, compared to the 174±45 value measured in the untreated Pgp⁺ tumor. At the same time, daunorubicin accumulation in the Pgp expressing tumors treated with the antibody or 10 mg/kg CsA alone did not increase significantly, compared to untreated mice (see Fig. 4 panels B and E). Without coadministration of the antibody, 5 times higher CsA concentration was required to reach effective pump inhibition (see Fig. 4, compare panels A and D). As Fig. 6 demonstrates, UIC2, when applied together with CsA, could readily penetrate into the compact solid tumors formed, intensively decorating cell surface Pgp. The binding of the antibody was specific, as significant labeling of the Pgp^" tumors was not detected (see Fig 7. panels D-E). LSC images demonstrate that the whole tumor section is labeled by UIC2 in the presence of CsA, while it barely labels the cells (at 5 mg/kg concentration), when added without CsA, as it is shown in Fig. 7 panels A and B. The localization of the areas with strong UIC2 binding correlates with the distribution pattern of the daunorubicin fluorescence intensity in the specimen (data not shown).

While the description above refers to particular embodiments of the present invention, it should be readily apparent to people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The accompanying claims are intended to cover such modifications as would fall within the true spirit and scope of the invention. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description. All changes that come within the meaning of and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A composition for inhibiting P-glycoprotein (Pgp) in a mammal, comprising: an antibody capable of recognizing an extracellular epitope of Pgp; and an antibody competition test positive (ACT⁺) modulator or substrate.

2. The composition of claim 1, wherein the antibody capable of recognizing the extracellular epitope of Pgp is selected from the group consisting of MRK16, MRK17, MC57, UIC2 and combinations thereof.

3. The composition of claim 1, wherein the antibody capable of recognizing the extracellular epitope of Pgp is UIC2.

4. The composition of claim 1, wherein the antibody capable of recognizing the extracellular epitope of Pgp is a type of antibody selected from the group consisting of a mammalian antibody, a mouse antibody, a humanized antibody, a human antibody and combinations thereof.

5. The composition of claim 1, wherein the ACT⁺ modulator or substrate is selected from the group consisting of cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m-amsacrine, ivermectin and combinations thereof.

6. A method for inhibiting P-glycoprotein (Pgp) in a mammal, comprising: providing an antibody capable of recognizing an extracellular epitope of Pgp; providing an antibody competition test positive (ACT⁺) modulator or substrate; and administering a therapeutically effective amount of the antibody and the ACT⁺ modulator or substrate to the mammal.

7. The method of claim 6, wherein the antibody capable of recognizing the extracellular epitope of Pgp is selected from the group consisting of MRKl 6, MRKl 7, MC57, UIC2 and combinations thereof.

8. The method of claim 6, wherein the antibody capable of recognizing the extracellular epitope of Pgp is UIC2.

9. The method of claim 6, wherein the antibody capable of recognizing the extracellular epitope of Pgp is a type of antibody selected from the group consisting of a mammalian antibody, a mouse antibody, a humanized antibody, a human antibody and combinations thereof.

10. The method of claim 6, wherein the ACT⁺ modulator or substrate is selected from the group consisting of cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m- amsacrine, ivermectin and combinations thereof.

11. The method of claim 6, wherein administering a therapeutically effective amount of the antibody and the ACT⁺ modulator or substrate to the mammal further comprises: administering a therapeutically effective amount of the antibody and the ACT⁺ modulator or substrate, either separately or together, to the mammal.

12. A kit for inhibiting P-glycoprotein (Pgp) in a mammal, comprising: an antibody capable of recognizing an extracellular epitope of Pgp; an antibody competition test positive (ACT⁺) modulator or substrate; and instructions to use the antibody and the ACT⁺ modulator or substrate to inhibit

PgP-

13. The kit of claim 12, wherein the antibody capable of recognizing the extracellular epitope of Pgp is selected from the group consisting of MRK16, MRK17, MC57, UIC2 and combinations thereof.

14. The kit of claim 12, wherein the antibody capable of recognizing the extracellular epitope of Pgp is UIC2.

15. The kit of claim 12, wherein the antibody capable of recognizing the extracellular epitope of Pgp is a type of antibody selected from the group consisting of a mammalian antibody, a mouse antibody, a humanized antibody, a human antibody and combinations thereof.

16. The kit of claim 12, wherein the ACT⁺ modulator or substrate is selected from the group consisting of cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m- amsacrine, ivermectin and combinations thereof.

17. The kit of claim 12, wherein the instructions to use the antibody and the ACT⁺ modulator or substrate comprise: instructions to administer a therapeutically effective amount of the antibody and the ACT⁺ modulator or substrate, either separately or together, to the mammal.

18. A method for detecting P-glycoprotein (Pgp) expressing cells, comprising: providing a sample comprising cells; providing an antibody capable of recognizing an extracellular epitope of Pgp; providing an antibody competition test positive (ACT⁺) modulator or substrate; providing a fluorescent substrate; placing the sample in contact with the antibody capable of recognizing the extracellular epitope of Pgp, the ACT⁺ modulator or substrate and the fluorescent substrate; and measuring the fluorescence of the sample to detect Pgp expressing cells.

19. The method of claim 18, wherein the sample is obtained from a mammal.

20. The method of claim 18, wherein the antibody capable of recognizing the extracellular epitope of Pgp is selected from the group consisting of MRKl 6, MRKl 7, MC57, UIC2 and combinations thereof.

21. The method of claim 18, wherein the antibody capable of recognizing the extracellular epitope of Pgp is UIC2.

22. The method of claim 18, wherein the antibody capable of recognizing the extracellular epitope of Pgp is a type of antibody selected from the group consisting of a mammalian antibody, a mouse antibody, a humanized antibody, a human antibody, and combinations thereof.

23. The method of claim 18, wherein the ACT⁺ modulator or substrate is selected from the group consisting of cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m- amsacrine, ivermectin and combinations thereof.

24. The method of claim 18, wherein the fluorescent substrate is selected from the group consisting of calcein, daunorubicin, rhodamine 123 and combinations thereof.

25. The method of claim 18, wherein the measuring the fluorescence from the sample is performed by a technique selected from the group consisting of cytofluorimetric analysis, flow cytometry, fluorescence microscopy, measuring the fluorescent signal, indirect immunofluorescence, confocal laser scanning microscopy, laser scanning cytometry and combinations thereof.

26. A kit for detecting P-glycoprotein (Pgp) expressing cells, comprising: an antibody capable of recognizing an extracellular epitope of Pgp; an antibody competition test positive (ACT⁺) modulator or substrate; a fluorescent substrate; instructions to use the antibody capable of recognizing the extracellular epitope of Pgp, the ACT⁺ modulator or substrate and the fluorescent substrate to detect Pgp expressing cells.

27. The kit of claim 26, wherein the antibody capable of recognizing the extracellular epitope of Pgp is selected from the group consisting of MRKl 6, MRKl 7, MC57, UIC2 and combinations thereof.

28. The kit of claim 26, wherein the antibody capable of recognizing the extracellular epitope of Pgp is UIC2.

29. The kit of claim 26, wherein the antibody capable of recognizing the extracellular epitope of Pgp is a type of antibody selected from the group consisting of a mammalian antibody, a mouse antibody, a humanized antibody, a human antibody and combinations thereof.

30. The kit of claim 26, wherein the ACT⁺ modulator or substrate is selected from the group consisting of cyclosporine A, vinblastine, valinomycin, PSC 833, o-amsacrine, m- amsacrine, ivermectin and combinations thereof.

31. The kit of claim 26, wherein the fluorescent substrate is selected from the group consisting of calcein, daunorubicin, rhodamine 123 and combinations thereof.

32. The kit of claim 26, wherein the instructions comprise: instructions to place a sample comprising cells in contact with the antibody capable of recognizing the extracellular epitope of Pgp, the ACT⁺ modulator or substrate and the fluorescent substrate; and instructions to measure the fluorescence of the sample.

33. The kit of claim 32, wherein the instructions to measure the fluorescence of the sample comprise instructions to use a technique selected from the group consisting of cytofluorimetric analysis, flow cytometry, fluorescence microscopy, measuring the fluorescent signal, indirect immunofluorescence, confocal laser scanning microscopy, laser scanning cytometry and combinations thereof.