JP2008504355A - Cancer treatment - Google Patents

Abstract

The present invention relates to novel drugs and preparations comprising anti-cancer agents effective with anti-Hsp90 antibodies that together enhance efficacy in the treatment of cancer and leukemia.

Description

  The present invention relates to novel agents and preparations, including pharmaceuticals effective with anti-Hsp90 antibodies that together enhance efficacy in the treatment of cancers including colorectal cancer. Another aspect of the invention relates to the treatment of leukemia.

Cancer Therapy and Leukemia Treatment A first aspect of the present invention relates to novel agents and preparations comprising anti-cancer agents effective with anti-Hsp90 antibodies that together enhance efficacy in the treatment of cancer.

  Members of the heat shock protein (Hsp) family of proteins have recently become apparent as having important roles in tumorigenesis and cell death. Indeed, heat shock proteins have been identified for many years as potential targets for cancer therapy (Whitesell L et al., PNAS USA, 1994 Aug 30, 91 (18): 8324-8; PMID: 8078881). ), Members of the ansamycin family (formerly called tyrosine kinase inhibitors) have been suggested to be useful in influencing cancer treatment (Neckers L et al., Invest New Drugs). , 1999, 17 (4): 361-73; PMID: 10759403; Schulte TW et al., Cancer Chemother Pharmacol., 1998, 42 (4): 273-9; PMID: 9744771).

  One heat shock protein, Hsp90, is breast cancer, prostate cancer, melanoma, leukemia and lymphoma, colon cancer and lung cancer (Banerji U et al., Curr Cancer Drug Targets, 2003 Oct; 3 (5): 385-90; PMID : 14529390), as well as being involved in thyroid carcinoma. The role of Hsp90 is to ensure the correct holding of “client proteins” involved in a wide range of cellular processes, such as signal transduction. Hsp90 client proteins include transcription factors such as mutant p53 and hypoxia-inducing factor 1α, and soluble kinases including v-Src, Akt, Raf-1 and Bcr-Abl. Hsp90 is structurally expressed at levels 2-10 times higher in tumor cells compared to levels in normal cells, suggesting that it may be important for tumor cell growth / survival ( Schwartz, J. et al., 2003, Semin. Hematol. 40: p87-96). Hsp90 is a pathway that regulates cellular effects, including cell proliferation, division, differentiation, migration and death, because the binding of client proteins to Hsp90 can regulate its conformation, stability and intracellular fate Can have major effects (Workman, P., Cancer Lett. 2004 Apr 8; 206 (2): 149-57; PMID: 15013520). The widespread role of Hsp90 in cellular processes means that the protein is now seen as a potential target for therapeutic drug development. By interacting specifically with a single molecular target, an Hsp90 inhibitor causes destabilization and thus degradation of the Hsp90 client protein.

  The second aspect of the present invention relates to novel agents and preparations comprising anticancer agents effective with anti-Hsp90 antibodies that together enhance efficacy in the treatment of leukemia.

  Leukemia is a cancer that affects the bone marrow. In people with leukemia, the bone marrow produces many abnormal white blood cells. Abnormal white blood cells are concentrated in the bone marrow, which prevents the bone marrow from producing enough normal red blood cells, white blood cells and platelets.

  Different types of leukemia can be classified by the rate of progression (acute or chronic) and the type of white blood cell affected (myeloid or lymphoid cells). Myeloid white blood cells are the first line of immune system defense against infection and are mainly found in the blood, swallowing and killing foreign organic matter. Lymphoid white blood cells are found in lymph nodes and blood.

  The four most common leukemia types include chronic lymphocytic (lymphocytic) leukemia (CLL), acute myeloid (myeloblastic) leukemia (AML), and acute lymphocytic (lymphoblastic) leukemia ( ALL), and chronic myelogenous leukemia (CML).

  CLL is not only a lymphocyte cancer but also develops more slowly than ALL. The disease is the most common form of leukemia that attacks adults and is very rare in children.

  AML is a cancer that primarily affects bone marrow cells known as granulocytes. This AML produces a large number of myeloblasts that can block blood vessels, but does not mature sufficiently to bone marrow cells. The disease occurs mainly in adults but can also affect children.

  CML (also called chronic granulocytic leukemia) is typically a slowly developing cancer of neutrophil cells that is rare in children and is generally more common among adult men than women . CML is usually easily diagnosed because leukemia cells in more than 95% of patients have a different cytogenetic abnormality, Philadelphia chromosome (Phl) (Kurzrock, R. et al., 2003, Ann. Intern). Med. 138 (10): p819-30, PMID: 12755554; Goldman, JM and Melo, JV, 2003, N. Engl. J. Med. 349 (15): p1451-64, PMID. : 14534339). Ph1 is the result of a reciprocal transition between the long arms of chromosomes 9 and 22 and is demonstrated in all hematopoietic progenitors (Deininger, MW et al., 2000, Blood 96 (10): p3343- 56, PMID: 11071626). This metastasis results in the transfer of the Abelson (abl) oncogene on chromosome 9 to a region of chromosome 22, called the breakpoint cluster region (BCR) (Deininger, MW et al., 2000, Blood 96 (10): p3343-56, PMID: 11071626). This in turn results in a fused BCR / ABL gene that encodes an 8.5 kb chimeric mRNA. The BCR / ABL gene is an oncogene sufficient to produce a CML-like disease in mice. The transcript of the BCR / ABL oncogene is translated to yield a 210 kDa or 190 kDa protein. Bcr-Abl protein is an abnormal tyrosine kinase protein that causes the disordered bone marrow hematopoiesis found in CML. CML progresses through different clinical stages called chronic, accelerated and blast crisis. Although the BCR / ABL oncogene is expressed at all stages, blast crisis is characterized by multiple additional genetic and molecular changes (Gorre, ME et al., 2002, Blood, 100 (8 ): P3041-3044).

  Ph1-negative CML results in the clinical characteristics of CML without cytogenetic or molecular (RT-PCR) evidence of t (9; 22) (q34; q11) metastases resulting in Bcr-Abl fusion mRNA It is a rare disease characterized. Ph1-negative CML is a poorly defined entity that is not clearly distinguished from other myeloproliferative syndromes. This figure is currently less than 5% when considering 5-10% of all clinical CML, along with the normal proximity of RT-PCR analysis for Bcr-Ablo transcripts. Interestingly, some patients with this condition may be the result of different fusions to AbI. TEL (ETV6) -ABL fusion as a result of t (9; 12) has been demonstrated in two cases of Ph-CML. Patients with Ph1 negative CML generally have poorer response to treatment and shorter survival compared to Ph1 positive patients (Onida, F. et al., 2002: Cancer 95 (8): p1673-84, PMID: 12365015 ).

  ALL is a cancer of immature lymphocytes known as lymphoblasts. This disease is the most common form of leukemia among young children, usually between 1 and 7 years of age, and very rare in adults. ALL generates a number of abnormal lymphocytes that push away normal red blood cells and platelets. The 185 kDa Bcr-Abl protein is directly involved in ALL development.

Two drugs that act as Hsp90 inhibitors, geldanamycin (GA) and 17-alkylamino, 17-desmethoxygeldanamycin (17-AAG), have promising biological and clinical activity in clinical trials. showed that. Indeed, the 210 kDa Bcr-Abl fusion protein (p210 ^Bcr-Abl ) relies on its interaction with Hsp90 for its stability, and treatment of cells with GA or 17-AAG is dependent on p210 ^Bcr-Abl . Leads to rapid collapse.

  Hsp90 inhibitors, such as 17AAG, in combination with conventional cytotoxic reagents or other novel reagents may also be therapeutically valuable in attacking multi-stage tumor formation (Workman P., Cancer). Lett. 2004 Apr 8; 206 (2): 149-57; PMID: 15013520). By inhibiting Hsp90 activity in cancer cells characterized by genetic instability, 17AAG releases various mutations that together prove “synthetic lethality” to the tumor. Normal cells without the genetic instability of tumor cells are relatively unaffected (Garber, K., 2002, Journal of the National Cancer Institute, Vol. 94, No. 22, p1666-1668). An obvious problem with 17AAG is that the drug is too toxic for long-term treatment and, as a result, non-toxic substitution is required (Banerji et al., Supra).

  Imatinib mesylate (Gleevec (RTM)) is a small molecule tyrosine kinase inhibitor that has a major impact in neoplastic disease as a single reagent. Originally designed as an inhibitor of Bcr-Abl tyrosine kinase properties of malignant organisms with pathogenic 9; 22 metastasis, imatinib has been shown to have mild specificity and has been shown to have chronic myeloid leukemia (CML) and filar Mainly affected in the treatment of Delphia chromosome positive (Ph1 +) ALL (Krystal, GW, 2004, Leukemia Research 28S1: pS53-S59). One problem associated with imatinib treatment of CML is drug resistance as a result of mutations in the Bcr-Abl tyrosine kinase. Importantly, CML cells that become resistant to imatinib in vivo maintain their Hsp90 dependence and thus maintain sensitivity to 17AAG.

  Recent publications suggest that tumor Hsp90 exists entirely in multiple chaperone complexes that promote malignant progression and that these are attractive targets for cancer therapy. In particular, it has been shown that Hsp90 in multiple chaperone complexes derived from tumor cells has a 100-fold higher binding affinity for 17AAG than Hsp90 from normal cells (ie, potentially uncomplexed Hsp90). This suggests that in multiple chaperone complexes, it may present epitopes that are not presented by potential uncomplexed Hsp90, particularly quaternary epitopes. Micograb (RTM) antibodies can bind to Hsp90 in its potentially uncomplexed state, and also in multiple chaperone complexes, without adversely affecting the binding kinetics.

  WO 01/76627 is a composition for the treatment of fungal infections comprising a combination of (i) a polyene or beta-glucan synthase inhibitor antifungal agent and (ii) an antibody specific for fungal Hsp90, In spite of its resistance to antifungal drugs in essence, it represents an effective composition against fungi causing infections.

According to a first aspect of the present invention, in a method for producing a medicament for the treatment of cancer,
(I) an antibody or antigen-binding fragment thereof specific for at least one epitope of Hsp90, and (ii) use of at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin Is done.

  Doxorubicin is an anthracycline antibiotic agent that has already been recognized as an antineoplastic agent.

  Epirubicin is a synthetic anthracycline antibiotic with low toxicity that has already been recognized as an antitumor agent.

  Daunorubicin is an antineoplastic agent used in a number of therapeutic areas, including anticancer agents.

  Herceptin (trastuzumab) is a monoclonal antibody used for the treatment of HER2 protein overexpressing metastatic breast cancer.

  Docetaxel is a recognized anticancer agent and a mitotic inhibitor.

  Cisplatin is a recognized anticancer agent and includes platinum complexes.

  As detailed in the experimental results below ("Experiment A"), doxorubicin and daunorubicin are particularly preferred and exhibit particularly good synergistic effects with anti-Hsp90 antibodies. Herceptin also shows good synergy with anti-Hsp90 antibodies. A synergistic effect is also observed with docetaxel and cisplatin when combined with anti-Hsp90 antibody. The synergistic effect between daunorubicin and antibody is particularly evident in estrogen receptor positive cells, so drugs and treatments using antibodies and daunorubicin are particularly (or administered) to cells that have estrogen receptor.

  The experiment ("Experiment A") also shows that when used with anti-Hsp90 antibodies, other anti-cancer agents show either no good or bad results (paclitaxel) or antagonism (imatinib). . This confirms the surprising / unexpected nature of the synergistic effect achieved with the anticancer agents described above when combined with anti-Hsp90 antibodies.

Also for simultaneous, separate or sequential use in the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin,
A combination preparation is provided.

In addition, the patient in need of a therapeutically effective amount of
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin,
There is provided a method of treating cancer comprising administering

  As used herein, the phrase “treatment” is intended to have a broad meaning unless specifically stated otherwise. Thus, “treatment” or “therapy” is designed to cure, alleviate, eliminate or reduce the symptoms of, or prevent or reduce, the symptoms of a disease or dysfunction in the human or animal body. Means any treatment to be taken. Thus, the phrase “treatment” means both treatment of the disease state as well as its prevention.

  The antibody or antigen-binding fragment thereof can be specific for an epitope presented by a peptide comprising the sequence of SEQ ID NO: 1.

  As discussed above, the quaternary epitope presented by Hsp90 in the multi-chaperone complex has been suggested to be a suitable target for therapy, but experiments (below) have actually demonstrated linear epitopes. Has been shown to be useful for therapy and an effective target.

  Antibodies, their production and use are well known and are described, for example, in Harlow, E and Lane, D.C. , Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.

  Antibodies may be produced using standard methods known in the art. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library, and antigen-binding fragments of antibodies.

  Antibodies may be raised in a variety of hosts, such as goats, rabbits, rats, mice, humans, and the like. These may be immunized by injection with fungal stress proteins, or any fragment or oligopeptide thereof having immunogenic properties. Depending on the host species, various adjuvants can be used to increase the immune response. Such adjuvants include, but are not limited to, floyd, mineral gels such as aluminum hydroxide, and lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Such surface active substrates are included. Of the adjuvants used in humans, BCG (Bacilt Calmette-Guerin) and Corynebacterium parvum are particularly useful.

  Monoclonal antibodies to fungal proteins, or any fragment or oligopeptide thereof, may be prepared using any technique provided to produce antibody molecules by continuous cell lines in culture. These include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV hybridoma technology (Koehler et al., 1975, Nature, 256: 495-497; Kosbor et al., 1983, Immunol. Today 4: 72, Cote et al., 1983, PNAS USA, 80: 2026-2030; Cole et al., 1985, Monoclonal Antibodies and Cancer Theory, Alan R. Lith Inc, New York, pp. 77-96). .

  Furthermore, techniques developed for the production of “chimeric antibodies”, splicing of mouse antibody genes to human antibody genes can be used to obtain molecules with appropriate antigen specificity and biological activity ( Morrison et al., 1984, PNAS USA, 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted using methods known in the art to produce fungal stress protein specific single chain antibodies. Antibodies with related specificity but different idiotype composition may be generated by shuffling chains from a random combinatorial immunoglobulin library (Burton, DR, 1991, PNAS USA, 88: 11120). -11123).

  Antibodies may be produced by inducing production in vivo in a lymphocyte population or by screening a recombinant immunoglobulin library or panel of highly specific binding reagents (Orlandi et al 1989, PNAS USA, 86: 3833-3837; Winter, G et al., 1991, Nature, 349: 293-299).

  Antigen binding fragments such as F (ab ′) 2 fragments that can be produced by pepsin digestion of antibody molecules, and Fab fragments that can be produced by reduction of disulfide bridges of F (ab ′) 2 fragments may also be produced. . Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., 1989, Science, 256: 1275-1281).

  A variety of immunoassays may be used for screening to identify antibodies with the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve the measurement of complex formation between a fungal stress protein or any fragment or oligopeptide thereof and its specific antibody. A two-site monoclonal-based immunoassay using monoclonal antibodies specific for two non-interfering fungal stress protein epitopes may be used, but a competitive binding assay may also be utilized (Maddox et al., 1983). , J. Exp. Med., 158: 1211-1216).

  For example, the antibody used in the composition or combination preparation can comprise the sequence of SEQ ID NO: 2.

  The present inventor has a group of breast cancer, prostate cancer, melanoma, leukemia, lymphoma, leukemia, colorectal cancer, testicular germ cell cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer and lung cancer that can be usually treated It has been discovered that fibrosarcomas and cancer are included, which are more selected.

According to a second aspect of the present invention, in a method for producing a medicament for the treatment of leukemia,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof,
Use of is provided.

Also, in a method for producing a drug for the treatment of leukemia,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
Use of is provided.

  Imatinib, a derivative of 2-phenylaminopyrimidine, is a small molecule antagonist with activity against protein tyrosine kinases and exhibits potent and specific inhibition of Bcr-Abl. Imatinib is indicated for the treatment of patients suffering from CML in the blast crisis, in the acute phase, or in the chronic phase after failure of IFN therapy.

  Paclitaxel is a chemotherapeutic agent considered as a treatment for several types of cancer. Most commonly used to treat ovarian cancer, breast cancer and non-small cell lung cancer.

  Docetaxel is a recognized anticancer agent and a mitotic inhibitor.

  Daunorubicin is an antineoplastic agent used in a number of therapeutic areas, including as an anti-cancer agent.

  Doxorubicin is an anthracycline antibiotic agent that has already been recognized as an antineoplastic agent.

  Hydroxyurea is an antineoplastic, ribonucleotide reductase inhibitor.

  As detailed in the experimental results below ("Experiment B"), doxetaxel and paclitaxel are particularly preferred and exhibit particularly good synergistic effects with anti-Hsp90 antibodies. A synergistic effect is also observed with imatinib, doxorubicin, daunorubicin and hydroxyurea when combined with anti-Hsp90 antibodies. When used with an anti-Hsp90 antibody, the anticancer drug cisplatin showed good or bad results. This confirms the surprising / unexpected nature of the synergistic effect achieved with the anti-cancer agents described above when combined with anti-Hsp90 antibodies.

Also for simultaneous, isolated or sequential use in the treatment of leukemia,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
A combination preparation is provided.

  Combination preparations include, for example, a pharmaceutical pack comprising (i) an antibody and (ii) at least one anticancer agent in separate doses (ie, not mixed together in a single preparation).

In addition, the patient in need of a therapeutically effective amount of
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
A method of treating leukemia is provided.

  The leukemia may be chronic myelogenous leukemia or acute lymphocytic leukemia and the at least one anticancer agent may be imatinib.

  The antibody or antigen-binding fragment thereof may be specific for an epitope presented by a peptide comprising the sequence of SEQ ID NO: 1.

  For example, the antibody used in the composition or combination preparation may comprise the sequence of SEQ ID NO: 2.

  The anticancer agent may be imatinib.

  The inventor presently treatable leukemia includes leukemia selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia and acute lymphocytic leukemia.

  The leukemia may be chronic myelogenous leukemia or acute lymphocytic leukemia.

  Chronic myelogenous leukemia is Ph1 positive or Ph1 negative, ie characterized by leukemia cells that contain the Philadelphia chromosome (Ph1 positive) or lack the Philadelphia chromosome (Ph1 negative).

  The inventor has discovered that chronic myeloid leukemia, which can usually be treated with imatinib, can be either Ph1-positive or Ph1-negative.

  The leukemia may be Ph1-positive chronic myeloid leukemia and the anticancer agent may be imatinib. In particular, the inventor has discovered that treatment of Ph1-positive CML can be affected by a combination of an antibody comprising the sequence of SEQ ID NO: 2 and imatinib. While not intending to adhere to any theory, Hsp90 is sequestered by an antibody comprising the sequence of SEQ ID NO: 2, and thus an abnormal Bcr-Abl tyrosine kinase (causing abnormal myelopoiesis found in CML) Means, for example, misfolded, targeted for proteolysis and / or preventing it from exerting its effect in the bone marrow hematopoietic pathway.

  This treatment is more effective in imatinib resistant CML Ph1-positive cells. Without intending to stick to any theory, possible resistance to imatinib is due to an aberrant congregation mutation in Bcr-Abl tyrosine kinase, which may prevent the drug from binding to the protein and / or interfere with the mode of activity of the drug, for example There is sex. In imatinib-resistant cells, sequestration of Hsp90 by an antibody comprising the sequence of SEQ ID NO: 2 causes, for example, misfolded aberrant tyrosine kinases to be misfolded, targeted for proteolysis, and / or exert their effects in the myelopoietic pathway Is prevented. Proving synthetic lethality to tumor cells together by sequestering Hsp90, which normally acts to “buffer” genetic mutations associated with cancer cells by binding to abnormal proteins and inhibiting their expression In addition, various mutations can be released. Normal cells that lack tumor cell genetic instability are relatively unaffected.

  Moreover, particularly surprisingly, the inventor has discovered that treatment of CML that is Ph1 negative can be affected by a combination of an antibody comprising the sequence of SEQ ID NO: 2 and imatinib.

  The leukemia may be Ph1-negative chronic myeloid leukemia and the anticancer agent may be imatinib.

  This finding is surprising because Ph1 negative CML cells lack the abnormal tyrosine kinase protein associated with Ph1 positive cells. However, while not intending to adhere to any theory, it is possible that the kinase in Ph1-negative cells is at low or basal levels (either abnormal or otherwise) and, as noted above, sequestration of Hsp90 Is that the tyrosine kinase protein is prevented from being misfolded, targeted for proteolysis and / or exerting its effect in the bone marrow hematopoietic pathway. means. Also, by sequestering Hsp90, various mutations can be released by tumor cells together to prove synthetic lethality.

  The surprising effect of imatinib and anti-Hsp90 antibodies in Ph1 negative cells may be due to the presence of TEL (ETV6) -ABL fusion, which has been demonstrated in two cases of Ph1 negative CML (Krystal, GW , 2004, Leukemia Research 28S1: pS53-S59), sensitive to imatinib.

  Leukemia can be characterized by imatinib resistant cells.

  The composition or preparation of the present invention may further comprise a known Hsp90 inhibitor, such as GA or 17-AAG.

  The third aspect of the invention (Experiment C) relates to novel agents and preparations comprising anti-cancer agents effective with anti-Hsp90 antibodies that together enhance efficacy in the treatment of colorectal or adenocarcinoma.

  Colorectal cancer is a malignant tumor of the large intestine or ileum. Colorectal cancer is a major cause of cancer morbidity and mortality. In the UK, it is the third most common cancer in men and the second most common cancer in women. Ninety-five percent of colorectal cancer is adenocarcinoma, which is a cancer of glandular cells that line the large intestine and ileum.

  Standard treatment for colorectal cancer is usually a combination of 5-fluorouracil and leucovorin (folinyl acid).

  5-Fluorouracil (5-FU) is used to treat a number of solid cancers, including gastrointestinal cancer and breast cancer. Commonly used with folinic acid in advanced colorectal cancer. 5-FU is converted to FdUMP in the cell and forms a complex with thymidine synthase (TS) that inhibits DNA, protein and RNA synthesis.

  Folinyl acid (leucovorin) is a vitamin given in combination with 5-FU. Folinic acid increases the response rate to 5-fluorouracil, is disease free and is accompanied by a significant improvement in overall survival. Folinyl acid increases intracellular folic acid and stabilizes the FdUMP / TS complex.

  Other drugs that have been found to be effective include irinotecan and oxaliplatin, which are licensed for initial use in patients with advanced colorectal cancer in combination with 5-fluorouracil and folinic acid. Irinotecan or raltitrex is licensed for use as a second-line monotherapy when fluorouracil-based treatment fails or is inappropriate.

  Oxaliplatin is a recognized anticancer agent and includes novel diaminocyclohexaneplatinum compounds that form crosslinks in DNA and thus inhibit DNA replication.

  “FOLFOX” is a commonly used combination chemotherapy of 5-fluorouracil, folinic acid and oxaliplatin.

  Irinotecan (CPT-11, Campto) inhibits topoisomerase I, a DNA unwinding enzyme essential for cell division, resulting in replication arrest that is interrupted in single-stranded DNA. In the UK, irinotecan is in combination with 5FU / FA for use in patients who have never received advanced colorectal cancer chemotherapy and in patients who have failed an established 5FU-based regimen. Licensed as a single agent for second-line chemotherapy.

  Raltitrexide (ZD 1694, Tomudex) inhibits the enzyme thymate synthase involved in DNA synthesis. This is the same enzyme targeted by 5FU. Raltitrex is licensed for palliative treatment of advanced colorectal cancer in the UK where a 5FU / FA based regimen is not acceptable or inappropriate.

  Tebutt et al. , 2002, European Journal of Cancer, 38: 1000-1015; Cutsem et al. , 2002, Best Practice and Research Clinical Gastroenterology, 16: 319-330; Beretta et al. , 2004, Surgical Oncology, 13: 63-73; NICE guidelines for irinotecan, xaliplatin and raltitrexide for advanced colorectal cancer, 2002.

According to a third aspect of the present invention, in a method for producing a medicament for the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
Use of is provided.

Alternatively, for simultaneous, separate or sequential use in the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
A combination preparation is provided.

According to yet a further aspect of the present invention, a therapeutically effective amount of a patient in need thereof,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
There is provided a method of treating cancer comprising administering

  Preferably, the cancer is colorectal cancer or adenocarcinoma.

  Most preferably, the anticancer agent 5-fluorouracil is further included and administered with folinyl acid (leucovorin).

  Additionally or alternatively, 5-fluorouracil, folinic acid and oxaliplatin are administered together.

  The composition or preparation according to any aspect of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient. Similarly, any manufacturing method of the present invention, or use thereof, may also include the use of a pharmacologically acceptable carrier, diluent or excipient. Examples of pharmacologically acceptable carriers, diluents and excipients are well known in the art, eg, see Remington's Pharmaceutical Sciences and US Pharmaceutical Company (1984, Mack Publishing Company, Easton, PA, USA). See

  The drug or combination preparation may be administered orally, for example, without implying that other routes of administration are excluded.

  An antibody or antigen-binding fragment thereof according to the present invention may be labeled with a detectable label using conventional procedures, or an effector molecule, such as a drug such as doxorubicin, daunorubicin, docetaxel or cisplatin or 5-fluorouracil, May be conjugated to anticancer agents such as oxaliplatin, irinotecan and raltitrex, or drugs useful in treating leukemias such as imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea, or toxic or enzymes such as ricin, The present invention extends to such labeled antibodies or antibody conjugates.

  Optionally, for example a mixture of antibodies recognizing different epitopes of two or more stress proteins according to the invention and / or a mixture of different classes of antibodies, eg the same or different epitope (s) of the invention A mixture of recognizing IgG and IgM antibodies may be used for diagnosis or treatment.

  As discussed above, the quaternary epitope presented by Hsp90 in multiple chaperone complexes has been suggested as an appropriate target for therapy, but experiments (below) have shown that It is shown to be a useful and effective target for therapy.

  The entire contents of each reference discussed herein, including the references cited therein, are hereby incorporated by reference.

  When a “PMID:” bibliography number is attached to a publication, a PubMed identification number is assigned by the US National Library of Medicine, so that complete bibliographic information for each publication and A summary is available at www. ncbi. nlm. nih. Available on gov. This also allows direct access to an electronic copy of the complete publication, especially in the case of PNAS, JBC and MBC publications, for example.

  The invention will be further apparent from the following description, which shows, by way of example only, specific embodiments of the composition and thereby the experiment.

Experiment first set of experiments ( "Experiments A") is detailed below, itself or anticancer agents doxorubicin, daunorubicin, docetaxel, trastuzumab, imatinib, are used in combination with cisplatin and paclitaxel, the sequence of SEQ ID NO: 2 Describes the investigation of the anti-cancer effect of anti-Hsp90 antibodies specific for the epitope presented by the peptide having the sequence SEQ ID NO: 1.

  The second set of experiments ("Experiment B") is described in detail below on the white chronic myeloid leukemia line K562, human myeloid leukemia cell line KU-812, or the anticancer drugs imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin Of the effect of an anti-Hsp90 antibody, specific for the epitope presented by the peptide having the sequence of SEQ ID NO: 1 and having the sequence of SEQ ID NO: 1 used in combination with paclitaxel, cisplatin and hydroxyurea Describes the survey.

  A third set of experiments ("Experiment C") is described in detail below on human colon cancer cell line HT29, either on its own or on the anticancer drugs 5-fluorouracil (5-FU) and folinic acid (leucovorin, LV) and / or oxalis. Describes the investigation of the effect of anti-Hsp90 antibodies specific for the epitope presented by the peptide having the sequence SEQ ID NO: 1 and having the sequence SEQ ID NO: 1 used in combination with platin .

General Materials and Methods Unless otherwise noted, all procedures were performed using standard protocols and the following instructions where applicable. Standard protocols for a variety of techniques, including PCR, molecular cloning, manipulation and sequencing, antibody production, epitope mapping and mimotope design, cell culture and phage display are described in McPherson, MJ et al. (1991, PCR: A practical approach, Oxford University Press, Oxford), Sambrook, J. et al. and Russell, D.A. , “Molecular Cloning: A Laboratory Manual”, Third Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 2001, Huynh and David. Ed. DM Glover), Sanger, F .; et al. (1977, PNAS USA 74 (12): 5463-5467), Harlow, E .; and Lane, D.C. ("Using Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1998), Jung, G. et al. and Beck-Sickinger, AG (1992, Angew. Chem. Int. Ed. Eng., 31 : 367-486), Harris, M .; A. and Rae, I .; F. ("General Techniques of Cell Culture", 1997, Cambridge University Press, ISBN 0521 JE, JB. And J. Phys. Academic Press Inc., 1996, ISBN 0-12-402380-0).

  In particular, reagents and equipment useful in the methods detailed herein include Amersham (www.amersham.co.uk), Boehringer Mannheim (www.boehringer-ingeltheim.com). , Clontech (www.clontech.com), Genosys (www.genosys.com), Millipore (www.millipore.com), Novagen (www.cov.movagen.com) -Perkin Elmer (www.perkinelmer.com), Pharmacia (www.far) macia.com), Promega (www.promega.com), Qiagen (www.qiagen.com), Sigma (www.sigma-aldrich.com), and Stratagene (Stratagene) www.stratagene.com).

Antibodies The antibodies used in Experiments A and B below are those disclosed in WO 01/76627 and are referred to herein as Mycograb (RTM). This antibody has the sequence of SEQ ID NO: 2 and is specific for the epitope presented by the peptide having the sequence of SEQ ID NO: 1. The basal antibody solution was a 4 mg / ml stock solution in water. Further dilution was performed in RPMI complete medium.

  Briefly, the DNA sequence of the original antibody specific for the Candida albicans Hsp90 epitope disclosed in British Patent No. 2240979 and European Patent No. 040629 is genetically determined by codon optimization. Modified and expressed in Escherichia coli (Operon Technologies Inc., Alameda, Calif., USA) and inserted into an E. coli expression vector. The amino acid sequence of the anti-Hsp90 antibody includes the sequence of SEQ ID NO: 2 (heavy chain, light chain and spacer domain). The antibody recognizes an epitope comprising the sequence of SEQ ID NO: 1.

Anti-Hsp90 antibody was expressed in an E. coli host and then purified to 95% purity by affinity chromatography and an imidazole exchange column. Standard molecular biology protocols were used (see, eg, Harlow & Lane, supra; Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual , 2nd Edition, Cold Spring Harbor Label LaborLabLab See Sambrook, J. & Russell, D., 2001, Molecular Cloning: A Laboratory Manual , 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

The drug cisplatin was obtained from Bristol-Myers Squibb, Mayne (Bristol-Myers Squibb, Mayne) and supplied at 1 mg / ml.

  Docetaxel was obtained from Sigma. First, 5 mg was diluted with dimethyl sulfoxide (DMSO) to 16 mg / ml.

  Doxorubicin was obtained from Pharmacia. 5 ml of doxorubicin hydrochloride as 2 mg / ml was supplied.

  Imatinib (Glivec (RTM)) obtained from Novartis was supplied as a 100 mg capsule. Imatinib was first diluted in water to make a 10 mg / ml stock solution.

  Paclitaxel was obtained from Sigma, reconstituted in 250 μl methanol and made up to 2.5 ml with water to give 2 mg / ml.

  Daunorubicin was obtained from Sigma and 5 mg was diluted in 2.5 ml water to give 2 mg / ml.

  Herceptin (RTM) (Trastuzumab) was obtained from Roche and reconstituted in 7.2 ml water to give 21 mg / ml.

  Hydroxyurea was obtained from Sigma and 1 g was diluted in 4 ml water to obtain 25 mg / ml.

  5-Fluorouracil (5-FU) was obtained from Sigma and 96 mg was reconstituted in 1 ml DMSO diluted 1/10 in complete RPMI medium to give 9.6 mg / ml.

  Folinyl acid (LV) was obtained from Sigma and 100 mg was reconstituted with 25 ml water to give a 4 mg / ml stock solution.

  Oxaliplatin was obtained from Sigma and 12.5 mg was reconstituted with 2.5 ml water to give 5 mg / ml.

  Each of the above drugs was further diluted in RPMI complete fold.

Cell concentration and viability determination Cells were counted and stained with an equal volume of 0.4% trypan blue solution (Sigma) before determining percent survival using a standard hemacytometer.

Cell viability assay Cell viability was assessed after each experiment using the cell titer blue assay (Promega). The medium was removed from the cells and 100 μl fresh complete medium was added followed by 20 μl cell titer blue reagent. This was incubated at 37 ° C. and 5% CO _{2 for} 4 hours, and the absorbance was 570 nm based on 600 nm. This assay measures the metabolic capacity of cells using the indicator dye resazurin (blue). Living cells reduce resazurin to resorufin (pink).

Data Interpretation Cell proliferation was assessed as described above. IC ₅₀ (dose required to cause cytotoxicity in 50% of cells) concentration was determined for each drug alone over a 48 hour incubation period.

Mean effect analysis, synergistic effects based on the Hill equation, additive effects, or antagonism measurements were performed using the Calcusyn product (BioSoft, Cambridge, UK-www.biosoft.com) from Chou and Tallaley. Determined by method. CI (combination index) reflecting synergistic effects below 1 and additive effects when equal to 1 and antagonism when greater than 1 was calculated at varying levels of drug effect. 10 fixed drugs that are greater or less than IC ₅₀ (concentration of drug required to exert 50% cytotoxic effect) in the range of 0.0156 N to 8 N, where N is a value near the IC _{50 of the} individual drug The ratio was investigated by incubating the drug mixture with the cells for 48 hours and then determining the degree of cytotoxicity. Fa50 is defined in that 50% of cells are affected. CI values are shown for Fa50.

Experiment A
This result shows that the antibody shows evidence of antagonism with imatinib and is unrelated to paclitaxel. Although synergistic with cisplatin and with docetaxel, the latter is a possibility at concentrations that are not clinically achievable. Doxorubicin synergizes with both cell lines at clinically achievable drug levels and does not depend on the presence of estrogen receptors. The results achieved with doxorubicin are evaluated as a very significant synergistic effect. Daunorubicin results are equally noticeable in cell lines with estrogen receptor, but lower in estrogen receptor negative cell lines, and synergy was limited to 6 and 12.5 mg / l. The results for Herceptin indicate that there was no synergistic effect on the estrogen receptor positive cell line, but a synergistic effect was observed in the estrogen receptor negative cell line.

Materials and Methods Cell Line and Culture Information Human Caucasian breast cancer cell line MCF7 expressing both wild type and variant estrogen receptors, as well as progesterone receptor, was obtained from ECACC (ECACC number-86012803).

Other cell lines used are as follows.
HS578T-ECACC # 86082104, human breast cancer, epithelium. Tumors form in immunosuppressed mice and colonize in semi-solid medium. Estrogen receptor negative.
SK-BR-3- (ATCC) human breast cancer. Estrogen receptor negative. Overexpress the HER2 / C-erb-2 gene.
UACC-812- (ATCC) ductal carcinoma. Prior to surgery, the patient received extensive chemotherapy. Estrogen receptor negative, progesterone receptor negative, P glycoprotein negative. Amplification of the HER-2 / neu oncogene sequence.
HCT-116-ATCC colorectal cancer. Positive for TGF beta 1 and beta 2 expression.

Cells were split using 0.25% trypsin / EDTA (Sigma), 10% fetal bovine serum, 1% non-essential amino acids, 2 mM glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin ( Maintained in RPMI medium without phenol red containing Sigma) at 37 ° C., 5% CO ₂ .

Experiment A
Effect of micograb on MCF7 cells Cell lines were fractionated and cells were counted. Cells were added to 12-well or 96-well flat bottom tissue culture plates. For 12 well plates, 1 ml of 4 × 10 ⁴ cells / ml was added along with 1 ml of medium. For 96 well plates, 100 μl of 4 × 10 ⁴ cells / ml were added, followed by an additional 100 μl of complete medium. These plates were incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. Then a fresh medium containing a doubled concentration of micograb (1.5-200 μg / ml) or formulation buffer, or only medium was added to the wells. Plates were returned to the incubator for 48 hours, followed by cell titer blue assay, or visual measurements were performed using a hemocytometer.

Effects of anticancer drugs doxorubicin, daunorubicin, herceptin, docetaxel, imatinib, paclitaxel and cisplatin on MCF7 cells Cell lines were fractionated and cells were counted. 100 μl of 4 × 10 ⁴ cells / ml was added to a 96-well flat bottom tissue culture plate and an additional 100 μl of complete medium was added to the plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. Various concentrations of test drugs (doxorubicin 0.55-600 μg / ml, daunorubicin 0.45-1000 μg / ml, Herceptin 0.2-200 μg / ml, docetaxel 0.75-800 μg / ml, imatinib 4.5-5000 μg / ml ml, fresh medium containing cisplatin 0.04-50 μg / ml, paclitaxel 1.8-1000 μg / ml), or medium alone was added to the wells. Plates were returned to the incubator for 48 hours followed by a cell titer blue assay.

Effect of micograb in combination with anticancer drugs doxorubicin, daunorubicin, herceptin, docetaxel, imatinib, paclitaxel and cisplatin on MCF7 cells The cell lines were split and the cells counted. 100 μl of 4 × 10 ⁴ cells / ml was added to a 96-well flat bottom tissue culture plate and an additional 100 μl of complete medium was added to the plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. 100 μl fresh medium was added to the plate and the mycograb vs. other drug checkerboard (using doxorubicin as an example) was set as outlined in Table 1 below, for a total volume of 200 μl per well.

  Plates were returned to the incubator for 48 hours followed by a cell titer blue assay.

  Experiments were also performed on HS578T cells using the above method. The results are shown below.

Results of Experiment A MCF7 cell line
Cisplatin IC ₅₀ was 6 mg / l and there was no effect in adding micograb except for synergistic effects at high concentrations. See Table 2.

Imatinib IC ₅₀ was 37.5 mg / l, and antagonism was observed with micograb and CIs in the range of 3.3-10 at imatinib concentrations below 37.5 mg / l.

Docetaxel IC ₅₀ was 225 mg / l, and there was a slight sign of synergy with micograb at high concentrations of docetaxel. See Table 3.

Paclitaxel IC ₅₀ was 225 mg / l, nothing at low concentrations of drug, and there was a high level and mild synergistic effect like 500 mg / l paclitaxel. These levels are outside of clinically relevant levels.

Doxorubicin IC ₅₀ was 1.75 mg / l. There was a clear synergistic effect with micograb over a wide range of drug concentrations. See Table 4.

Daunorubicin IC ₅₀ was 1 mg / l. Signs of synergy with micograb were seen over a wide range of drug concentrations. See Table 5.

Detectable activity by Herceptin Herceptin is absent, there was no sign of synergy.

HS578T cell line This cell line was insensitive to mycograb even at increasing concentrations up to 400 mg / l. This is not surprising since these tumors are steroid sensitive and therefore may not inherently respond to Hsp90 inhibitors such as micograb. However, there was an unexpected synergistic effect in combination with anthracycline doxorubicin and daunorubicin and herceptin.

Doxorubicin IC ₅₀ was 1 mg / l. Signs of synergy with micograb were seen over a wide range of drug concentrations. See Table 6.

Daunorubicin IC ₅₀ was 1 mg / l. There were slight signs of synergy with micograb, but it was almost unrelated. See Table 7.

In Herceptin HS578T, single Herceptin failed to kill 50% of cells at concentrations up to 200 mg / l, but see synergistic effect in the presence of micograb, see Table 8.

For the docetaxel cell line HS578T, an IC ₅₀ of ₅₀ mg / l was given and showed no signs of synergy with micograb.

Cisplatin Cisplatin had an IC _{50 of} 12.5 mg / l for the cell line HS578T and there was no sign of synergy with micograb.

Conclusion There were signs of antagonism with imatinib and was independent of paclitaxel. Cisplatin and docetaxel had a slight synergistic effect, but the latter is probably at a concentration that is not clinically achievable. Doxorubicin showed synergistic effects at clinically achievable drug levels in both cell lines, regardless of the presence of the estrogen receptor. The results achieved with doxorubicin were evaluated as a very significant synergistic effect. Daunorubicin results are similarly noticeable in cell lines with estrogen receptor, but less in estrogen negative cell lines, and the interaction was limited to 6 and 12.5 mg / l.

  These surprising synergistic effects were observed with antibodies and certain anticancer agents, but not with other anticancer agents such as imatinib.

Experiment B
A second set of experiments (described below) was carried out against the white chronic myeloid leukemia line K562, the human myeloid leukemia cell line KU-812, or the anticancer drugs imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin, paclitaxel, cisplatin and To investigate the effect of anti-Hsp-90 antibody, which is specific for the epitope presented by the peptide having the sequence of SEQ ID NO: 1 and having the sequence of SEQ ID NO: 1 used in combination with hydroxyurea It is detailed.

  From this result, it can be seen that the antibody showed signs of synergy with imatinib, paclitaxel, docetaxel, and daunorubicin. The antibody showed slight signs of synergy with daunorubicin and hydroxyurea. This result shows that the antibody showed signs that it was unrelated to cisplatin.

  The results achieved with docetaxel and paclitaxel are evaluated as a very significant synergistic effect.

Materials and Methods Cell lines and culture information The human myeloid leukemia cell line KU-812 was obtained from ECACC (ECACC number 90071807). The Philadelphia chromosome (Ph1) has been detected in this cell line. Cells are a morphological characteristic of basophils.

  Human Caucasian chronic myeloid leukemia cells K562 were obtained from ECACC (ECACC No. 89121407). K562 was established from pleural effusion of a 53 year old woman with chronic myelogenous leukemia in a terminal blast crisis. Nuclear studies in the various K-562 sublines have been divided into three groups (A, B, C) (Dimery, IW et al., 1983, Exp. Hematol .; 11 (7): p601-10). The strain used in this experiment was K562B. Experiments indicate that these strains are genetically similar in terms of morphology, growth kinetics in liquid suspension culture, cloning efficiency in soft agar culture, binding of anti-K562 monoclonal antibodies and cell surface proteins It was shown that. K562B was compared to K562A and K562C with respect to growth rate, cell surface protein markers, surface antigens, cytogenetics and hemoglobin production. Differences were observed between cells, the most important difference being that more than 90% of K562A or C cells appeared to be Ph1 positive and less than 15% of K562B cells contained Ph1 (Dimery, IW, et al., 1983, Exp. Hematol .; 11 (7): p601-10). Cytotoxicity tests do not reveal the presence of a pure Ph1 chromosome, but K562 appears to contain a portion of the Ph1 chromosome and is amplified at least 4-fold. This part of the Ph1 chromosome encodes a chimeric bcr / c-abl transcript, which when transcribed yields a bcr / c-abl fusion protein (Grosveld, G., et al., 1986, Mol. Cell Biol.6, No. 2: p607-616). The bcr / b-abl fusion protein has activated tyrosine kinase activity that is associated with the pathogenesis of CML.

Cells were cultured in RPMI medium 1640 without phenol red, 10% fetal calf serum, 2 mM glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin (Sigma) at 37 ° C., 5% CO ₂ . Maintained between 2 × 10 ^{6 and} 9 × 10 ⁶ cells / ml.

Effect of micograb on experimental K562 cells Cell lines were counted. Cells were added to 96 well flat bottom tissue culture plates using 100 μl aliquots containing 4 × 10 ⁵ cells / ml. Fresh medium containing two concentrations of various concentrations of micograb (RTM) (1.5-200 μg / ml), or medium alone, was added to the wells. Plates were returned to the incubator for 48 hours, followed by cell titer blue assay, or live counts determined using a hemocytometer.

Effect of anticancer drugs doxorubicin, daunorubicin, docetaxel, paclitaxel, imatinib, cisplatin and hydroxyurea on K562 cells Cell lines were counted. 100 μl of 2 × 10 ⁵ or 4 × 10 ⁵ cells / ml was added to a 96 well flat bottom tissue culture plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . Various concentrations of test drugs (doxorubicin 0.55-600 μg / ml, daunorubicin 0.07-100 μg / ml, docetaxel 0.75-800 μg / ml, paclitaxel 0.5-500 μg / ml, imatinib 4.5-5000 μg / ml ml, fresh medium containing cisplatin 0.04-50 μg / ml), or medium alone was added to the wells. Plates were returned to the incubator for 48 hours followed by a cell titer blue assay.

Effect of anticancer drugs doxorubicin, daunorubicin, docetaxel, paclitaxel, imatinib, cisplatin and hydroxyurea on K562 cells Cell lines were counted. 100 μl of 2 × 10 ⁵ or 4 × 10 ⁵ cells / ml was added to a 96 well flat bottom tissue culture plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . 100 μl of fresh medium was added to the plate and the crossover of mycograb versus other drugs was set as outlined below in Table 9 (using daunorubicin as an example) to give a total volume of 200 μl per well.

  Experiments were also performed on KU-812 cells using the above method. The results are shown below.

Results Effect of micograb (RTM) on K562 cells In cell line K562, at 12.5 μg / ml, micograb itself showed a 40% reduction in cell viability.

Effects of micograb (RTM) and anticancer drugs on K562 cells
Imatinib IC ₅₀ was 16 μg / ml. There was a slight indication of synergy between imatinib and micograb (RTM) over a wide range of drug concentrations (see Table 10).

Doxorubicin IC ₅₀ was 1 μg / ml. At some drug concentrations, there were slight signs of synergy between doxorubicin and micograb (RTM), but it had little to do with micograb (RTM).

Daunorubicin IC ₅₀ was 0.75 μg / ml. There were few signs of synergy between daunorubicin and micograb (RTM) at low drug concentrations (see Table 11).

Docetaxel IC ₅₀ was 70 μg / ml. There was a clear indication of synergy between docetaxel and micograb (RTM) over a wide range of drug concentrations (see Table 12).

Paclitaxel IC ₅₀ was 32 μg / ml. There was a clear sign of synergy between paclitaxel and micograb (RTM) over a wide range of drug concentrations (see Table 13).

Cisplatin IC ₅₀ was 12.5 μg / ml. There were no signs of synergy between cisplatin and micograb (RTM).

Hydroxyurea IC ₅₀ did not reach hydroxyurea as a single agent. However, there were few signs of synergy between hydroxyurea and micograb (RTM) at low drug concentrations (see Table 14).

Effect of micograb (RTM) on KU-812 cell line KU-812 cells In cell line KU-812, micograb itself showed a 40% reduction in cell viability at 50 μg / ml.

Effects of micograb (RTM) and anticancer drugs on KU-812 cells
Imatinib IC ₅₀ was 0.12 μg / ml. There were few signs of synergy between imatinib and micograb (RTM) at low drug concentrations (see Table 15).

Summary There were signs of synergy with imatinib, paclitaxel and docetaxel using the K562 cell line. There were slight signs of synergy with daunorubicin, doxorubicin and hydroxyurea. There was no association with cisplatin.

  There were slight signs of synergy with imatinib using the KU-812 cell line.

CONCLUSION The data presented herein clearly show that mycograb (RTM) antibodies can themselves reduce the viability of both Ph1-positive and Ph1-negative CML cell lines. Furthermore, there is a surprising synergistic effect between anti-cancer agents including imatinib and anti-Hsp90 antibodies in the Ph1-positive CML cell line. The data also show that there is a synergistic effect between anti-Hsp90 antibody and paclitaxel, docetaxel, daunorubicin, doxorubicin, hydroxyurea in Ph1-positive leukemia cell lines. These results allow the use of a composition comprising an anti-cancer agent such as imatinib together with an anti-Hsp90 antibody (Micograb, RTM) for the treatment of CML. The synergistic effect demonstrated by the combination of anticancer agent and micograb (RTM) antibody is essentially at lower treatment doses, or the same dose, which, in particular imatinib, can give the significant toxicity of many anticancer agents. Allowing any of the more effective and longer treatments, thus reducing undesirable side effects.

  Among the clinical significance of the present invention is (i) the production of a synergistic combination of an anti-Hsp90 antibody and an anti-cancer agent such as imatinib in the treatment of CML should be the treatment of choice, possibly due to this Leads to a decrease in mortality to CML; (ii) Imatinib is toxic, and the synergistic effect provided by the present invention allows the use of low doses of imatinib while maintaining efficacy and at the same time reducing toxicity And (iii) The toxicity-sparing effect of anti-hsp90 enables the clinical effects of higher doses of imatinib exposed and is further involved in improving clinical outcomes.

Experiment C
A third set of experiments ("Experiment C") was conducted against the human colon adenocarcinoma cell line HT29 itself or with the anticancer drugs 5-fluorouracil (5-FU) and folinic acid (leucovorin, LV) and / or oxaliplatin. Detailed investigation of the effect of anti-Hsp90 antibodies, specific for the epitope presented by the peptide having the sequence of SEQ ID NO: 1 and having the sequence of SEQ ID NO: 1 used in combination.

  This result shows that the antibody showed signs of synergy with 5-FU and folinic acid and with oxaliplatin. In addition, signs of synergistic effects were seen with 5-FU, folinic acid and oxaliplatin and a drug combination of mycograb / anti-Hsp90 antibody. Concentrations of more than 75 μg / ml of 5-FU and 10.5 μg / ml of oxaliplatin have been found to be particularly useful.

Materials and Methods Cell Line and Culture Information The human Caucasian colon adenocarcinoma grade II cell line HT29 was obtained from ECACC (ECACC number 91072201).

Cells are fractionated using 0.25% trypsin / EDTA (Sigma) and in MoCoy's 5a medium containing 10% fetal calf serum, 2 mM glutamine, 100 U penicillin, 0.1 mg streptomycin (Sigma), Maintained at 37 ° C., 5% CO ₂ .

  Other cell lines included HCT116.

Experimental Effect of Micograb (RTM) on HT29 cells Cell lines were fractionated and cells were counted. Cells were added to 12-well or 96-well flat bottom tissue culture plates. For 12-well plates, 1 ml of 4 × 10 ⁴ cells / ml or 4 × 10 ⁵ cells / ml was added to each well and 1 m of complete MoCoy's 5a medium was further added. For 96-well plates, 100 μl of 4 × 10 ⁴ cells / ml or 4 × 10 ⁵ cells / ml were added, followed by another 100 μl of complete McCoy's 5a medium to each well. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. Then fresh medium containing a doubled concentration of micograb (1.5-200 μg / ml), or medium alone, was added to the wells. Plates were returned to the incubator for 48 hours and a cell titer blue assay was performed or viability measurements were performed using a hemocytometer.

Effect of anticancer agent 5FU and folinyl acid and oxaliplatin on HT29 cells Cell lines were fractionated and cells were counted. 100 μl of 4 × 10 ⁴ cells / ml or 4 × 10 ⁵ cells / ml was added to a 96 well flat bottom tissue culture plate and another 100 μl of complete McCoy's 5a medium was added to the plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. Then 100 μl of fresh complete RPMI medium containing 2-fold increasing concentration of test drug (5-FU4.5-2400 μg / ml + 1 mg / ml folinic acid or oxaliplatin 1-500 μg / ml), or medium alone (vs. 5-FU control) Medium + 2.5% DMSO) was added to the wells. Plates were returned to the incubator for 48 hours followed by a cell titer blue assay.

Effect of mycograb (RTM) in combination with anticancer agent 5-FU and folinyl acid or oxaliplatin on HT29 cells. Cell lines were fractionated and cells were counted. 100 μl of 4 × 10 ⁴ cells / ml or 4 × 10 ⁵ cells / ml was added to a 96 well flat bottom tissue culture plate and another 100 μl of complete McCoy's 5a medium was added to the plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. 100 μl of fresh RPMI medium is added to the plate and crossed of Micograb (RTM) vs. test drug ((5-FU4.5-2400 μg / ml + 1 mg / ml folinic acid or oxaliplatin 1-500 μg / ml) or medium alone (5- Medium + 2.5 DMSO relative to FU control)) was set up as outlined in Table 16 for a total volume of 200 μl per well.

Effect of mycograb (RTM) in combination with anticancer agent 5-FU and folinyl acid and oxaliplatin on HT29 cells. Cell lines were fractionated and cells were counted. 100 μl of 4 × 10 ⁴ cells / ml or 4 × 10 ⁵ cells / ml was added to a 96 well flat bottom tissue culture plate, and another 100 μl of complete McCoy's 5a medium was added to the plate. The plates were then incubated overnight at 37 ° C., 5% CO ₂ . The next day, the cells were observed under a phase contrast microscope to ensure they adhered to the plate and the supernatant medium was removed by aspiration. 100 μl of fresh RPMI medium is added to the plate and crossed with Micograb (RTM) vs. test drug ((5-FU: folinylate: oxaliplatin = 3: 1: 0.42) or medium alone (vs. 5-FU control) Medium + 2.5 DMSO)) was set as outlined in Table 16 for a total volume of 200 μl per well.

Results Effect of micograb (RTM) on HT29 cells In the cell line HT29, micograb (RTM) itself showed a 50% reduction in cell viability at 125 μg / ml.

Effect of micograb (RTM) on HT29 cells in combination with anticancer agent 5FU or oxaliplatin alone and in combination with anticancer agent 5FU or oxaliplatin
5-FU:
The IC ₅₀ of 5-fluorouracil was 150 μg / ml. There was a clear indication of synergy between 5-FU and micograb (RTM) over a wide range of drug concentrations. See Tables 17-20.

Oxaliplatin:
The IC ₅₀ of oxaliplatin was 16 μg / ml. There was a slight indication of synergy between oxaliplatin and micograb (RTM) over a wide range of drug concentrations. Table 18.

Effect of micograb (RTM) in combination with anticancer drug 5FU and oxaliplatin on HT29 cells The IC ₅₀ of LV / 5FU / Ox was 25/75 / 10.5 μg / ml. There was a slight indication of synergy between 5-FU and oxaliplatin and mycograb (RTM) over a wide range of drug concentrations. See Tables 22-24.

Summary From this result, the antibody was found to be synergistic with 5-FU and 5-FU at concentrations of 5-FU greater than 75 μg / ml and oxaliplatin greater than 10.5 μg / ml. It can be seen that and in combination with folinic acid and oxaliplatin showed slight signs of synergy with the four drugs. There were signs of synergy with 5-FU and oxaliplatin. Table 25 summarizes the CI values for ED ₅₀ , ED ₇₅ and ED ₉₀ .

CONCLUSION The data presented herein demonstrates that mycograb (RTM) antibodies themselves can reduce the viability of colon adenocarcinoma cell lines. There was a synergistic effect between anti-cancer drugs, including 5-fluorouracil and oxaliplatin, and anti-Hsp90 antibodies in colon adenocarcinoma cell lines. The data also demonstrate a synergistic effect between 5-fluorouracil and oxaliplatin with anti-HSP90 in colon adenocarcinoma lines.

Claims (33)

In a method for producing a medicament for the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin,
Use of. For simultaneous, separate or sequential use in the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin,
A combination preparation comprising:   Use or combination preparation according to any of claims 1 or 2, wherein the antibody or antigen-binding fragment thereof is specific for an epitope presented by a peptide having the sequence of SEQ ID NO: 1.   4. The use or combination preparation according to any one of claims 1 to 3, wherein the antibody comprises the sequence of SEQ ID NO: 2.   The cancer is selected from the group consisting of fibrosarcoma, breast cancer, prostate cancer, melanoma, leukemia, lymphoma, colon cancer, testicular germ cell, pancreatic cancer, ovarian cancer, endometrial tumor, thyroid cancer and lung cancer; Use or combination preparation according to any one of claims 1 to 4. A therapeutically effective amount for the patient in need
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin,
A method of treating cancer comprising administering In a method for producing a medicament for the treatment of leukemia,
(I) Use of an antibody or antigen-binding fragment thereof specific for at least one epitope of Hsp90. In a method for producing a medicament for the treatment of leukemia,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
Use of. For simultaneous, isolated or sequential use in the treatment of leukemia,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
A combination preparation comprising:   10. Use or combination preparation according to any one of claims 7 to 9, wherein the antibody or antigen-binding fragment thereof is specific for an epitope presented by a peptide having the sequence of SEQ ID NO: 1.   11. Use or combination preparation according to any one of claims 7 to 10, wherein the antibody comprises the sequence SEQ ID NO: 2.   The use or combination preparation according to any one of claims 7 to 11, wherein the leukemia is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia.   13. A private or combined preparation according to any one of claims 7 to 12, wherein the at least one anticancer agent is imatinib.   14. Use or combination preparation according to any one of claims 7 to 13, wherein the leukemia is chronic myeloid leukemia or acute lymphoblastic leukemia.   15. Use or combination preparation according to claim 14, wherein the leukemia is characterized by Philadelphia chromosome positive cells or Philadelphia chromosome positive negative cells.   15. Use or combination preparation according to claim 14, wherein the anticancer agent is imatinib and the leukemia is characterized by Philadelphia chromosome positive cells.   15. Use or combination preparation according to claim 14, wherein the anticancer agent is imatinib and the leukemia is characterized by Philadelphia chromosome negative cells.   18. Use or combination preparation according to any one of claims 7 to 17, wherein the leukemia is characterized by imatinib resistant cells. A therapeutically effective amount for the patient in need
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of imatinib, paclitaxel, docetaxel, daunorubicin, doxorubicin and hydroxyurea;
A method of treating leukemia, comprising administering   20. The method of claim 19, wherein the leukemia is chronic myelogenous leukemia and the at least one anticancer agent is imatinib. In a method for producing a medicament for the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
Use of. For simultaneous, separate or sequential use in the treatment of cancer,
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
A combination preparation comprising:   23. Use or combination preparation according to any of claims 21 or 22, wherein the antibody or antigen-binding fragment thereof is specific for an epitope presented by a peptide having the sequence of SEQ ID NO: 1.   24. Use or combination preparation according to any one of claims 21 to 23, wherein the antibody comprises the sequence SEQ ID NO: 2.   The cancer is selected from the group consisting of fibrosarcoma, breast cancer, prostate cancer, melanoma, leukemia, lymphoma, colon cancer, testicular germ cell, pancreatic cancer, ovarian cancer, endometrial tumor, thyroid cancer and lung cancer; Use or combination preparation according to any one of claims 21 to 24.   26. Use or combination preparation according to claim 25, wherein the cancer is colorectal cancer or adenocarcinoma. A therapeutically effective amount for the patient in need
(I) an antibody specific for at least one epitope of Hsp90 or an antigen-binding fragment thereof, and (ii) at least one anticancer agent selected from the group consisting of 5-fluorouracil, oxaliplatin, irinotecan and raltitrexide;
A method of treating cancer comprising administering   28. Use, combination preparation or method according to any one of claims 21 to 27, wherein the anticancer agent is 5-fluorouracil and further comprises folinic acid (leucovorin).   29. Use, combination preparation or method according to claim 28, wherein the anticancer agent comprises 5-fluorouracil, folinic acid (leucovorin) and oxaliplatin.   30. The method of any one of claims 6, 19, 20, 27, 28, and 29, wherein the composition or combination preparation is administered orally.   31. Use, combination preparation or method according to any one of claims 1 to 30, wherein the antibody or antigen-binding fragment thereof is labeled with a detectable label.   32. Use, combination preparation or method according to any one of claims 1-31, wherein the antibody or antigen-binding fragment thereof is conjugated to an effector molecule.   The invention as essentially described herein with reference to the examples.