Epothilone resistant cell lines

Epothilone resistant cells lines are disclosed. The invention also discloses methods for identifying substances which are cytotoxic to epothilone resistant cells or which are chemosensitizers or analogs of epothilone. The invention further discloses methods for identifying epothilone resistant cells and for inhibiting the growth of epothilone resistant cells in vitro and in vivo. The invention also discloses antibodies specific for epothilone resistant cells. Also disclosed is a method to identify microtubule stabilizing agents using the epothilone resistant cell lines disclosed.

Patent Agent: Novartis Corporate Intellectual Property - East Hanover, NJ, US
Patent Inventors: Peter Wisdom Atadja, Markus Wartmann, Yan Yan-Neale, Dalia Cohen
Applicaton #: 20070134744 Class: 435007230 (USPTO)
Related Patents: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving A Micro-organism Or Cell Membrane Bound Antigen Or Cell Membrane Bound Receptor Or Cell Membrane Bound Antibody Or Microbial Lysate, Animal Cell, Tumor Cell Or Cancer Cell
Brief Patent Description - Full Patent Description - Patent Application Claims

[0001] This invention relates to newly identified epothilone A and epothilone B resistant cell lines.

BACKGROUND OF THE INVENTION

[0002] Multidrug resistance refers to the process whereby cells acquire resistance to certain cytotoxic agents and also demonstrate acquired cross-resistance to other, sometimes structurally and functionally unrelated drugs. Acquired resistance to chemotherapeutic agents in this way is a major obstacle to the effective treatment of cancer. Although the best characterized mechanism of resistance to anticancer drugs involves the overexpression of proteins of the P-glycoprotein (P-gp) and multidrug related protein (MRP) family, which are plasma membrane ATP-dependent pumps that efflux a large variety of cytotoxic drugs out of the cell, not all multidrug resistant cells overexpress these proteins, indicating the existence of non-P-gp/MRP mediated mechanisms of multidrug resistance.

[0003] The epothilones, especially epothilones A and B, represent a new class of microtubule stabilizing cytotoxic agents (see Gerth, K. et al., J. Antibiot. 49, 560-3 (1996); or Hoefle et al., DE 41 38 042), e.g. with the formulae: wherein R is hydrogen (epothilone A) or methyl (epothilone B).

[0004] The epothilones are macrolide polyketides that, like paclitaxel (TAXOL.RTM.), have the ability to arrest cells in mitosis, bind directly to tubulin and preferentially microtubules, cause formation of bundles of intracellular microtubules in non-mitotic cells and induce the formation of hyperstable tubulin polymers. In contrast to TAXOL.RTM., however, epothilones A and B do not appear to be substrates for P-gp, in that they retain activity against P-gp expressing multidrug resistant cancer cell lines. Additionally, epothilone-mediated microtubule stabilization does not trigger endotoxin signaling, an effect that is thought to mediate some non-hematological effects of TAXOL.RTM. in cancer therapy (see Muhlardt, P. F. and Sasse, F. (1997) Cancer Research 57:3344-3346).

[0005] In addition, epothilones show better water solubility, than TAXOL.RTM. and are thus more appropriate for formulation. Since epothilones are not subject to P-gp or MRP mediated multi-drug resistance, they can inhibit the proliferation of cells that otherwise show resistance to treatment with other chemotherapeutics due to the activity of the P-glycoprotein efflux pump; incubation of cells with epothilones induces stabilization of microtubules followed by apoptosis (see Bollag, D. M., et al., "Epothilones, a new class of microtubule-stabilizing agents with a TAXOL.RTM.-like mechanism of action", Cancer Research 55, 2325-33 (1995); and Bollag D. M., Exp. Opin. Invest. Drugs 6, 867-73 (1997)). Furthermore, despite apparently sharing the same, or a sterically proximal binding site on the microtubule, the epothilones have been shown to be active against a TAXOL.RTM.-resistant ovarian carcinoma cell line that carry a mutation in a .beta.-tubulin isoform (see Kowalski, R. J., et al., J. Biol. Chem. 272(4), 2534-2541 (1997)).

[0006] While two epothilone-resistant human ovarian carcinoma cell lines have been previously described in the literature (Giannakakou et al., PNAS 97(6): 2904-2909 (2000)), data disclosed herein indicate that the cell lines of the present invention are phenotypically distinct from the epothilone-resistant cells previously described. For example, while the cell lines disclosed in Giannakakou et al. display resistance to both epothilone A and B, the EA150 cells disclosed herein appear to exhibit very little resistance to TAXOL.RTM. and epothilone B. Similarly, while the EpoA8 cell lines in Giannakakou et al. show epothilone resistance, but limited paclitaxel resistance, both the C5/0 and D4/40 cell lines disclosed herein display full cross-resistance to paclitaxel. Furthermore, the D4/40 cell lines disclosed herein display not only drug resistance but also drug dependency.

[0007] In addition, the epothilone-resistant cell lines disclosed in Giannakakou et al. were shown to contain acquired .beta.-tubulin mutations which affect the ability of epothilones to induce tubulin polymerization as well as inhibit cell growth. Both the C5/0 and D4/40 cell lines disclosed herein carry a single point mutation (Threonine at position 274 replaced by Proline) in the HM40 tubulin isoform. While the site of mutation is identical to that reported for the EpoA8 cell line, the nature of the mutation (Threonine at position 274 replaced by Isoleucine) is not. Thus, while a similar mutation may be, in part, involved in the mechanism of resistance seen in the cell lines disclosed herein, phenotypic differences between the cell lines of the instant invention and those described by Giannakakou et al. indicate that the mechanism of action of epothilone resistance and associated genotypic alterations, which remain to be fully characterized, may not be the same in all resistant cell lines. As such, it is believed that the cells lines of the present invention are phenotypically and genotypically distinct from the epothilone resistant cells previously reported.

SUMMARY OF THE INVENTION

[0008] One aspect of the invention provides an epothilone resistant subline of the MDA-MB-435 breast adenocarcinoma cell line. In one embodiment the epothilone resistant cell line is resistant to 10 nM epothilone A and is the EA10 cell line. In another embodiment the epothilone resistant cell line is resistant to 20 nM epothilone A and is the EA20 cell line. In yet another embodiment the epothilone resistant cell line is resistant to 40 nM epothilone A and is the EA40 cell line. In still another embodiment the epothilone resistant cell line is resistant to 60 nM epothilone A and is the EA60 cell line. In another embodiment the epothilone resistant cell line is resistant to 150 nM epothilone A and is the EA150 cell line.

[0009] Another aspect of the invention provides epothilone resistant sublines of the KB-31 carcinoma cell line. In one embodiment the epothilone resistant cell line is the carcinoma cell line 297/C5/0. In another embodiment the epothilone resistant cell line is the carcinoma cell line 298/D4/40. In a further embodiment the epothilone resistant cell line is the carcinoma cell line 315/sc5.9.

[0010] Another aspect of the invention is a method to identify agents which display improved cytotoxicity to epothilone resistant cells in comparison to epothilone, said method comprising the steps of: incubating said epothilone resistant cells and sensitive cells with an agent to be tested; determining the cytotoxicity of said agent for epothilone resistant cells and sensitive cells; and, identifying agents which display a reduced resistance factor (IC.sub.50 value for resistant cells divided by IC.sub.50 value for parental cells) compared to epothilone.

[0011] A further aspect of the invention provides a method to identify agents which display selective cytotoxicity to epothilone resistant cells in comparison to epothilone, said method comprising the steps of: incubating said epothilone resistant cells and sensitive cells with an agent to be tested; determining the cytotoxicity of said agent for epothilone resistant cells and sensitive cells; and, identifying agents which display a resistance factor (IC.sub.50 value for resistant cells divided by IC.sub.50 value for parental cells)<1.

[0012] In yet another aspect, the invention provides the agents identified according to the methods disclosed herein which are selectively cytotoxic to epothilone resistant cells.

[0013] Another aspect of the invention is a method to selectively inhibit the growth of epothilone resistant cells in vitro or in vivo, said method comprising contacting said resistant cells with one or more of said cytotoxic agents identified according to the methods disclosed herein.

[0014] Another aspect of the invention is a method to identify agents which are chemosensitizers of epothilones, said method comprising the steps of incubating epothilone resistant cells with an epothilone to which the cells are resistant, in the presence and absence of an agent to be tested and determining the cytotoxicity of the epothilone for the cells, wherein increased cytotoxicity in cultures incubated in the presence of the agent compared to cultures incubated in the absence of the agent indicates that the agent is a chemosensitizer.

[0015] In yet another aspect, the invention provides the chemosensitizers of epothilones identified according to the methods disclosed herein.

[0016] In a still further aspect, the invention provides a method to inhibit the growth of epothilone resistant cells in vitro or in vivo, said method comprising contacting said epothilone resistant cells with one or more epothilones and one or more of the chemosensitizers identified according to the method disclosed herein.

[0017] Another aspect of the invention is a method to identify potential epothilone resistant cells, including tumor cells, said method comprising the steps of preparing total mRNA from test cells, preparing CDNA from the total mRNA of said test cells, performing gene expression analysis of said cDNA samples by conventional methods, for example using chip array techniques; and comparing the expression patterns derived from said test cells to that obtained for epothilone resistant cells, wherein similar patterns of expression suggest that the test cells are potentially epothilone resistant cells. As an extension of this method, conventional methods, for example chip array techniques, could be applied to obtain a differential gene expression pattern by comparing diseased tissue or test cells with non-diseased control tissue or cells, and then compare the pattern of differentially expressed genes with that obtained by comparison of epothilone resistant cells with parental epothilone-sensitive cells. A similar differential gene expression pattern may suggest that the diseased tissue or test cells are potentially resistant to epothilone.

[0018] In still another aspect, the invention provides purified antibodies specific for the epothilone resistant cells of the present invention. Additionally, the invention provides pharmaceutical compositions comprising one or more of these purified antibodies in conjunction with a pharmaceutically acceptable carrier or diluent.

[0019] In another aspect, the invention provides a method of identifying epothilone resistant cell lines, said method comprising the steps of labelling one or more antibodies disclosed herein, exposing test cells to said one or more labelled antibodies; and measuring binding of said one or more labelled antibodies thereto.

[0020] In a further aspect, the invention provides a method of isolating epothilone resistant cell lines, said method comprising labelling one or more antibodies disclosed herein with a fluorescent dye; [0021] incubating cell mixtures with said one or more fluorescently labelled antibodies; and, [0022] isolating epothilone resistant cells fluorescently labelled with one or more antibodies by use of a fluorescence activated cell sorter. An alternative method of separating antibody-reactive epothilone-resistant cells from normal, non-reactive cells would be to covalently link one or more claimed antibodies with magnetic beads and then to isolate epothilone resistant cells by immobilizing them using a magnetic source while washing away unbound, non-reactive cells.

[0023] In another aspect, the invention provides a method for killing epothilone resistant cells, said method comprising attaching a substance having cytotoxic or therapeutic activity to one or more antibodies specific for epothilone-resistant cells; administering said one or more antibodies to cell culture (or a patient); and, targeting the cytotoxic drug to epothilone resistant cells, selectively killing these cells.

[0024] In yet another aspect, the invention provides a method for diagnosing an epothilone resistant cancer in vivo, comprising labelling one or more of the antibodies disclosed herein, administering said one or more labelled antibodies to a patient or a patient tissue specimen; and identifying tissue to which said one or more labelled antibodies bind.

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