Patent Application
20110091524
The present invention relates, in general, to methods for determining if a malignant or neoplastic cell in a subject or any medical condition in a subject mediated by IGF1R is sensitive to an IGF1R inhibitor.
The insulin-like growth factors, also known as somatomedins, include insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) (Klapper, et al., (1983) Endocrinol. 112:2215 and Rinderknecht, et al., (1978) Febs. Lett. 89:283). These growth factors exert mitogenic activity on various cell types, including tumor cells (Macaulay, (1992) Br. J. Cancer 65:311), by binding to a common receptor named the insulin-like growth factor receptor-1 (IGF1R) (Sepp-Lorenzino, (1998) Breast Cancer Research and Treatment 47:235). There are several available anti-cancer therapies which target IGF1R; however, due to factors including, e.g., individual genetic variability which can render a particular patient non-responsive to a given therapy some patients are not fully responsive to the therapy. The use of biomarkers for responsiveness to a given therapy is, thus, a useful tool for quickly and conveniently determining the responsiveness of a patient before a course of treatment is initiated. Biomarkers include, for example, the expression of a given gene or post-translational modification of a protein (e.g., phosphorylation) in a patient (e.g., in the cells of a cancer patient's tumor), e.g., at a level greater or less than that of a known responder or known non-responder.
Often, early, successful treatment of a given cancer is critical to the patient's clinical outcome. The use of biomarkers can aid in this process by quickly helping to identify treatments likely to be effective in a given patient and/or helping to eliminate treatments likely to be ineffective in a given patient.
Another benefit of the use of biomarkers relates to patient compliance. Patients assured that a given IGF1R inhibitor therapy will likely be effective against their specific tumor will exhibit an enhanced likelihood of continuing with the prescribed IGF1R inhibitor-based regimen over time.
The present invention provides a method for evaluating sensitivity of malignant or neoplastic cells (e.g., from an in vitro or in vivo source) to an IGF1R inhibitor (e.g., with about 70% certainty, e.g., about 72.5% or 75.7%) comprising determining if said cells exhibit high expression of one or more genes set forth in table 1 or low expression of one or more genes set forth in table 3 relative to that of a cell resistant to said inhibitor; wherein said cells are determined to be sensitive if said high expression or said low expression is observed. In an embodiment of the invention, the method comprises (a) obtaining a sample of one or more malignant or neoplastic cells from the body of a subject; (b) evaluating expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) or table 3 in the malignant or neoplastic cells; and (c) comparing said expression level to that of cells resistant to said IGF1R inhibitor; wherein the cells are determined to be sensitive to the inhibitor if expression of one or more genes in table 1 is higher than that of a cell resistant to said inhibitor or if expression of one or more genes in table 3 is lower than that of a cell resistant to said inhibitor. In an embodiment of the invention, the method further comprising administering a therapeutically effective dose of said inhibitor, optionally in association with a further therapeutic agent, to the body of a subject comprising said malignant or neoplastic cells if the cells are determined to be sensitive.
The present invention also provides a method for selecting a subject with malignant or neoplastic cells for treatment with an IGF1R inhibitor comprising evaluating sensitivity of the malignant or neoplastic cells to said inhibitor by the method for evaluating sensitivity discussed above; wherein said subject is selected if said cells are determined to be sensitive.
The present invention further provides a method for treating a tumor or cancerous condition with an IGF1R inhibitor comprising evaluating sensitivity of malignant or neoplastic cells, which are in said tumor or which mediate said cancerous condition, to said inhibitor by the method for evaluating sensitivity discussed above and, if said cells are determined to be sensitive, continuing or commencing treatment by administering, to the subject, a therapeutically effective dose of the inhibitor.
The present invention also provides a method for selecting a therapy for a subject with one or more malignant or neoplastic cells comprising evaluating sensitivity of the cells to an IGF1R inhibitor by the method for evaluating sensitivity discussed above; wherein said inhibitor is selected as the therapy if said cells are determined to be sensitive to the inhibitor.
The present invention further provides a method of advertising an IGF1R inhibitor or a pharmaceutically acceptable composition thereof or a therapeutic regimen comprising administration of said inhibitor or composition comprising promoting, to a target audience, the use of the inhibitor or composition for treating a patient or patient population whose tumors or cancerous conditions are mediated by malignant or neoplastic cells that exhibit increased expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 1, relative to cells resistant to said inhibitor.
The scope of the present invention further includes an article of manufacture comprising, packaged together, an IGF1R inhibitor or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent or pharmaceutical composition is indicated for treating patients having a tumor comprising malignant or neoplastic cells or a cancerous condition mediated by malignant or neoplastic cells that exhibit increased expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 3, relative to cells resistant to said inhibitor.
Also provided by the present invention is a method for manufacturing an IGF1R inhibitor or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier said method comprising combining, in a package, the inhibitor or composition; and a label conveying that the inhibitor or composition is indicated for treating patients having a tumor comprising malignant or neoplastic cells or a cancerous condition mediated by malignant or neoplastic cells that exhibit increased expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 3, relative to cells resistant to said inhibitor.
In an embodiment of any of the inventions discussed herein an IGF1R inhibitor is administered in association with a further chemotherapeutic agent. For example, a further therapeutic agent is, in an embodiment of the invention, any member selected from the group consisting of everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR, KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380,
SB-556629, chlamydocin, JNJ-16241199,
vorinostat, etoposide, gemcitabine, doxorubicin, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, irinotecan; a combination of irinotecan, 5-fluorouracil and leucovorin; PEG-labeled irinotecan, FOLFOX regimen, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258,
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(But) 6, Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 acetate [C59H84N18O14.(C2H4O2)x where x=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, sunitinib, sunitinib malate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, lonafarnib,
BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, hydroxyurea, idarubicin, ifosfamide, imatinib, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, cremophor-free paclitaxel, docetaxel, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, 5-fluorouracil, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa.
Also, in an embodiment of any of the inventions set forth herein, an IGF1R inhibitor is any member selected from the group consisting of any antibody or antigen-binding fragment thereof which binds specifically to IGF1R,
For example, such an antibody or fragment can, in an embodiment of the invention, comprise one or more complementarity determining regions (CDRs) selected from the group consisting of:
e.g., a light chain immunoglobulin comprising CDRs comprising the amino acid sequences of SEQ ID NOs: 99-101;
LGNFYYGMDV (SEQ ID NO: 104); e.g., a heavy chain immunoglobulin comprising a CDR comprising the amino acid sequence of SEQ ID NO: 102 or 107, a CDR comprising the amino acid sequence of SEQ ID NO: 103 and a CDR comprising the amino acid sequence of SEQ ID NO: 104;
or a mature fragment of a light chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8; and/or a mature fragment of a heavy chain immunoglobulin which comprises the amino acid sequence of SEQ ID NO: 10 or 12.
Embodiments of the present invention includes those wherein the malignant or neoplastic cells are in a tumor or mediate a cancerous condition which tumor or condition is selected from the group consisting of osteosarcoma, rhabdomyosarcoma, neuroblastoma, any pediatric cancer, kidney cancer, leukemia, renal transitional cell cancer, Werner-Morrison syndrome, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, benign prostatic hyperplasia, bone cancer, lung cancer, gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, tumor angiogenesis, head and neck cancer, squamous cell carcinoma, multiple myeloma, solitary plasmacytoma, renal cell cancer, retinoblastoma, germ cell tumors, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing Sarcoma, chondrosarcoma, haemotological malignancy, chronic lymphoblastic leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, chronic myeloblastic leukemia, Hodgekin's disease, non-Hodgekin's lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, hairy cell leukemia, mast cell leukemia, mast cell neoplasm, follicular lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, Burkitt Lymphoma, mycosis fungoides, seary syndrome, cutaneous T-cell lymphoma, chronic myeloproliferative disorders, a central nervous system tumor, brain cancer, glioblastoma, non-glioblastoma brain cancer, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, a myeloproliferative disorder, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, germ cell tumors and liver cancer.
Embodiments of the present invention also includes those wherein expression of one or more of said genes is identified by Northern blot analysis.
The present invention relates e.g., to methods for selecting patients for treatment with an IGF1R inhibitor. Such patients comprise one or more malignant or neoplastic cells and, in an embodiment of the invention, suffer from a disease or medical condition which is mediated by such a malignant or neoplastic cell. Malignant or neoplastic cells include, for example, cancerous cells. Malignant cells include, for example, cells exhibiting anaplasia, metastasis, invasiveness, tendency to form a tumor and/or a tendency to lead to death (e.g., due to cancer caused by a tumor including such malignant cells). Neoplastic cells include, for example, cells which abnormally divide at a supra-normal level (e.g., high numbers of lifetime divisions or division at a high rate) and/or to exhibit low mortality or immortality. In an embodiment of the invention, said malignant and/or neoplastic properties are mediated by IGF1R activity or expression in the cell.
The term “subject” or “patient” includes any animal including, e.g., a mammal such as a human.
The term IGF1R inhibitor resistant cell or the like includes any cell that is resistant to an IGF1R inhibitor, e.g., with respect to its growth and/or proliferation and/or survival. For example, in an embodiment of the invention, an IGF1R inhibitor sensitive cell or cell line exhibits 50% or more tumor growth inhibition (e.g., reduction in tumor volume and/or tumor mass) in a mouse xenograft system (wherein the tested cells form the tumor) wherein, when the inhibitor is an anti-IGF1R antibody or antigen-binding fragment thereof, the mouse is administered 0.5 mg of antibody or fragment twice a week for about 3 weeks. In an embodiment of the invention, the cell is resistant if less than 50% in vivo tumor growth inhibition is exhibited. In an embodiment of the invention, a cell or cell line is sensitive to an IGF1R inhibitor if, in vitro, the cell or cell line exhibits 30% or more growth inhibition, wherein, when the inhibitor is an anti-IGF1R antibody or antigen-binding fragment thereof, the cell or cell line is exposed to about 20 nM to about 100 nm of the antibody or fragment, e.g., by a luminescent cell viability assay such as a CellTiter Glo assay. In an embodiment of the invention, the cell or cell line is resistant when it exhibits less than 30% in vitro growth inhibition.
The terms “IGF1R inhibitor” or “IGF1R antagonist” or the like include any substance that decreases the expression, ligand binding (e.g., binding to IGF-1 and/or IGF-2), kinase activity (e.g., autophosphorylation activity) or any other biological activity of IGF1R (e.g., mediation of anchorage-independent cellular growth) e.g., that will elicit a biological or medical response of a tissue, system, subject or patient that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes any measurable alleviation of the signs, symptoms and/or clinical indicia of cancer (e.g., tumor growth) and/or the prevention, slowing or halting of progression or metastasis of cancer to any degree.
In an embodiment of the invention, the IGF1R inhibitor is any isolated antibody or antigen-binding fragment thereof that binds specifically to insulin-like growth factor-1 receptor (e.g., human IGF1R) or any soluble fragment thereof (e.g., monoclonal antibodies (e.g., fully human monoclonal antibodies), polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single chain Fv antibody fragments, dsFv antibody fragments, humanized antibodies or chimeric antibodies) such as any of those disclosed in any of Burtrum et. al Cancer Research 63:8912-8921 (2003); in French Patent Applications FR2834990, FR2834991 and FR2834900 and in PCT Application Publication Nos. WO 03/100008; WO 03/59951; WO 04/71529; WO 03/106621; WO 04/83248; WO 04/87756, WO 05/16970; and WO 02/53596.
In an embodiment of the invention, an IGF1R inhibitor is an isolated anti-insulin-like growth factor-1 receptor (IGF1R) antibody comprising a mature 19D12/15H12 Light Chain (LC)-C, D, E or F and a mature 19D12/15H12 heavy chain (HC)-A or B (e.g., mature LCB/mature HCB, mature LCC/mature HCB or mature LCF/mature HCA). In an embodiment of the invention, an IGF1R inhibitor that is administered to a patient in a method according to the invention is an isolated antibody that specifically binds to IGF1R that comprises one or more complementarity determining regions (CDRs) of 19D12/15H12 Light Chain-C, D, E or F and/or 19D12/15H12 heavy chain-A or B (e.g., all 3 light chain CDRs and/or all 3 heavy chain CDRs).
The amino acid and nucleotide sequences of the some antibody chains of the invention are shown below. Dotted, underscored type indicates the signal peptide. Solid underscored type indicates the CDRs. Plain type indicates the framework regions. Mature fragments lack the signal peptide.
Plasmids comprising a CMV promoter operably linked to the 15H12/19D12 light chains and heavy chains have been deposited at the American Type Culture Collection (ATCC); 10801 University Boulevard; Manassas, Va. 20110-2209 on May 21, 2003. The deposit name and the ATCC accession numbers for the cell lines are set forth below:
CMV promoter-15H12/19D12 LCC (κ)—
Deposit name: “15H12/19D12 LCC (κ)”;
ATCC accession No.: PTA-5217
CMV promoter-15H12/19D12 LCD (κ)—
Deposit name: “15H12/19D12 LCD (κ)”;
ATCC accession No.: PTA-5218
CMV promoter-15H12/19D12 LCE (κ)—
Deposit name: “15H12/19D12 LCE (κ)”;
ATCC accession No.: PTA-5219
CMV promoter-15H12/19D12 LCF (κ)—
Deposit name: “15H12/19D12 LCF (κ)”;
ATCC accession No.: PTA-5220
CMV promoter-15H12/19D12 HCA (γ4)—
Deposit name: “15H12/19D12 HCA (γ4)”
ATCC accession No.: PTA-5214
CMV promoter-15H12/19D12 HCB (γ4)—
Deposit name: “15H12/19D12 HCB (γ4)”
ATCC accession No.: PTA-5215
CMV promoter-15H12/19D12 HCA (γ1)—
Deposit name: “15H12/19D12 HCA (γ1)”;
ATCC accession No.: PTA-5216
The present invention includes methods and compositions (e.g., any disclosed herein) comprising anti-IGF1R antibodies and antigen-binding fragments thereof comprising any of the light and/or heavy immunoglobulin chains or mature fragments thereof located in any of the foregoing plasmids deposited at the ATCC.
In an embodiment of the invention, the IGF1R inhibitor is an isolated antibody or antigen-binding fragment thereof comprising one or more (e.g., 3) of the following CDR sequences:
For example, in an embodiment of the invention, a light chain immunoglobulin comprises 3 CDRs and/or a heavy chain immunoglobulin comprises 3 CDRs.
In an embodiment, an antibody that binds “specifically” to human IGF1R binds with a Kd of about 10−8 M or 10−7 M or a lower number; or, in an embodiment of the invention, with a Kd of about 1.28×10−10 M or a lower number by Biacore measurement or with a Kd of about 2.05×10−12 or a lower number by KinExA measurement. In another embodiment, an antibody that binds “specifically” to human IGF1R binds exclusively to human IGF1R and to no other protein at significant or at detectable levels.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2002/53596 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 47 and 51 as set forth in WO 2002/53596 and/or a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 45 and 49 as set forth in WO 2002/53596. In an embodiment, the antibody comprises a heavy and/or light chain selected from that of antibody 2.12.1; 2.13.2; 2.14.3; 3.1.1; 4.9.2; and 4.17.3 in WO 2002/53596.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2003/59951 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 61 and 65 as set forth in WO 2003/59951 and/or a heavy chain variable region comprising an amino acids sequence selected from the group consisting of SEQ ID NOs: 69, 75, 79 and 83 as set forth in WO 2003/59951.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2004/83248 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 and 143 as set forth in WO 2004/83248 and/or a heavy chain variable region comprising an amino acids sequence selected from the group consisting of SEQ ID NOs: 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140 and 142 as set forth in WO 2004/83248. In an embodiment, the antibody comprises a light and/or heavy chain selected from that of PINT-6A1; PINT-7A2; PINT-7A4; PINT-7A5; PINT-7A6; PINT-8A1; PINT-9A2; PINT-11A1; PINT-11A2; PINT-11A3; PINT-11A4; PINT-11A5; PINT-11A7; PINT-12A1; PINT-12A2; PINT-12A3; PINT-12A4 and PINT-12A5 in WO 2004/83248.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2003/106621 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-12, 58-69, 82-86, 90, 94, 96, 98, as set forth in WO 2003/106621 and/or a heavy chain variable region comprising an amino acids sequence selected from the group consisting of SEQ ID NOs: 7, 13, 70-81, 87, 88, 92 as set forth in WO 2003/106621.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2004/87756 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 2 as set forth in WO 2004/87756 and/or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 1 as set forth in WO 2004/87756.
In an embodiment of the invention, the IGF1R inhibitor comprises any light chain immunoglobulin and/or a heavy chain immunoglobulin as set forth in Published International Application No. WO 2005/16970 which is herein incorporated by reference in its entirety. For example, in an embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO: 6 or 10 as set forth in WO 2005/16970 and/or a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2 as set forth in WO 2005/16970.
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region comprising an amino acid sequence selected from the group consisting of:
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain variable region comprising an amino acid sequence selected from the group consisting of:
In an embodiment of the invention, the anti-IGF1R antibody comprises a light chain immunoglobulin, or a mature fragment thereof (i.e., lacking signal sequence), or variable region thereof, comprising the amino acid sequence of:
wyqq kpgkapkrli y
gv psrfsgsgsg teftltissl
qpedfatyyc
f gqgtkveikr tvaapsvfif ppsdeqlksg
wyqq kpgkapkrli y
gv psrfsgsgsg teftltissl
qpedfatyyc
f gqgteveiir tvaapsvfif ppsdeqlksg
wyqq kpgkapkrli y
gv psrfsgsgsg teftltissl
qpedfatyyc
f gqgtkleikr tvaapsvfif ppsdeqlksg
wyqq kpgkapkrli y
gv psrfsgsgsg teftltissl
qpedfatyyc
f gqgtkleikr tvaapsvfif ppsdeqlksg
In an embodiment of the invention, the signal sequence is amino acids 1-22 of SEQ ID NOs: 25-28. In an embodiment of the invention, the mature variable region is underscored. In an embodiment of the invention, the CDRs are in bold/italicized font. In an embodiment of the invention, the anti-IGF1R antibody or antigen-binding fragment thereof of the invention comprises one or more CDRs (e.g., 3 light chain CDRS) as set forth above.
In an embodiment of the invention, the anti-IGF1R antibody comprises a heavy chain immunoglobulin or a mature fragment thereof (i.e., lacking signal sequence), or a variable region thereof, comprising the amino acid sequence of:
wirqap gkglewvs
rftis rdnaknslyl
qmnslraedt avyycar
wgqg ttvtvssast
wirqap gkglewvs
rftis rdnaknslyl
qmnslraedt avyycvr
wgqgttv tvssastkgp
wvrqap gkglewvs
rftis rdnskntlyl
qmnslraedt avyycak
wgqgttv tvssastkgp
wvrqap gkglewvs
rftis rdnsrttlyl
qmnslraedt avyycak
wgqgttv tvssastkgp
In an embodiment of the invention, the signal sequence is amino acids 1-19 of SEQ ID NOs: 29-32. In an embodiment of the invention, the mature variable region is underscored. In an embodiment of the invention, the anti-IGF1R antibody or antigen-binding fragment thereof of the invention comprises one or more CDRs (e.g., 3 light chain CDRS) as set forth above.
In an embodiment of the invention, the anti-IGF1R antibody comprises a light chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 19-24 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 13-18, respectively. In an embodiment of the invention, the anti-IGF1R antibody comprises a mature light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 25 or 26 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 29 or 30. In an embodiment of the invention, the anti-IGF1R antibody comprises a mature light chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 27 or 28 paired with a heavy chain variable region comprising an amino acid sequence of any of SEQ ID NOs: 31 or 32.
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain or mature fragment or variable region of 2.12.1 fx (SEQ ID NO: 33) (in an embodiment of the invention, the leader sequence is underscored; in an embodiment of the invention, the CDRs are in bold/italicized font):
mefglswvfl vaiikgvqcq vqlvesgggl vkpggslrls caas
wirqap gkglewvs
rftis rdnaknslyl
wgqgttv tvssastkgp
In an embodiment of the invention, the anti-IGF1R antibody or antigen-binding fragment thereof comprises amino acids 20-470 of 2.12.1 fx (SEQ ID NO: 33).
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises mature immunoglobulin heavy chain variable region 2.12.1 fx (amino acids 20-144 or SEQ ID NO: 33; SEQ ID NO: 34):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises an immunoglobulin light chain or mature fragment or variable region 2.12.1 fx (SEQ ID NO: 35) (in an embodiment of the invention, the leader sequence is underscored; in an embodiment of the invention, the CDRs are in bold/italicized font):
mdmrvpaqll gllllwfpga rcdiqmtqsp sslsasvgdr vtitc
wyqq kpgkapkrli y
gv psrfsgsgsg teftltissl
f gqgtkveikr tvaapsvfif ppsdeqlksg
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises amino acids 23-236 of 2.12.1 fx (SEQ ID NO: 35).
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises mature immunoglobulin light chain variable region 2.12.1 fx (amino acids 23-130 of SEQ ID NO: 35; SEQ ID NO: 36):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises or consists of a light chain immunoglobulin chain comprising or consisting of amino acids 23-236 of 2.12.1 fx (SEQ ID NO: 35) and a heavy chain immunoglobulin chain comprising or consisting of amino acids 20-470 of 2.12.1 fx (SEQ ID NO: 33).
In an embodiment of the invention, the anti-IGF1R antibody or antigen-binding fragment thereof comprises one or more 2.12.1 fx CDRs (e.g., 3 light chain CDRs and/or 3 heavy chain CDRs) as set forth above.
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof or antigen-binding fragment thereof comprises a humanized 7C10 immunoglobulin light chain variable region; version 1 (SEQ ID NO: 37):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises humanized 7C10 immunoglobulin light chain variable region; version 2 (SEQ ID NO: 38):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises a humanized 7C10 immunoglobulin heavy chain variable region; version 1 (SEQ ID NO: 39):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises the humanized 7C10 immunoglobulin heavy chain variable region; version 2 (SEQ ID NO: 40):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises the humanized 7C10 immunoglobulin heavy chain variable region; version 3 (SEQ D NO: 41):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises A12 immunoglobulin heavy chain variable region (SEQ ID NO: 42):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises A12 immunoglobulin light chain variable region (SEQ ID NO: 43):
or
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises 1A immunoglobulin heavy chain variable region (SEQ ID NO: 44):
; optionally including one or more of the following mutations: R30, S30, N31, S31, Y94, H94, D104, E104.
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises 1A immunoglobulin light chain variable region (SEQ ID NO: 45):
; optionally including one or more of the following mutations: P96, I96, P100, Q100, R103, K103, V104, L104, D105, E105
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 8A1 (SEQ ID NO: 46):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 9A2 (SEQ ID NO: 47):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 11A4 (SEQ ID NO: 48):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 7A4 (SEQ ID NO: 49):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 11A1 (SEQ ID NO: 50):
In an embodiment of the invention, an anti-IGF1R antibody or antigen-binding fragment thereof comprises single chain antibody (fv) 7A6 (SEQ ID NO: 51)
In an embodiment of the invention, an anti-IGF1R antibody or an antigen-binding fragment thereof (e.g., a heavy chain or light chain immunoglobulin) comprises one or more complementarity determining regions (CDR) selected from the group consisting of:
In an embodiment of the invention, an anti-IGF1R antibody or an antigen-binding fragment thereof comprises a heavy chain immunoglobulin variable region selected from the group consisting of:
The scope of the present invention includes embodiments wherein the variable region of an anti-IGF1R antibody is linked to any immunoglobulin constant region. In an embodiment, the light chain variable region is linked to a κ chain constant region. In an embodiment, the heavy chain variable region is linked to a γ1, γ2, γ3 or γ4 chain constant region. Any of the immunoglobulin variable regions set forth herein, in embodiments of the invention, can be linked to any of the foregoing constant regions.
Furthermore, the scope of the present invention comprises any antibody or antibody fragment comprising one or more CDRs (3 light chain CDRs and/or 3 heavy chain CDRs) and/or framework regions of any of the light chain immunoglobulin or heavy chain immunoglobulins set forth herein as identified by any of the methods set forth in Chothia et al., J. Mol. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985) or Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)).
In an embodiment of the invention, the term “monoclonal antibody,” as used herein, includes an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they may be synthesized by a hybridoma culture, essentially uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As mentioned above, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method described by Kohler, et al., (1975) Nature 256: 495.
In an embodiment of the invention, a polyclonal antibody is an antibody which was produced among or in the presence of one or more other, non-identical antibodies. In general, polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes which produced non-identical antibodies. Usually, polyclonal antibodies are obtained directly from an immunized animal.
In an embodiment of the invention, a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992) J. Immunol. 148:1547-1553. In addition, bispecific antibodies may be formed as “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as “Janusins” (Traunecker, et al., (1991) EMBO J. 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).
In an embodiment of the invention, the term “fully human antibody” refers to an antibody which comprises human immunoglobulin protein sequences only (lacking non-human sequences). A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” refers to an antibody which comprises mouse immunoglobulin protein sequences only.
The present invention includes “chimeric antibodies”; in an embodiment of the invention, an antibody which comprises a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken). These antibodies may be used e.g., to modulate the expression or activity of IGF1R in a non-human species.
“Single-chain Fv” or “sFv” antibody fragments have, in an embodiment of the invention, the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. Techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786; 5,132,405 and 4,946,778) can be adapted to produce anti-IGF1R-specific single chain antibodies. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).
In an embodiment of the invention, “disulfide stabilized Fv fragments” and “dsFv” refer to immunoglobulins comprising a variable heavy chain (VH) and a variable light chain (VL) which are linked by a disulfide bridge.
Antigen-binding fragments of antibodies within the scope of the present invention also include F(ab)2 fragments which may, in an embodiment of the invention, be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)2 with dithiothreitol or mercaptoethylamine. A Fab fragment is, in an embodiment of the invention, a VL-CL chain appended to a VH-CH1 chain by a disulfide bridge. A F(ab)2 fragment is, in an embodiment of the invention, two Fab fragments which, in turn, are appended by two disulfide bridges. The Fab portion of an F(ab)2 molecule includes, in an embodiment of the invention, a portion of the Fc region between which disulfide bridges are located.
In an embodiment of the invention, an FV fragment is a VL or VH region.
Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. As discussed herein, any such antibody or antigen-binding fragment thereof is within the scope of the present invention.
The anti-IGF1R antibodies of the invention may, in an embodiment of the invention, be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionuclide or a cytotoxic factor. In an embodiment of the invention, the chemical moiety is a polymer which increases the half-life of the antibody or antigen-binding fragment thereof in the body of a subject. Polymers include, but are not limited to, polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).
The antibodies and antibody fragments may, in an embodiment of the invention, be conjugated with labels such as 99Tc, 90Y, 111In, 32P, 14C, 125I, 3H, 131I, 11C, 15O, 13N, 18F, 35S, 51Cr, 57To, 226Ra, 60Co, 59Fe, 57Se, 152Eu, 67Cu, 217Ci, 211At, 212Pb, 47Sc, 109Pd, 234Th, and 40K, 157Gd, 55Mn, 52Tr, and 56Fe.
The antibodies and antibody fragments may also be, in an embodiment of the invention, conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, 152Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.
The antibodies and antibody fragments may also be, in an embodiment of the invention, conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.
Any method known in the art for conjugating the antibodies or antigen-binding fragments thereof of the invention to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies are conventional and very well known in the art.
In an embodiment of the invention, an IGF1R inhibitor is
In an embodiment of the invention, an IGF1R inhibitor is provided in association with erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR, KRX-0402, lucanthone, LY 317615, neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763 or AT-9263.
Abraxane is an injectable suspension of paclitaxel protein-bound particles comprising an albumin-bound form of paclitaxel with a mean particle size of approximately 130 nanometers. Abraxane is supplied as a white to yellow, sterile, lyophilized powder for reconstitution with 20 mL of 0.9% Sodium Chloride Injection, USP prior to intravenous infusion. Each single-use vial contains 100 mg of paclitaxel and approximately 900 mg of human albumin. Each milliliter (mL) of reconstituted suspension contains 5 mg paclitaxel. Abraxane is free of solvents and is free of cremophor (polyoxyethylated castor oil).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with romidepsin
chlamydocin
or vorinostat
In an embodiment of the invention, an IGF1R inhibitor is provided in association with etoposide
In an embodiment of the invention, an IGF1R inhibitor is provided in association with gemcitabine
In an embodiment of the invention, an IGF1R inhibitor is provided in association with any compound disclosed in published U.S. patent application no. U.S. 2004/0209878A1 (e.g., comprising a core structure represented by
or doxorubicin
including Caelyx or Doxil® (doxorubicin HCl liposome injection; Ortho Biotech Products L.P; Raritan, N.J.). Doxil® comprises doxorubicin in STEALTH® liposome carriers which are composed of N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-DSPE); fully hydrogenated soy phosphatidylcholine (HSPC), and cholesterol.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with 5′-deoxy-5-fluorouridine
In an embodiment of the invention, an IGF1R inhibitor is provided in association with vincristine
In an embodiment of the invention, an IGF1R inhibitor is provided in association with temozolomide
any CDK inhibitor such as ZK-304709, Seliciclib (R-roscovitine)
any MEK inhibitor such as PD0325901
AZD-6244; capecitabine (5′-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine); or L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate
Pemetrexed disodium heptahydrate).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with camptothecin
Stork et al., J. Am. Chem. Soc. 93(16): 4074-4075 (1971); Beisler et al., J. Med. Chem. 14(11): 1116-1117 (1962)), irinotecan
sold as Camptosar®; Pharmacia & Upjohn Co.; Kalamazoo, Mich.); a combination of irinotecan, 5-fluorouracil and leucovorin; or PEG-labeled irinotecan.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with the FOLFOX regimen (oxaliplatin
together with infusional fluorouracil
and folinic acid
(Chaouche et al., Am. J. Clin. Oncol. 23(3):288-289 (2000); de Gramont et al., J. Clin. Oncol. 18(16):2938-2947 (2000)).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with an antiestrogen such as
(tamoxifen; sold as Nolvadex® by AstraZeneca Pharmaceuticals LP; Wilmington, Del.) or
(toremifene citrate; sold as Fareston® by Shire US, Inc.; Florence, Ky.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with an aromatase inhibitor such as
(anastrazole; sold as Arimidex® by AstraZeneca Pharmaceuticals LP; Wilmington, Del.),
(exemestane; sold as Aromasin® by Pharmacia Corporation; Kalamazoo, Mich.) or
(letrozole; sold as Femara® by Novartis Pharmaceuticals Corporation; East Hanover, N.J.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with an estrogen such as DES (diethylstilbestrol),
(estradiol; sold as Estrol® by Warner Chilcott, Inc.; Rockaway, N.J.) or conjugated estrogens (sold as Premarin® by Wyeth Pharmaceuticals Inc.; Philadelphia, Pa.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with anti-angiogenesis agents including bevacizumab (Avastin™; Genentech; San Francisco, Calif.), the anti-VEGFR-2 antibody IMC-1C11, other VEGFR inhibitors such as: CHIR-258
any of the inhibitors set forth in WO2004/13145 (e.g., comprising the core structural formula:
WO2004/09542 (e.g., comprising the core structural formula:
WO00/71129 (e.g., comprising the core structural formula:
WO2004/09601 (e.g., comprising the core structural formula:
WO2004/01059 (e.g., comprising the core structural formula:
WO01/29025 (e.g., comprising the core structural formula:
WO02/32861 (e.g., comprising the core structural formula:
or set forth in WO03/88900 (e.g., comprising the core structural formula
3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone; Vatalanib
and the VEGF trap (AVE-0005), a soluble decoy receptor comprising portions of VEGF receptors 1 and 2.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with a LHRH (Lutenizing hormone-releasing hormone) agonist such as the acetate salt of [D-Ser(But) 6, Azgly 10] (pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 acetate [C59H84N18O14.(C2H4O2)x where x=1 to 2.4];
(goserelin acetate; sold as Zoladex® by AstraZeneca UK Limited; Macclesfield, England),
(leuprolide acetate; sold as Eligard® by Sanofi-Synthelabo Inc.; New York, N.Y.) or
(triptorelin pamoate; sold as Trelstar® by Pharmacia Company, Kalamazoo, Mich.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with sunitinib or sunitinib malate
In an embodiment of the invention, an IGF1R inhibitor is provided in association with a progestational agent such as
(medroxyprogesterone acetate; sold as Provera® by Pharmacia & Upjohn Co.; Kalamazoo, Mich.),
(hydroxyprogesterone caproate; 17-((1-Oxohexyl)oxy)pregn-4-ene-3,20-dione;), megestrol acetate or progestins.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with selective estrogen receptor modulator (SERM) such as
(raloxifene; sold as Evista® by Eli Lilly and Company; Indianapolis, Ind.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with an anti-androgen including, but not limited to:
(bicalutamide; sold at CASODEX® by AstraZeneca Pharmaceuticals LP; Wilmington, Del.);
(flutamide; 2-methyl-N-[4-nitro-3 (trifluoromethyl)phenyl] propanamide; sold as Eulexin® by Schering Corporation; Kenilworth, N.J.);
(nilutamide; sold as Nilandron® by Aventis Pharmaceuticals Inc.; Kansas City, Mo.) and
(Megestrol acetate; sold as Megace® by Bristol-Myers Squibb).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with one or more inhibitors which antagonize the action of the EGF Receptor or HER2, including, but not limited to,
erlotinib, Hidalgo et al., J. Clin. Oncol. 19(13): 3267-3279 (2001)), Lapatanib
GW2016; Rusnak et al., Molecular Cancer Therapeutics 1:85-94 (2001); N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsulfonypethyl]amino}methyl)-2-furyl]-4-quinazolinamine; PCT Application No. WO99/35146), Canertinib
Erlichman et al., Cancer Res. 61(2):739-48 (2001); Smaill et al., J. Med. Chem. 43(7):1380-97 (2000)), ABX-EGF antibody (Abgenix, Inc.; Freemont, Calif.; Yang et al., Cancer Res. 59(6):1236-43 (1999); Yang et al., Crit. Rev Oncol Hematol. 38(1):17-23 (2001)), erbitux (U.S. Pat. No. 6,217,866; IMC-C225, cetuximab; Imclone; New York, N.Y.), EKB-569
Wissner et al., J. Med. Chem. 46(1): 49-63 (2003)), PKI-166
CGP-75166), GW-572016, any anti-EGFR antibody and any anti-HER2 antibody.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with:
(Ionafarnib; Sarasar™; Schering-Plough; Kenilworth, N.J.). In another embodiment, one of the following FPT inhibitors is provided in association with an IGF1R inhibitor:
Other FPT inhibitors, that can be provided in association with an IGF1R inhibitor include BMS-214662
Hunt et al., J. Med. Chem. 43(20):3587-95 (2000); Dancey et al., Curr. Pharm. Des. 8:2259-2267 (2002); (R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine)) and R155777 (tipifarnib; Garner et al., Drug Metab. Dispos. 30(7):823-30 (2002); Dancey et al., Curr. Pharm. Des. 8:2259-2267 (2002); (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone];
sold as Zarnestra™; Johnson & Johnson; New Brunswick, N.J.).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with
(suberoyl analide hydroxamic acid),
(Valproic acid; Michaelis et al., Mol. Pharmacol. 65:520-527 (2004)),
(trichostatin A),
(SU11248; Mendel et al., Clin. Cancer Res. 9(1):327-37 (2003)),
(BAY43-9006; sorafenib),
(Anastrozole; sold as Arimidex by AstraZeneca Pharmaceuticals LP; Wilmington, Del.); Asparaginase; Bacillus Calmette-Guerin (BCG) vaccine (Gamido et al., Cytobios. 90(360):47-65 (1997));
(Busulfan; 1,4-butanediol, dimethanesulfonate; sold as Busulfex® by ESP Pharma, Inc.; Edison, N.J.);
(Carboplatin; sold as Paraplatin® by Bristol-Myers Squibb; Princeton, N.J.);
(Imatinib; sold as Gleevec® by Novartis Pharmaceuticals Corporation; East Hanover, N.J.);
(Melphalan; sold as Alkeran® by Celgene Corporation; Warren, N.J.);
octreotide
Katz et al., Clin Pharm. 8(4):255-73 (1989); sold as Sandostatin LAR® Depot; Novartis Pharm. Corp; E. Hanover, N.J.); edotreotide (yttrium-90 labeled or unlabeled); oxaliplatin
sold as Eloxatin™ by Sanofi-Synthelabo Inc.; New York, N.Y.);
(Pamidronate; sold as Aredia® by Novartis Pharmaceuticals Corporation; East Hanover, N.J.);
(Pentostatin; sold as Nipent® by Supergen; Dublin, Calif.);
(Porfimer; sold as Photofrin® by Axcan Scandipharm Inc.; Birmingham, Ala.);
Rituximab (sold as Rituxan® by Genentech, Inc.; South San Francisco, Calif.);
or 13-cis-retinoic acid
In an embodiment of the invention, an IGF1R inhibitor is provided in association with one or more of any of: phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5-deooxyuridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, vairubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin, diftitox, gefitinib, bortezimib, paclitaxel, docetaxel, epithilone B, BMS-247550 (see e.g., Lee et al., Clin. Cancer Res. 7:1429-1437 (2001)), BMS-310705, droloxifene (3-hydroxytamoxifen), 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene (CP-336156), idoxifene, TSE-424, HMR-3339, ZK186619, topotecan, PTK787/ZK 222584 (Thomas et al., Semin Oncol. 30(3 Suppl 6):32-8 (2003)), the humanized anti-VEGF antibody Bevacizumab, VX-745 (Haddad, Curr Opin. Investig. Drugs 2(8):1070-6 (2001)), PD 184352 (Sebolt-Leopold, et al. Nature Med. 5: 810-816 (1999)), any mTOR inhibitor, rapamycin
sirolimus), 40-O-(2-hydroxyethyl)-rapamycin, CCI-779
temsirolimus; Sehgal et al., Med. Res. Rev., 14:1-22 (1994); Elit, Curr. Opin. Investig. Drugs 3(8):1249-53 (2002)),
LY294002, LY292223, LY292696, LY293684, LY293646 (Vlahos et al., J. Biol. Chem. 269(7): 5241-5248 (1994)), wortmannin, BAY-43-9006, (Wilhelm et al., Curr. Pharm. Des. 8:2255-2257 (2002)), ZM336372, L-779,450, any Raf inhibitor disclosed in Lowinger et al., Curr. Pharm Des. 8:2269-2278 (2002); flavopiridol (L86-8275/HMR 1275; Senderowicz, Oncogene 19(56): 6600-6606 (2000)) or UCN-01 (7-hydroxy staurosporine; Senderowicz, Oncogene 19(56): 6600-6606 (2000)).
In an embodiment of the invention, an IGF1R inhibitor is provided in association with one or more of any of the compounds set forth in U.S. Pat. No. 5,656,655, which discloses styryl substituted heteroaryl EGFR inhibitors; in U.S. Pat. No. 5,646,153 which discloses bis mono and/or bicyclic aryl heteroaryl carbocyclic and heterocarbocyclic EGFR and PDGFR inhibitors; in U.S. Pat. No. 5,679,683 which discloses tricyclic pyrimidine compounds that inhibit the EGFR; in U.S. Pat. No. 5,616,582 which discloses quinazoline derivatives that have receptor tyrosine kinase inhibitory activity; in Fry et al., Science 265 1093-1095 (1994) which discloses a compound having a structure that inhibits EGFR (see FIG. 1 of Fry et al.); in U.S. Pat. No. 5,196,446 which discloses heteroarylethenediyl or heteroarylethenediylaryl compounds that inhibit EGFR; in Panek, et al., Journal of Pharmacology and Experimental Therapeutics 283: 1433-1444 (1997) which disclose a compound identified as PD166285 that inhibits the EGFR, PDGFR, and FGFR families of receptors-PD166285 is identified as 6-(2,6-dichlorophenyl)-2-(4-(2-diethylaminoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3-d)pyrimidin-7-one.
In an embodiment of the invention, an IGF1R inhibitor is provided in association with one or more of any of: pegylated or unpegylated interferon alfa-2a, pegylated or unpegylated interferon alfa-2b, pegylated or unpegylated interferon alfa-2c, pegylated or unpegylated interferon alfa n−1, pegylated or unpegylated interferon alfa n−3 and pegylated, unpegylated consensus interferon or albumin-interferon-alpha.
The term “interferon alpha” as used herein means the family of highly homologous species-specific proteins that inhibit cellular proliferation and modulate immune response. Typical suitable interferon-alphas include, but are not limited to, recombinant interferon alpha-2b, recombinant interferon alpha-2a, recombinant interferon alpha-2c, alpha 2 interferon, interferon alpha-n1 (INS), a purified blend of natural alpha interferons, a consensus alpha interferon such as those described in U.S. Pat. Nos. 4,897,471 and 4,695,623 (especially Examples 7, 8 or 9 thereof), or interferon alpha-n3, a mixture of natural alpha interferons.
Interferon alfa-2a is sold as ROFERON-A® by Hoffmann-La Roche (Nutley, N.J.).
Interferon alfa-2b is sold as INTRON-A® by Schering Corporation (Kenilworth, N.J.). The manufacture of interferon alpha 2b is described, for example, in U.S. Pat. No. 4,530,901.
Interferon alfa-n3 is a mixture of natural interferons sold as ALFERON N INJECTION® by Hemispherx Biopharma, Inc. (Philadelphia, Pa.).
Interferon alfa-n1 (INS) is a mixture of natural interferons sold as WELLFERON® by Glaxo-Smith-Kline (Research Triangle Park, N.C.).
Consensus interferon is sold as INFERGEN® by Intermune, Inc. (Brisbane, Calif.).
Interferon alfa-2c is sold as BEROFOR® by Boehringer Ingelheim Pharmaceutical, Inc. (Ridgefield, Conn.).
A purified blend of natural interferons is sold as SUMIFERON® by Sumitomo; Tokyo, Japan.
The term “pegylated interferon alpha” as used herein means polyethylene glycol modified conjugates of interferon alpha, preferably interferon alpha-2a and alpha-2b. The preferred polyethylene-glycol-interferon alpha-2b conjugate is PEG 12000-interferon alpha-2b. The phrases “12,000 molecular weight polyethylene glycol conjugated interferon alpha” and “PEG 12000-IFN alpha” as used herein include conjugates such as are prepared according to the methods of International Application No. WO 95/13090 and EP1039922 and containing urethane linkages between the interferon alpha-2a or -2b amino groups and polyethylene glycol having an average molecular weight of 12000. The pegylated inteferon alpha, PEG 12000-IFN-alpha-2b is available from Schering-Plough, Kenilworth, N.J.
Pegylated interferon alfa-2b is sold as PEG-INTRON® by Schering Corporation (Kenilworth, N.J.).
Pegylated interferon-alfa-2a is sold as PEGASYS® by Hoffmann-La Roche (Nutley, N.J.).
Other interferon alpha conjugates can be prepared by coupling an interferon alpha to a water-soluble polymer. A non-limiting list of such polymers includes other polyalkylene oxide homopolymers such as polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. As an alternative to polyalkylene oxide-based polymers, effectively non-antigenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides, polyvinyl alcohols, carbohydrate-based polymers and the like can be used. Such interferon alpha-polymer conjugates are described, for example, in U.S. Pat. No. 4,766,106, U.S. Pat. No. 4,917,888, European Patent Application No. 0 236 987 or 0 593 868 or International Publication No. WO 95/13090.
Pharmaceutical compositions of pegylated interferon alpha suitable for parenteral administration can be formulated with a suitable buffer, e.g., Tris-HCl, acetate or phosphate such as dibasic sodium phosphate/monobasic sodium phosphate buffer, and pharmaceutically acceptable excipients (e.g., sucrose), carriers (e.g. human plasma albumin), toxicity agents (e.g., NaCl), preservatives (e.g., thimerosol, cresol or benzyl alcohol), and surfactants (e.g., tween or polysorbates) in sterile water for injection. The pegylated interferon alpha can be stored as lyophilized powder under refrigeration at 2°-8° C. The reconstituted aqueous solutions are stable when stored between 2° and 8° C. and used within 24 hours of reconstitution. See for example U.S. Pat. Nos. 4,492,537; 5,762,923 and 5,766,582. The reconstituted aqueous solutions may also be stored in prefilled, multi-dose syringes such as those useful for delivery of drugs such as insulin. Typical, suitable syringes include systems comprising a prefilled vial attached to a pen-type syringe such as the NOVOLET® Novo Pen available from Novo Nordisk or the REDIPEN®, available from Schering Corporation, Kenilworth, N.J. Other syringe systems include a pen-type syringe comprising a glass cartridge containing a diluent and lyophilized pegylated interferon alpha powder in a separate compartment.
The scope of the present invention also includes compositions comprising an IGF1R inhibitor in association with one or more other anti-cancer chemotherapeutic agents (e.g., as described herein) in association with one or more antiemetics including, but not limited to, casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine (sold as Benadryl® by Pfizer; New York, N.Y.), hydroxyzine (sold as Atarax® by Pfizer; New York, N.Y.), metoclopramide (sold as Reglan® by AH Robins Co,; Richmond, Va.), lorazepam (sold as Ativan® by Wyeth; Madison, N.J.), alprazolam (sold as Xanax® by Pfizer; New York, N.Y.), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, N.J.), droperidol (Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as Decadron® by Merck and Co.; Rahway, N.J.), methylprednisolone (sold as Medrol® by Pfizer; New York, N.Y.), prochlorperazine (sold as Compazine® by Glaxosmithkline; Research Triangle Park, N.C.), granisetron (sold as Kytril® by Hoffmann-La Roche Inc.; Nutley, N.J.), ondansetron (sold as Zofran® by Glaxosmithkline; Research Triangle Park, N.C.), dolasetron (sold as Anzemet® by Sanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban® by Novartis; East Hanover, N.J.).
Compositions comprising an antiemetic are useful for preventing or treating nausea; a common side effect of anti-cancer chemotherapy. Accordingly, the present invention also includes methods for treating or preventing cancer in a subject by administering an IGF1R inhibitor optionally in association with one or more other chemotherapeutic agents (e.g., as described herein) and/or optionally in association with one or more antiemetics.
Other side effects of cancer treatment include red and white blood cell deficiency. Accordingly, the present invention includes compositions comprising an IGF1R inhibitor optionally in association with an agent which treats or prevents such a deficiency, such as, e.g., pegfilgrastim, erythropoietin, epoetin alfa or darbepoetin alfa.
The present invention further comprises a method for treating or preventing any stage or type of any medical condition set forth herein by administering an IGF1R inhibitor in association with a therapeutic procedure such as surgical tumorectomy or anti-cancer radiation treatment; optionally in association with a further chemotherapeutic agent and/or antiemetic, for example, as set forth above.
The term “in association with” indicates that the components of a composition of the invention (e.g., anti-IGF1R antibody or antigen-binding fragment thereof along with imatinib) can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e.g., a kit). Furthermore, each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at several intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route (e.g., wherein an anti-IGF1R antibody is administered parenterally and gosrelin acetate is administered orally).
The present invention provides methods for determining the expression levels of any of the genes set forth in table 1 or table 3 in a subject receiving IGF1R inhibitor therapy. In an embodiment of the invention, the subject suffers from a medical condition mediated by cellular IGF1R expression or activity or the expression or activity of any member of the IGF1R pathway (including e.g., IRS-1, PI3 kinase, ERK2 or AKT). In an embodiment of the invention, the medical condition is any of the following: osteosarcoma, rhabdomyosarcoma, neuroblastoma, any pediatric cancer, kidney cancer, leukemia, renal transitional cell cancer, Werner-Morrison syndrome, acromegaly, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, benign prostatic hyperplasia, breast cancer, prostate cancer, bone cancer, lung cancer, gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, diarrhea associated with metastatic carcinoid, vasoactive intestinal peptide secreting tumors, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels and inappropriate microvascular proliferation, head and neck cancer, squamous cell carcinoma, multiple myeloma, solitary plasmacytoma, renal cell cancer, retinoblastoma, germ cell tumors, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing Sarcoma, chondrosarcoma, haemotological malignancy, chronic lymphoblastic leukemia, chronic myelomonocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, chronic myeloblastic leukemia, Hodgekin's disease, non-Hodgekin's lymphoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, hairy cell leukemia, mast cell leukemia, mast cell neoplasm, follicular lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, Burkitt Lymphoma, mycosis fungoides, seary syndrome, cutaneous T-cell lymphoma, chronic myeloproliferative disorders, a cental nervous system tumor, brain cancer, glioblastoma, non-glioblastoma brain cancer, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma and choroid plexus papilloma, a myeloproliferative disorder, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer, germ cell tumors, liver cancer, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels, inappropriate microvascular proliferation, acromegaly, gigantism, psoriasis, atherosclerosis, smooth muscle restenosis of blood vessels or inappropriate microvascular proliferation, Grave's disease, multiple sclerosis, systemic lupus erythematosus, Hashimoto's Thyroiditis, Myasthenia Gravis, auto-immune thyroiditis and Bechet's disease.
The IGF1R inhibitors discussed herein (e.g., anti-IGF1R antibodies and antigen-binding fragments thereof) and compositions thereof are, in an embodiment of the invention, administered at a therapeutically effective dosage. The term “therapeutically effective amount” or “therapeutically effective dosage” means that amount or dosage of an IGF1R inhibitor or composition thereof that will elicit a biological or medical response of a tissue, system, patient, subject or host that is being sought by the administrator (such as a researcher, doctor or veterinarian) which includes any measurable alleviation of the signs, symptoms and/or clinical indicia of a medical disorder, such as cancer (e.g., tumor growth and/or metastasis) including the prevention, slowing or halting of progression of the medical disorder to any degree whatsoever. For example, in one embodiment of the invention, a “therapeutically effective dosage” of any anti-IGF1R antibody or antigen-binding fragment thereof discussed herein (e.g., an anti-IGF1R antibody comprising mature LCC, LCD, LCE or LCF light chain and/or mature HCA or HCB heavy chain) is between about 0.3 and 20 mg/kg of body weight (e.g., about 0.3 mg/kg of body weight, about 0.6 mg/kg of body weight, about 0.9 mg/kg of body weight, about 1 mg/kg of body weight, about 2 mg/kg of body weight, about 3 mg/kg of body weight, about 4 mg/kg of body weight, about 5 mg/kg of body weight, about 6 mg/kg of body weight, about 7 mg/kg of body weight, about 8 mg/kg of body weight, about 9 mg/kg of body weight, about 10 mg/kg of body weight, about 11 mg/kg of body weight, about 12 mg/kg of body weight, about 13 mg/kg of body weight, about 14 mg/kg of body weight, about 15 mg/kg of body weight, about 16 mg/kg of body weight, about 17 mg/kg of body weight, about 18 mg/kg of body weight, about 19 mg/kg of body weight, about 20 mg/kg of body weight), about once per week to about once every 3 weeks (e.g., about once every 1 week or once every 2 weeks or once every 3 weeks). The therapeutically effective dosage of an IGF1R inhibitor or any further therapeutic agent is, when possible, as set forth in the Physicians' Desk Reference.
Genes upregulated in sensitive cells relative to resistant cells are:
C14orf132;
C7orf41;
Genes downregulated in sensitive cells relative to resistant cells are:
C6orf192;
C19orf54;
hqp0376
Embodiments of the present include those in which any of the foregoing genes comprise any of the following nucleotide sequences or any allelic variant thereof (e.g., a sequence conserved variant or a functionally conserved variant thereof):
Gene: Homo sapiens transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila) (TLE4)
Gene: Homo sapiens bone morphogenetic protein 7 (osteogenic protein 1) (BMP7)
Gene: Homo sapiens protocadherin gamma subfamily C, 3 (PCDHGC3)
Gene: Homo sapiens autism susceptibility candidate 2 (AUTS2)
Gene: Homo sapiens chromosome 14 open reading frame 132 (C14orf132)
Gene: Homo sapiens ceramide kinase (CERK)
Gene: Homo sapiens hepatoma-derived growth factor, related protein 3 (HDGFRP3)
Gene: Homo sapiens transcription factor 4 (TCF4)
Gene: Homo sapiens Meis homeobox 2 (MEIS2)
Gene: Homo sapiens echinoderm microtubule associated protein like 4 (EML4)
Gene: Homo sapiens chromosome 7 open reading frame 41 (C7orf41; ELLS1)
Gene: Homo sapiens mRNA for KIAA1450 protein
Gene: Homo sapiens zinc finger protein 136 (ZNF136)
Gene: Homo sapiens D15F37 gene (D15F37)
Gene: Homo sapiens cyclin-dependent kinase 6 (CDK6)
Gene: Homo sapiens protein tyrosine phosphatase type IVA, member 1 (PTP4A1)
Gene: Homo sapiens TIMP metallopeptidase inhibitor 1 (TIMP1)
Gene: Homo sapiens clusterin (CLU)
Gene: Homo sapiens acetyl-Coenzyme A acetyltransferase 1 (acetoacetyl Coenzyme A thiolase) (ACAT1), nuclear gene encoding mitochondrial protein
Gene: Homo sapiens aldolase C, fructose-bisphosphate (ALDOC)
Gene: Homo sapiens chromosome 6 open reading frame 192 (C6orf192),
Gene: Homo sapiens collagen, type IV, alpha 5 (Alport syndrome) (COL4A5)
Gene: Homo sapiens complement component 1, q subcomponent binding protein (C1QBP), nuclear gene encoding mitochondrial protein
Gene: Homo sapiens cysteine-rich protein 1 (intestinal) (CRIP1)
Gene: Gene: Homo sapiens deaminase domain containing 1 (DEADC1)
Gene: Homo sapiens glutathione S-transferase kappa 1 (GSTK1),
Gene: Homo sapiens glutathione S-transferase omega 2 (GSTO2)
Gene: Homo sapiens phosphatidic acid phosphatase type 2 domain containing 1B (PPAPDC1B)
Gene: Homo sapiens transmembrane protein 107 (TMEM107)
Gene: Homo sapiens Josephin domain containing 3 (JOSD3)
Gene: Homo sapiens transmembrane protein 77 (TMEM77)
Gene: Homo sapiens macrophage stimulating 1 (hepatocyte growth factor-like) (MST1)
Gene: Homo sapiens metallothionein 1E (MT1E)
Gene: Homo sapiens parvin, beta (PARVB)
Gene: Homo sapiens peroxiredoxin 4 (PRDX4)
Gene: Homo sapiens RasGEF domain family, member 1A (RASGEF1A)
Gene: Homo sapiens ribosomal protein L14 (RPL14)
Gene: Homo sapiens interferon, gamma-inducible protein 30 (IFI30)
Gene: Homo sapiens activating transcription factor 1 (ATF1)
Gene: Homo sapiens acyl-Coenzyme A dehydrogenase, very long chain (ACADVL), nuclear gene encoding mitochondrial protein
Gene: Homo sapiens F-box protein 6 (FBXO6)
Gene: Homo sapiens NAD(P)H dehydrogenase, quinone 2 (NQO2)
Gene: Homo sapiens transmembrane protein 64 (TMEM64)
Gene: Homo sapiens zinc finger, AN1-type domain 1 (ZFAND1)
Gene: Homo sapiens transmembrane emp24 protein transport domain containing 5 (TMED5)
Gene: Homo sapiens protein disulfide isomerase family A, member 5 (PDIA5)
Gene: Homo sapiens myosin IC (MYO1C)
Gene: Homo sapiens N-acetylglucosamine-1-phosphate transferase, alpha and beta subunits (GNPTAB)
Gene: Homo sapiens lactamase, beta 2 (LACTB2).
Gene: Homo sapiens ribosomal protein L22 (RPL22)
Gene: Homo sapiens tetraspanin 4 (TSPAN4)
Gene: Homo sapiens ribosomal protein L15 (RPL15)
Gene: Homo sapiens propionyl Coenzyme A carboxylase, beta polypeptide (PCCB)
Gene: Homo sapiens crystallin, zeta (quinone reductase) (CRYZ)
Gene: Homo sapiens DnaJ (Hsp40) homolog, subfamily C, member 10 (DNAJC10)
Gene: Homo sapiens chromosome 19 open reading frame 54 (C19orf54)
Gene: Homo sapiens heat shock 10 kDa protein 1 (chaperonin 10) (HSPE1)
Gene: Homo sapiens hqp0376 protein
The present invention comprises methods by which a practitioner may evaluate the level of expression of any of the foregoing biomarkers or any allele or variant or fragment thereof for the purpose of predicting the sensitivity of a cell from which the biomarker was measured to any IGF1R inhibitor.
In an embodiment of the invention, the method comprises determining that a cell is sensitive if it expresses higher levels of one or more of the biomarkers taken from table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) than that of any cell known to be resistant to the IGF1R inhibitor. Similarly, in an embodiment of the invention, the method comprises determining that a cell is sensitive if it expresses lower levels of one or more of the biomarkers taken from table 2 than that of any cell known to be resistant to the IGF1R inhibitor. In an embodiment of the invention, a cell characterized by one of such genes exhibiting said comparatively high or low expression is characterized as possessing one biomarker for IGF1R inhibitor sensitivity; similarly, a cell characterized by, e.g., four or five or more of such genes exhibiting said comparatively high or low expression is characterized as possessing biomarkers for IGF1R inhibitor sensitivity.
In an embodiment of the invention, the level of expression of a gene in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) or table 3 in a sensitive cell is, e.g., higher or lower, respectively, by any detectable and/or significant degree, e.g., at least about 1% (e.g., at least about 2%, 3%, 4%, 5%, 10%, 25%, 50%, 75%, 100%, 200%, 300%, 500% or 700%) higher or lower, respectively, than that of a resistant cell. In an embodiment of the invention, a sensitive cell possesses more than one biomarker for IGF1R inhibitor sensitivity. For example, in an embodiment of the invention, the sensitive cell comprises all of the biomarkers for IGF1R inhibitor sensitivity described in tables 1 and 3. In an embodiment of the invention, one or more of the biomarkers for IGF1R inhibitor sensitivity possessed by a sensitive cell exhibit levels of expression, when compared to that of a resistant cell, similar to that set forth in any of tables 1, 3, 5, or 7 (a-k).
In an embodiment of the invention, the magnitude of overexpression of one or more of the biomarkers in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to that of an IGF1R resistant cell is approximately as set forth in table 1 or more (i.e., greater magnitude of overexpression). For example, in an embodiment of the invention, a malignant cell is determined to be sensitive to an IGF1R inhibitor if the ratio of the TRE2 expression level in the cell being evaluated divided by the TRE2 expression level of an IGF1R inhibitor resistant cell is at least about 3.8. In an embodiment of the invention, the resistant cell used in this comparison is 22rv1, 2774 or H838. In an embodiment of the invention, a one or more genes other than TRE2 or one or more other genes in addition to TRE2 are evaluated. Similarly, the magnitude of underexpression of one or more of the biomarkers in table 3, relative to that of an IGF1R inhibitor resistant cell is approximately as set forth in table 3 or more (i.e., greater magnitude of underexpression).
In an embodiment of the invention, a sensitive cell can be evaluated for possession of one or more biomarkers for IGF1R inhibitor sensitivity by comparison of the expression levels of one or more of the genes set forth in Tables 1 and 3 to that of any of the following resistant cell lines: 22rv1, 2774 and H838. In an embodiment of the invention, a sensitive cell overexpresses one or more of the genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) and/or underexpresses one or more of the genes set forth in table 3 when compared to that of 22rv1, 2774 and H838. Cell line 22rv1 is a human prostate carcinoma cell line (see ATCC deposit no. CRL-2505). Cell line 2774 is an ovarian cancer cell line. H838 is a non-small cell lung cancer cell line (see ATCC deposit no. CRL-5844). These cells are known in the art.
The term “overexpress” or “high expression”, when used on the context of a comparison of gene expression levels in a cell and a reference cell, relates to cells characterized by expression of a given gene at a higher level than that of a reference cell. For example, in the present invention, IGF1R sensitive cells express the TRE2 gene at a higher level than that of resistant cells; thus, the TRE2 is overexpressed or exhibits high expression in sensitive cells. Similarly, the acetyl-coenzyme A acetyltransferase 1 gene is “underexpressed” or exhibits “low expression” in IGF1R inhibitor sensitive cells. In an embodiment of the invention, the terms overexpress, underexpress, high expression or low expression refer to expression of mRNA encoded by the biomarker gene. In an embodiment of the invention, the terms refers to expression of protein encoded by the biomarker gene.
Specifically, the present invention includes a method for evaluating sensitivity of malignant cells to an IGF1R inhibitor (e.g., an anti-IGF1R antibody) comprising determining if said cells exhibit high expression (e.g., RNA or protein expression (transcription or translation)) of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) or low expression of one or more genes set forth in table 3 relative to that of a cell resistant to said inhibitor. The present method may be used to evaluate sensitivity of in vitro cells, e.g., a cell line, or to evaluate sensitivity of cells derived from the body of a subject suffering from cancer. The cells evaluated under this method are determined to be sensitive if said high expression or said low expression is observed. In a more specific embodiment of the invention, the method comprises the steps of (a) obtaining a sample of one or more malignant cells from the body of a subject (e.g., a biopsy of tumor tissue or a blood sample from a suffering from a blood cancer such as leukemia); optionally transferring such a sample to a testing facility such as a laboratory for: (b) evaluating expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever) or table 3 in the malignant cells; and (c) comparing said expression level to that of cells resistant to said IGF1R inhibitor; wherein the cells are determined to be sensitive to the inhibitor if expression of one or more genes in table 1 is higher than that of a cell resistant to said inhibitor or if expression of one or more genes in table 3 is lower than that of a cell resistant to said inhibitor.
Patient selection methods are also within the scope of the present invention. Such methods are beneficial, e.g., for the efficient targeting of subjects with cancer that is likely to be responsive to a given IGF1R inhibitor therapy. Specifically, the present invention provides a method for selecting a subject with malignant cells for treatment with an IGF1R inhibitor comprising evaluating sensitivity of the malignant cells to said inhibitor, e.g., by the method discussed above; wherein said subject is selected if said cells are determined to be sensitive. Moreover, the present invention provides a method for identifying a subject with malignant cells sensitive to an IGF1R inhibitor comprising evaluating sensitivity of the malignant cells to said inhibitor, e.g., by the method discussed above; wherein said subject is identified if said cells are determined to be sensitive.
Methods of treating cancer with an IGF1R inhibitor including selecting, e.g., pre-selecting, subjects with cancers sensitive or likely to be sensitive to the inhibitor are also provided herein. For example, the present invention provides a method for treating a tumor or cancerous condition with an IGF1R inhibitor comprising evaluating sensitivity of malignant cells, which are in said tumor or which mediate said cancerous condition, to said inhibitor, e.g., by the method discussed above, and, if said cells are determined to be sensitive, commencing or continuing treatment by administering, to the subject, a therapeutically effective dose of the inhibitor. In an embodiment of the invention, the evaluation may be performed after treatment has been commenced and, if the malignant cells in the body of the subject being tested are determined to be sensitive, treatment may be continued at the same or a different dose.
The present invention also provides methods for selecting a therapy suitable for treatment of cancer by prescreening the subject's malignant cells for IGF1R inhibitor sensitivity. For example, the present invention provides a method for selecting a therapy for a subject with one or more malignant cells comprising evaluating sensitivity of the cells to an IGF1R inhibitor, e.g., by the method discussed above; wherein said inhibitor is selected as the therapy if said cells are determined to be sensitive to the inhibitor.
The scope of the present invention also provides a method of advertising an IGF1R inhibitor or a pharmaceutically acceptable composition thereof or a therapeutic regimen comprising administration of said inhibitor or composition comprising promoting, to a target audience, the use of the inhibitor or composition for treating a patient or patient population whose tumors or cancerous conditions are mediated by malignant cells that exhibit increased expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 3, relative to cells resistant to said inhibitor. Such uses may be promoted by any medium including, e.g., television, print or radio.
The present invention also provides articles of manufacture including one or more IGF1R inhibitors and literature explaining the relationship between the biomarkers of the present invention and sensitivity of a subject's cancer to the inhibitor. Specifically, the present invention provides an article of manufacture comprising, packaged together, an IGF1R inhibitor or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent or pharmaceutical composition is indicated for treating patients having a tumor comprising malignant cells or a cancerous condition mediated by malignant cells that exhibit increased expression of one or more genes set forth in table 1 (e.g., ELLS1 and/or AUTS2 and/or TCF4 and/or TLE; e.g., all 4, 3, 2 or 1 in any combination whatsoever), relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 3, relative to cells resistant to said inhibitor. Methods of making such articles also form a part of the present invention. For example, the present invention provides a method for manufacturing an IGF1R inhibitor or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier said method comprising combining, in a package, the inhibitor or composition; and a label conveying that the inhibitor or composition is indicated for treating patients having a tumor comprising malignant cells or a cancerous condition mediated by malignant cells that exhibit increased expression of one or more genes set forth in table 1, relative to cells resistant to said inhibitor; or that exhibit decreased expression of one or more genes set forth in table 3, relative to cells resistant to said inhibitor.
An aspect of the invention includes determining whether a patient exhibits elevated or decreased levels of RNA or protein encoding various genes. Gene expression can be quantitated in a patient by any of the numerous methods known in the art. Expression can be quantited, for example, by simply hiring or contracting with a commercial laboratory to perform an assay wherein the patient's or subject's sample is harvested/biopsied and transferred to the lab. Alternatively, the practitioner can perform the assay himself. In an embodiment of the invention, expression is quantitated by a northern blot analysis, gene chip expression analysis, RT-PCR (real-time polymerase chain reaction), radioimmunoassay (RIA) (see e.g., Smith et al., J. Clin. Endocrin. Metab. 77(5): 1294-1299 (1993); Cohen et al., J. Clin. Endocrin. Metab. 76(4): 1031-1035 (1993); Dawczynski et al., Bone Marrow Transplant. 37:589-594 (2006); and Clemmons et al., J. Clin. Endocrin. Metab. 73:727-733 (1991)), western blot (WLB) or by ELISA (enzyme linked immunosorbent assay).
Any method for determining biomarker expression, e.g., as discussed herein, may be used to compare the expression level of the sample being evaluated (e.g., malignant or cancerous cells or an extract thereof) and the expression level of a resistant cell sample or an extract thereof so as to determine if the biomarker is overexpressed or underexpressed in the sample relative to that of the resistant cell. The quantity of cell samples being evaluated may be normalized against e.g., total cellular protein or RNA to ensure an accurate and meaningful comparison.
Northern blot analysis of biomarker transcription in a sample is, in an embodiment of the invention, performed. Northern analysis is a standard method for detection and quantitation of mRNA levels in a sample. Initially, RNA is isolated from a sample to be assayed (e.g., tumor tissue). In the analysis, the RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. In an embodiment of the invention, Northern hybridization involves polymerizing radiolabeled or nonisotopically detectably labeled DNA, in vitro, or generation of oligonucleotides as hybridization probes. In an embodiment of the invention, the membrane holding the RNA sample is prehybridized or “blocked” prior to probe hybridization to prevent the probe from coating the membrane and, thus, to reduce non-specific background signal. After hybridization, typically, unhybridized probe is removed by washing in several changes of buffer. Stringency of the wash and hybridization conditions can be designed, selected and implemented by any practitioner of ordinary skill in the art. If a radiolabeled probe was used, the blot can be wrapped in plastic wrap to keep it from drying out and then immediately exposed to film for autoradiography e.g, in the presence of a scintillant. If a nonisotopic probe was used, the blot must, generally, be treated with nonisotopic detection reagents, to develop the detectable probe signal, prior to film exposure. The relative levels of expression of the genes being assayed can be quantified using, for example, densitometry or visual estimation.
In an embodiment of the invention, expression of one or more biomarkers is determined in a gene chip analysis procedure. Such a procedure, in an embodiment of the invention, includes the following steps: target preparation, target hybridization, probe array washing and staining, probe array scan and data analysis. Target preparation entails, in an embodiment of the invention, preparing a biotinylated target RNA obtained from the sample to be tested. In an embodiment of the invention, the target hybridization step includes preparing a hybridization cocktail, including the fragmented target, probe array controls, BSA, and herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation. In an embodiment of the invention, immediately following hybridization, the probe array undergoes an automated washing and staining. In an embodiment of the invention, in the scanning and analysis step, the hybridized probe array is stained with streptavidin phycoerythrin conjugate and scanned for light emission at 570 nm wavelength. The amount of light emitted at 570 nm is proportional to the bound target at each location on the probe array. Computer analysis using commercially available equipment and software is possible (Affymetrix; Santa Clara, Calif.). Modifications to this general scheme, which are known in the art, form part of the present invention.
Biomarker expression is determined, in an embodiment of the invention, using RT-PCR. RT-PCR allows detection of the progress of a PCR amplification of a target gene in real time. Design of the primers and probes required to detect expression of a biomarker of the invention is within the skill of a practitioner of ordinary skill in the art. RT-PCR can be used to determine the level of RNA encoding a biomarker of the invention in a sample. In an embodiment of the invention, RNA from the tissue sample is isolated, under RNAse free conditions, then converted to DNA by treatment with reverse transcriptase. Methods for reverse transcriptase conversion of RNA to DNA are well known in the art. Reverse transcription may be performed prior to RT-PCR analysis or simultaneously, within a single reaction vessel (e.g., tube).
RT-PCR probes depend on the 5′-3′ nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon (biomarker gene). RT-PCR probes are oligonucleotides that have a fluorescent reporter dye attached to the 5′ end and a quencher moiety coupled to the 3′ end (or vice versa). These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR amplification, when the polymerase replicates a template on which an RT-PCR probe is bound, the 5′-3′ nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, in a manner proportional to the amount of probe cleavage. Fluorescence signal emitted from the reaction can be measured or followed over time using equipment which is commercially available using routine and conventional techniques. Quantitation of biomarker RNA in a sample being evaluated may be performed by comparison of the amplification signal to that of one or more standard curves wherein known quantities of RNA were evaluated in a similar manner. Such methods are known in the art.
In an embodiment of the invention, western blots are performed as follows: A sample comprising an extract of a tumor tissue source is electrophoresed on 10% polyacrylamide-sodium dodecyl sulfate (SDS-PAGE) gel and electroblotted onto nitrocellulose or some other suitable membrane. The membrane is then incubated with a primary antibody which binds to the protein product of the gene being evaluated, optionally washed and then incubated with a detectably labeled secondary antibody that binds to the primary antibody and optionally washed again. The presence of the secondary antibody is then detected. For example, if the secondary antibody is labeled with a chemilluminescence label, the label is developed with a developing agent, then the membrane is exposed to film and then the film is developed. In an embodiment of the invention, each lane of the autoradiograph is scanned and analyzed by densitometer.
In an embodiment of the invention, an ELISA assay employs an antibody specific for a biomarker coated on a 96-well plate. Standards and samples are pipetted into the wells and biomarker present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-biomarker antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of biomarker bound. Stop solution added to the reaction changes the color from blue to yellow, and the intensity of the color is measured at 450 nm (see e.g., Human IGF-BP-2 ELISA Kit from RayBiotech, Inc.; Norcross, Ga.; and Angervo M et al., Biochemical and Biophysical Research Communications 189: 1177-83 (1992); Kratz et al., Experimental Cell Research 202: 381-5 (1992); and Frost et al. Journal of Biological Chemistry 266: 18082-8 (1991)). A standard ELISA curve using known concentrations of biomarker can be plotted and the concentration of biomarker in the unknown sample (e.g., the serum of a patient) can be determined by comparing the signal observed therein with the signal observed in the standard.
Radioimmunoassay (RIA) is a scientific method used to detect the presence of a given antigen, e.g., encoded by a biomarker gene. RIA involves mixing known quantities of radioactive antigen (e.g., labeled with gamma-radioactive isotopes of iodine attached to tyrosine) with antibody to that antigen, then adding unlabeled or “cold” antigen and measuring the amount of labeled antigen displaced. Initially, the radioactive antigen is bound to the antibodies. When “cold” (unlabeled) antigen is added, the two compete for antibody binding sites—at higher concentrations of “cold” antigen, more of it binds to the antibody, displacing the radioactive variant. The bound antigens are then separated from the unbound ones and the quantitiy of labeled bound antigen is then quantitated. The bound antigen can be separated from unbound antigen in several ways; for example, by precipitating the antigen-antibody complexes by adding a secondary antibody directed against the primary antibody. In another embodiment of the invention, the antigen-specific antibodies can be coupled to the inner walls of a test tube or microtiter well or to some other solid substrate. After incubation, the contents are removed and the tube, well or substrate, which is washed leaving bound, labeled antibody/antigen complexes; and, then, the radioactive label present in the tube or well of both is measured.
This section is intended to further describe the present invention and should not be construed to further limit the invention. Any composition or method set forth herein constitutes part of the present invention.
Xenograft samples. The xenografts used in this study (and their tissue of origin) are: H322 and H838 (both derived from non-small cell lung carcinoma), SK-N-AS and SK-N-FI (both derived from neuroblastomas), 22rv1 (derived from prostate), 2774 (derived from ovarian) and SJSA-1 (derived from an osteosarcoma). The 22rv1, 2774 and H838 cell lines are resistant to anti-IGF1R antibody (mature Ig fragments of SEQ ID NOs: 8 and 10/κ light chain, γ1 heavy chain) mediated growth inhibition. Cells were injected into nude mice and tumors were allowed to reach approximately 200 mm3 in size before harvesting. Mice were treated with one intraperitoneal injection of either 0.1 mg of anti-IGF-1R antibody (mature Ig fragments of SEQ ID NOs: 8 and 10/κ light chain, γ1 heavy chain) (for xenografted mice bearing SK-N-AS and SK-N-FI tumors) or 0.5 mg of the anti-IGF1R antibody (for xenografted mice bearing SJSA-, H322, H388, 22rv1 and 2774 tumors). Tumors were harvested 48 hrs after antibody treatment and were cut in half and snap-frozen. Half were processed for RNA as described below and the other half were processed for protein identification.
Chip hybridization. RNA was made from cells using the Trizol reagent (Molecular Research Center, Inc.; Cincinnati, Ohio) followed by purification over an RNAeasy column (Qiagen; Valencia, Calif.). Five micrograms of total RNA was used to make probes as described in the Affymetric Expression Analysis Technical Manual (www.affymetrix.com/support/technical/manual/expression_manual.affx) (Affymetrix, Inc; Santa Clara, Calif.). Probes were hybridized to the Affymetrix human (U133 Plus 2.0) high-density oligonucletide arrays as described in the manual.
Microarray analysis. Data analysis was performed using an S+ based program licensed from Insightful Corp. (Seattle, Wash.). Data was filtered so that measurements for probe sets with the prefix AFFX (Affymetrix control probe sets), probe sets which were called “absent” in all experiments, or where less than 7 probe pairs registered data were dropped from the analysis. All data was Log 2 transformed. The data was then normalized to the median inter-quartile range for each probe set. The normalized data was then filtered as follows: an expression percentage restriction was applied so that only conditions where the raw data had a value of 100 in at least three chips were included. Statistical analysis was done in a pair-wise fashion using the multiple testing t-test (with a p value of <0.05) in conjunction with a Benjamin-Hochberg multi-test correction. Data from each sensitive xenograft model was compared to data from each resistant model. Statistically significant gene lists generated from each of the pair-wise comparisons were then overlapped using Venn diagrams to find the genes that were up or down regulated in all sensitive xenografts when compared to all resistant xenografts.
The data from these analyses are set forth below in tables 1-4. In these tables, the name of the gene/biomarker analyzed is set forth along with the Genbank accession number for each gene. Also shown in tables 1 and 3 are the average expression levels of each biomarker in the sensitive (Savg) and resistant (Ravg) cell lines analyzed along with a ratio thereof (Savg/Ravg). The normalized expression levels of each biomarker in each of the resistant (R) and sensitive (S) cell lines are set forth below in tables 2 and 4. The normalized data in table 2 corresponds to the data set forth in table 1 and the normalized data in table 4 corresponds to the data in table 3.
Homo sapiens hqp0376 protein mRNA,
In addition to the biomarkers set forth above, the expression levels of the additional biomarkers, CDK6, T1MP, CLu and PRL1, were evaluated in all 7 xenografts discussed above. The results of these analyses are set forth below in tables 5 and 6. The normalized data in table 6 corresponds to the data in table 5.
The analyses of the genes set forth above (cdk6, TIMP, CLu, PRL1) were extended to a larger panel of 24 xenografts. Seven of the xenograft data points used in the studies above were repeated in this study. Expression of each gene analyzed was determined by RT-PCR (Taqman). Expression levels of each gene analyzed in the 22 xenografts which are shown, below, in Tables 7a-7k, are relative to the expression level in the known anti-IGF1R sensitive cell line, H322. The tables set forth the cell line name, its origin and the resistant/sensitive status of the cell line along with the % tumor growth inhibition (% TGI) associated with each cell line.
In this example, the biomarkers set forth herein were statistically analyzed in order to assess their value with respect to predicting the sensitivity of a cell to an IGF1R inhibitor. The biomarkers ELLS1, AUTS2, TCF4 and TLE were found to have a particularly high predictive value.
Based on the gene expression data from RT-PCR (Taqman; see above in Tables 7a-7k), two classification methods, Diagonal Linear Discriminant Analysis (DLDA) and Support Vector Machines (SVM), were used to develop multiplex marker assays for prediction of cell line sensitivity to the IGF1R inhibitor (anti-IGF1R antibody LCF/HCA (κ/γ1)).
DLDA is the simplest case of the maximum likelihood discriminant rule, in which the class densities are supposed to have the same diagonal covariance matrix. In the special case of binary classification, the DLDA scheme can be viewed as the “weighted voting scheme” proposed by Golub et al. (Science (1999) 286:531-537).
SVM has been recognized as the most powerful classifier in various applications of pattern classification. For binary classification, SVM learns the classifier by mapping the input training samples into a possibly high-dimensional feature space and seeking a hyperplane in this space which separates the two types of examples with the largest possible margin, i.e. distance to the nearest points. If the training set is not linearly separable, SVM finds a hyperplane, which optimizes a trade-off between good classification and large margin. (Cristianini N, Shawe-Taylor J., An Introduction to Support Vector Machines, Cambridge University Press, Cambridge, UK 2000).
Both DLDA and SVM approaches use the feature selection criterion similar to that described in Golub et al. (Science (1999) 286:531-537) and Slonim et al (“Class Prediction and discovery using gene expression data”, in Proceedings of the 4th Annual International Conference on Computational Molecular Biology (RECOMD), Univeral Academy Press, Tokyo, Japan, pp. 263-272 (2000)). We started with a dataset S consisting of m expression vector xi=(xi1, . . . , xin), 1≦i≦m, where m is the number of xenografts (23) and n is the number of genes measured (57). Each sample is labeled with Yε{+1, −1} (e.g., sensitive vs resistant). In order to find genes that discriminant between the two classes, we calculated a score
where μj+ (resp. μj−) is the mean expression for gene j using only the xenografts labeled +1 (resp. −1), and σj+ and σj− are the standard deviations respectively. The F(xj) score, which is closely related to Fisher criterion score, gave the highest score to those genes whose expression levels differ most on average in the two classes while also favoring those with small deviations in scores in the respective classes.
In order to evaluate the generalization power of each of the classification methods and to estimate their prediction capabilities for unknown samples, we used a standard 10-fold cross-validation technique and split the data randomly and repeatedly into training and test sets. The training sets consisted of randomly chosen subsets containing 90% of each class (resistant and sensitive); the remaining 10% of the samples from each class were left as test sets. The overall accuracy was defined by
where TP, FP, TN and FN refer to the number of true positives, false positives, true negatives and false negatives proteins, respectively. In order to keep computing times reasonable, we reported the average of overall accuracy estimates over 100 runs.
We also used the cross validation method to optimize the feature selection and select models that maximize the classification performance. Genes were first ranked based on F(xj) scores. Then, genes with high Pearson correlation coefficients (≧0.9) with top ranked genes were removed to reduce the redundancy. Genes with least F(xj) scores were further recursively removed and predictive accuracies of classifiers were estimated.
For the DLDA approach, the best classification was achieved using four genes, ELLS1, AUTS2, TCF4 and TLE, in the model. The prediction accuracy was estimated as 72.5%. For the SVM approach, the best prediction accuracy was estimated as 75.7%, with three genes, ELLS1, AUTS2 and TCF4, included in the model.
Statistical analysis was performed using R package which is freely available for example at www.r-project.org.
These data demonstrate that ELLS1, AUTS2, TCF4 and TLE are highly useful biomarkers which can be used to predict sensitivity or resisance of any cell to an IGF1R inhibitor, e.g., with about 70% certainty (e.g., about 72.5% or about 75.5% certainty or a range of from about 72.5% to about 75.5% certainty). The present invention includes methods of evaluating expression of all 4, 3, 2 or just 1 of these biomarkers in any combination whatsoever. Methods of evaluating sensitivity can be used, in turn, e.g., for selecting a patient for IGF1R inhibitor therapy.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.
This application claims the benefit of U.S. provisional patent application no. 61/014,556, filed Dec. 18, 2007; U.S. provisional patent application No. 61/015,938, filed Dec. 21, 2007; and U.S. provisional patent application No. 61/022,909, filed Jan. 23, 2008; each of which is herein incorporated by referenced in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2008/087240 | 12/17/2008 | WO | 00 | 9/7/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 61014556 | Dec 2007 | US | |
| 61015938 | Dec 2007 | US | |
| 61022909 | Jan 2008 | US |
