EP2531185A2 - Combination therapy for treating cancer comprising an igf-1r inhibitor and an hdac inhibitor - Google Patents

Abstract

The present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an IGF-1R inhibitor in a first treatment procedure and a second amount of a histone deacetylase (HDAC) inhibitor or a pharmaceutically acceptable salt or hydrate thereof in a second treatment procedure. The first and second amounts together comprise a therapeutically effective amount.

Description

COMBINATION THERAPY FOR TREATING CANCER COMPRISING AN IGF-1R

INHIBITOR AND AN HDAC INHIBITOR

This Application claims the benefit of U.S. Provisional Patent Application no.

61/300,643, filed February 2, 2010; which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method of treating cancer by administering an IGF-1R specific antibody in combination with an anti-cancer agent exemplified by a histone deacetylase (HDAC) inhibitor. The first and second amounts together comprise a therapeutically effective amount.

BACKGROUND OF THE INVENTION

Cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms that normally govern proliferation and differentiation. Lung carcinomas are responsible for the majority of deaths from cancer among men and are overtaking breast carcinomas as the most f equent cause of cancer death among women. The current prognosis for patients with lung cancer is poor. The mortality rate attendant lung cancer deaths have increased ten-fold in both men and women since 1 30, primarily due to an increase in cigarette smoking, but also due to an increased exposure to arsenic, asbestos, chromates, chloromethyl ethers, nickel, polycyclic aromatic hydrocarbons and other agents. See Scott, Lung Cancer: A Guide to Diagnosis and Treatment, Addicus Books (2000) and Alberg et al., in Kane et al. (eds.) Biology of Lung Cancer, pp. 11-52, Marcel Dekker, Inc. (1998). The American Cancer Society estimates there will be over 173,550 new cases of lung cancer in 2004.

Additionally, there will be an estimated 160,440 deaths from lung cancer in 2004. ACS Website: cancer with the extension org of the world wide web.

Lung cancer may result from a primary tumor originating in the lung or a secondary tumor which has spread from another organ such as the bowel or breast. Although there are over a dozen types of lung cancer, over 90% fall into two categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). See Scott, supra. 70-80% are diagnosed as NSCLC. The term non-small cell lung carcinoma ("NSCLC") includes the following cell types:

epidermoid carcinoma cells, adenocarcinoma cells, and large undifferentiated carcinoma cells. A diagnosis of lung cancer is usually confirmed by biopsy of the tissue.

Treatment approaches and natural history differ for these two diseases. The majority (80%) of cases of lung cancer in the United States are NSCLC. Although advances in the understanding of important clinical and prognostic factors for both NSCLC and SCLC have been made in the past 20 years, there have been minimal improvements in therapeutic results. NSCLS is generally divided into three types: squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Both squamous cell cancer and adenocarcinoma develop from the cells that line the airways; however, adenocarcinoma develops from the goblet cells that produce mucus. Large cell lung cancer has been thus named because the cells look large and rounded when viewed microscopically, and generally are considered relatively undifferentiated. See Yesner, Atlas of Lung Cancer, Lippincott-Raven (1998). Non-small cell cancer may be divided into four stages. Stage I is highly localized cancer with no cancer in the lymph nodes. Stage II cancer has spread to the lymph nodes at the top of the affected lung. Stage in cancer has spread near to where the cancer started. This can be to the chest wall, the covering of the lung (pleura), the middle of the chest (mediastinum) or other lymph nodes. Stage IV cancer has spread to another part of the body. Stage I-III cancer is usually treated with surgery, with or without chemotherapy. Stage IV cancer is usually treated with chemotherapy and or palliative care.

A number of chromosomal and genetic abnormalities have been observed in lung cancer. In NSCLC, chromosomal aberrations have been described on 3p, 9p, 1 lp, 15p and I7p, and chromosomal deletions have been seen on chromosomes 7, 11 , 13 and 19. See Skarin (ed.), Mulumodality Treatment of Lung Cancer, Marcel Dekker, Inc. (2000); Gemmill et al., pp.465- 502, in Kane, supra; Bailey-Wilson et al., pp. 53-98, in Kane, supra. Chromosomal abnormalities have been described on 1p, 3p, 5q, 6q, 8q, 13q and 17p in SCLC. Id. In addition, the loss of the short arm of chromosome 3p has also been seen in greater than 90% of SCLC tumors and approximately 50% of NSCLC tumors. Id.

A number of oncogenes and tumor suppressor genes have been implicated in lung cancer. See Mabry, pp. 391-412, in Kane, supra and Sclafani et al., pp. 295-316, in Kane, supra. In both SCLC and NSCLC, the p53 tumor suppressor gene is mutated in over 50% of lung cancers. See Yesner, supra. Another tumor suppressor gene, FHIT, which is found on chromosome 3p, is mutated by tobacco smoke. Id.; Skarin, supra. In addition, more than 95% of SCLCs and approximately 20-60% of NSCLCs have an absent or abnormal retinoblastoma (Rb) protein, another tumor suppressor gene. The ras oncogene (particularly K-ras) is mutated in 20-30% of NSCLC specimens and the c-erbB2 oncogene is expressed in 18% of stage 2 NSCLC and 60% of stage 4 NSCLC specimens. See Van Houtte, supra. Other tumor suppressor genes that are found in a region of chromosome 9, specifically in the region of 9p21 , are deleted in many cancer cells, including p1 .sup.INK4A and p15.sup.INK4B. See Bailey- Wilson, supra Sclafani et al., supra. These tumor suppressor genes may also be implicated in lung cancer pathogenesis.

In addition, many lung cancer cells produce growth factors that may act in an autocrine or paracrine fashion on lung cancer cells. See Siegfried et al., pp. 317-336, in Kane, supra, Moody, pp. 337-370, in Kane, supra and Heasley et al., 371 -390, in Kane, supra. Many NSCLC tumors express epidermal growth factor (EGF) receptors, allowing NSCLC cells to proliferate in response to EGF. Insulin-like growth factor (IGF-1) is elevated in greater than 80% of NSCLC tumors; it is thought to function as an autocrine growth factor. Although the majority of lung cancer cases are attributable to cigarette smoking, most smokers do not develop lung cancer. Epidemiological evidence has suggested that susceptibility to lung cancer may be inherited in a Mendelian fashion, and thus have an inherited genetic component. Bailey-Wilson, supra. Thus, it is thought that certain allelic variants at some genetic loci may affect susceptibility to lung cancer.

Current therapies for lung cancer are quite limited. Generally, patient options comprise surgery, radiation therapy, and chemotherapy.

Most cases of lung carcinomas are incurable by chemotherapy and radiation therapy. Depending on the type and stage of a lung cancer, surgery may be used to remove the tumor along with some surrounding lung tissue. A lobectomy refers to a lobe (section) of the lung being removed. If the entire lung is removed, the surgery is called a pneumonectomy. Removing only part of a lobe is known as a segmentectomy or wedge resection.

Indeed, the only curative option for patients with NSCLC is local therapy (surgical excision or local irradiation) in patients with early stage disease (I & II) when the tumor is still localized. At diagnosis however, the majority of patients with NSCLC present with advanced disease, which is not curable by surgery alone. In advanced stages of disease, systemic chemotherapy and/or irradiation can produce objective responses and palliation of symptoms, however, they offer only modest improvements in survival. The median survival of patients with non-resectable disease is 6 - 12 months. Two-year survival rates for stages IIIB and IV NSCLC are 10.8 and 5.4 percent respectively. Likewise, five-year survival rates are 3.9 and 1.3 percent.

If the cancer has spread to the brain, benefit may be gained from removal of the brain metastasis. This involves a craniotomy (surgery through a hole in the skull).

For radiation therapy several methods exist External beam radiation therapy uses radiation delivered from outside the body that is focused on the cancer. This type of radiation therapy is most often used to treat a primary lung cancer or its metastases to other organs.

Additionally, radiation therapy can be used as a post surgical treatment to kill very small deposits of cancer that cannot be seen or removed during surgery. Radiation therapy can also be used to palliate (relieve) symptoms of lung cancer such as pain, bleeding, difficulty swallowing, and problems caused by brain metastases.

For chemotherapy, cisplatin or a related drug, carboplatin, are the chemotherapy agents most often used in treating NSCLC. Other new chemical entities available for the treatment of NSCLC including paclitaxel (Taxol), docetaxel (Taxotere), topotecan, irinotecan, vinorelbine, and gemcitabine. While these drugs are improvements over prior chemotherapeutic agents (etoposide, cisplatin and carboplatin), the overall cure rate remains low.

The insulin-like growth factor-1 has been implicated in an array of physiologic processes through insulin-like growth factor-1 receptor (IGF-1R) activation, including cellular

proliferation, apoptosis, metabolism, organism size, and longevity. Dysregulation of IGF-1R signaling is linked to several oncologic processes, specifically carcinogenesis, cancer progression, and metastasis. Overexpression of IGF-1 or IGF1-R has been detected in diverse tumor cell lines, and high plasma concentrations of IGF-1 correlate with an increased risk of prostate, breast, and colorectal cancer. IGF-1R signaling is also implicated in resistance to radiation and chemotherapy. Refer to Prakash Chinnaiyan, Seminars in Radiation Oncology, 16: 59-64 (2006).

Indeed, Insulin-like growth factors (IGF), e.g., insulin-like growth factor -I and - II have been implicated in exerting mitogenic activity on various cell types such as tumor cells. IGFs are structurally similar to insulin, and have been implicated as a therapeutic tool in a variety of diseases and injuries. Insulin-like growth factor-I (IGF-I) is a 7649-dalton polypeptide with a pi of 8.4 that circulates in plasma in high concentrations and is detectable in most

tissues(Rinderknecht and Humbel, Proc. Natl. Acad. Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253: 2769 (1978)). IGF-I stimulates cell differentiation and cell proliferation, and is required by most mammalian cell types for sustained proliferation. These cell types include, among others, human diploid fibroblasts, epithelial cells, smooth muscle cells, T lymphocytes, neural cells, myeloid cells, chondrocytes, osteoblasts and bone marrow stem ceils. Each of these growth factors exerts its mitogenic effects 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). See also lapper, et al., (1 83) Endocrinol. 112:2215 and Rinderknecht, et al., (1978) Febs. Lett. 89:283. There is a large body of literature on the actions and activities of IGFs (IGF-1 , IGF-2, and IGF variants). See Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974); Binoux, Ann. Endocrinol., 41 : 157 (1980); Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1 81); Baxter, Adv. Clin. Chem., 25:49 (1986); U.S. Pat. No. 4,988,675; WO 91/03253; WO 93/23071).

The IGF system is composed of membrane-bound receptors for IGF-1, IGF-2, and insulin. The Type 1 IGF receptor (IGF-1R) is closely related to the insulin receptor (IR) in structure and shares some of its signaling pathways (Jones and Clemmons, Endocr. Rev., 16: 3- 34 (1995); Ullrich et al., Cell 61 : 203 212, 1990), and is structurally similar to the insulin receptor (Ullrich et al., EMBO J. 5: 25032512, 1986)). The IGF-I receptor is composed of two types of subunits: an alpha subunit (a 130 135 kD protein that is entirely extracellular and functions in ligand binding) and a beta subunit (a 95-kD transmembrane protein, with

transmembrane and cytoplasmic domains). The IGF-IR is initially synthesized as a single chain proreceptor polypeptide which is processed by glycosylation, proteolytic cleavage, and covalent bonding to assemble into a mature 460-kD heterotetramer comprising two alpha-subunits and two beta-subunits. The beta subunit(s) possesses ligand-activated tyrosine kinase activity. This activity is implicated in the signaling pathways mediating ligand action which involve autophosphorylation of the beta-subunit and phosphorylation of IGF-IR substrates.

IGF-IR binds IGF I and IGF II with nanomolar affinity, e.g., Kd of 1 x 10-9nM but is capable of binding to insulin with an affinity 100 to 1000 times less. Representative nanomolar affinity values may be found in FEBS Letters, 565: 19-22 (2004), the entire content of which is incorporated by reference herein.

There is considerable evidence for a role for IGF-I and/or IGF-IR in the maintenance of tumor cells in vitro and in vivo. For example, individuals with "high normal" levels of IGF-I have an increased risk of common cancers compared to individuals with IGF-I levels in the "low normal" range (Rosen et al., Trends Endocrinol. Metab. 10: 13641, 1999). For a review of the role IGF-I IGF-I receptor interaction plays in the growth of a variety of human tumors, see Macaulay, Br. J. Cancer, 65: 311 320, 1992. In addition to playing a key role in normal cell growth and development, IGF-1R signaling has also been implicated as playing a critical role in growth of tumor cells, cell transformation, and tumorigenesis. See Baserga, Cancer Res., 55:249-252 (1995); for a review, see Khandwala et al., Endocr. Rev. 21 : 215-244 (2000));

Daughaday and Rotwein, Endocrine Rev., 10:68-91 (1989).

Recent data impel the conclusion that IGF-IR is expressed in a great variety of tumors and of tumor lines and the IGFs amplify the tumor growth via their attachment to IGF-IR.

Indeed, the crucial discovery which has clearly demonstrated the major role played by IGF-IR in the transformation has been the demonstration that the R- cells, in which the gene coding for IGF-IR has been inactivated, are totally refractory to transformation by different agents which are usually capable of transforming the cells, such as the E5 protein of bovine papilloma virus, an overexpression of EGFR or of PDGFR, the T antigen of SV 40, activated ras or the combination of these two last factors (Sell C. et al., Proc. Natl. Acad. Sci., USA, 90: 11217- 11221, 1993; Sell C. et al., Mol. Cell. Biol., 14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; Coppola D. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T et al., J. Cell. Physiol., 164:214-221, 1995). Other key examples supporting this hypothesis include loss of metastatic phenotype of murine carcinoma cells by treatment with anti sense RNA to the IGF-1R (Long et al., Cancer Res., 55:1006-1009 (1995)) and the in vitro inhibition of human melanoma cell motility (Stracke et al., J. Biol. Chem., 264:21554-21559 (1989)) and of human breast cancer cell growth by the addition of IGF-1R antibodies (Rohlik et al., Biochem. Biophys. Res. Commun., 149:276-281 (1987)).

Other arguments in favor of the role of IGF-IR in carcinogenesis come from studies using murine monoclonal antibodies directed against the receptor or using negative dominants of IGF- IR. In effect, murine monoclonal antibodies directed against IGF-IR inhibit the proliferation of numerous cell lines in culture and the growth of tumor cells in vivo (Arteaga C. et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys. Res. Com., 196:92-98, 1993; Zia F et al., J. Cell. Biol., 24:269-275, 1996; Scotlandi et al., Cancer Res., 58:4127-4131, 1998). It has likewise been shown in the works of Jiang et al. (Oncogene, 18:6071 -6077, 1 99) that a negative dominant of IGF-IR is capable of inhibiting tumor proliferation. IGF-IR levels are also elevated in tumors of lung (Kaiser et al., J. Cancer Res. Clin. Oncol. 119: 665668, 1993; Moody et al., Life Sciences 52: 1161 1173, 1993; Macauley et al., Cancer Res., 50: 2511 2517, 1990). Even though a wide range of anticancer agents have been developed, many patients with advanced solid tumors still have a poor prognosis. For the treatment of advanced lung cancer, there are many anticancer agents in clinical use, such as cisplatin, carboplatin, docetaxel, paclitaxel, vinorelbine, gemicitabine, fluorouracil (5-FU) derivatives, and irinotecan, they still have little effect on recurrent disease. Likewise, a number of combination therapy regimens employing platinum compounds have proven to be effective and are widely applied for the initial treatment of inoperative non-small cell lung cancer (NSCLC). In addition, docetaxel,

pemetrexed, and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been reported to be effective in the context of second-line chemotherapy for NSCLC. However, the effect of these therapies on improving patient survival remains far from satisfactory. For example, use of anti-EOFR molecules, for example, Gefitinib (ZD1839, Iressa) and Erlotinib, are effective in about 10-20% of individuals, particularly for patients whose cancer is associated with prominent EGFR activity often associated with an activation mutation of EGFR. As such, there remains an unmet medical need to find more appropriate therapeutic opportunities for NSCLC.

Histone deacetylase (HDAC) and histone acetyl ase catalyze deacetylation and

acetylation, respectively, of histone in eukaryotes, whose dynamic balance is important for the accurate regulation of gene expression in eukaryotes. There are 18 HDACs in humans. These enzymes are not redundant in function. Eleven of the HDACs are zinc dependent, classified on the basis of homology to yeast HDACs: Class I includes HDACs 1 , 2, 3, and 8; Class IIA includes HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and 10; and Class IV, HDAC 11. In addition to histones, HDACs have many nonhistone protein substrates which have a role in regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis.

There are several lines of evidence that histone acetylation and deacetylation are mechanisms by which transcriptional regulation in a cell is achieved (Grunstein, M. (1997) Nature 389:349-52). These effects are thought to occur through changes in the structure of chromatin by altering the affinity of histone proteins for coiled DNA in the nucleosome.

Chromatin remodeling is a key step in the regulation of gene expression, consequently affecting cell function, differentiation, and proliferation. Chromatin structure affects transcription by opening or closing the access of transcriptional factors to their target sequences. The key mechanism in chromatin remodeling is thought to be the modification of NH2-terminal tails of histones, which contributes to a 'histone code' determining the transcription of target genes. 'Closed' chromatin is not transcribed and consists of nucleosomes in which the lysine residues of the histone tails become deacetylated. Acetylation of those regions neutralizes the positive charge on lysine residues and changes the nucleosome structure, leading to 'opened' chromatin in which transcription factors have easy access, and resulting in gene expression. Acetylation of histone tails is regulated by the opposing activities of HATs and HDACs, and aberrant deacetylation due to HDACs is associated with certain types of human cancer. There are five types of histones that have been identified (designated H1, H2A, H2B, H3 and H4). Histones H2A, H2B, H3 and H4 are found in the nucleosomes and H1 is a linker located between nucleosomes. Each nucleosome contains two of each histone type within its core, except for H1, which is present singly in the outer portion of the nucleosome structure. It is believed that when the histone proteins are hypoacetylated, mere is a greater affinity of the histone to the DNA phosphate backbone This affinity causes DNA to be tightly bound to the histone and renders the DNA inaccessible to transcriptional regulatory elements and machinery. The regulation of acetylated states occurs through the balance of activity between two enzyme complexes, histone acetyl transferase (HAT) and histone deacetylase (HDAC). The hypoacetylated state is thought to inhibit transcription of associated DNA. This hypoacetylated state is catalyzed by large multiprotein complexes that include HDAC enzymes. In particular, HDACs have been shown to catalyze the removal of acetyl groups from the chromatin core histones.

Imbalance in these key enzymes can bring disorder to proliferation and differentiation in normal cells, which, in turn, can lead to tumor initiation. Altered expression of HDACs has been reported in association with a number of human cancers. See, for example, Glozak et al,

Histone deacetylases and cancer. Oncogene 26: 5420-5432 (2007); Jones et al., Cell 128: 683- 69292007); Xu et al. -Histone deacetylase inhibitors: Molecular mechanisms of action.

Oncogene 26: 5541-5552 (200); Dokmanovic et al., Histone deacetylase inhibitors: Overview and perspectives.; Mol Cancer Res 5: 981-989 (2007); Blackwell et al., The use of diversity profiling to characterize chemical modulators of the histone deacetylases.; Life Sci 82: 1050- 1058 (2008).

Indeed, it has been shown in several instances that the disruption of HAT or HDAC activity is implicated in the development of a malignant phenotype. For instance, in acute promyelocytic leukemia, the oncoprotein produced by the fusion of PML and RAR alpha appears to suppress specific gene transcription through the recruitment of HDACs (Lin, R. J., Nagy, L., Inoue, S., et al. (1998) Nature 391 :811-14). In this manner, the neoplastic cell is unable to complete differentiation and leads to excess proliferation of the leukemic cell line. As a consequence, the role of histone deacetylases (HDAC) and the potential of these enzymes as therapeutic targets for cancer is an area of rapidly expanding investigation (Jones et al., Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.; Science 321: 1801-1806 (2008)).

HDACi cause accumulation of acetylated forms of proteins which can alter their structure and function. HDACi can induce different phenotypes in various transformed cells, including growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death. Normal cells are relatively resistant to HDACi induced cell death. The result of HDAC inhibition is not believed to have a generalized effect on the genome, but rather, only affects a small subset of the genome (Van Lint, C., Emiliani, S., Verdin, E. (1996) Gene Expression 5:245-53).

Evidence provided by DNA microarrays using malignant cell lines cultured with an HDACi shows that there are a finite (1-2%) number of genes whose products are altered. For example, cells treated in culture with HDACis show a consistent induction of the cyclin-dependent kinase inhibitor p21 (Archer, S. Shufen, M. Shei, A., Hodin, R. (1998) PNAS 95:6791-96). This protein plays an important role in cell cycle arrest. HDACi's are thought to increase the rate of transcription of p21 by propagating the hyperacetylated state of hi stones in the region of the p21 gene, thereby making the gene accessible to transcriptional machinery. Genes whose expression is not affected by HDACi's do not display changes in the acetylation of regional associated histones ( ressel, U., Renkawitz, R., Baniahmad, A. (2000) Anticancer Research 20(2A):1017- 22).

Several HDACi's are in various stages of development, including clinical trials as monotherapy and in combination with other anti-cancer drugs and radiation. A large number of structurally diverse HDACi have been synthesized that often inhibit the activity of all eleven class I and II HDACs. Various HDACi's have been reported to exhibit antitumor activities against hematologic, breast, and bladder malignancies. Although the antitumor activity of HDACis against NSCLC has been indicated previously, these prior studies have been somewhat limited in relation to the number of cell types examined. Also, while these agents demonstrate many features required for anti-cancer activity, such as low toxicity against normal cells and an ability to inhibit tumor cell growth, their mechanisms of action are very complex and largely unknown . Naoki et al., Oncology Reports 15: 187-191 (2006).

The first HDACi approved by the FDA for cancer therapy is suberoylanilide hydroxamic acid (SAH A, vorinostat, Zolinza), approved for treatment of cutaneous T-cell lymphoma. It belongs to a class of agents that have the ability to induce tumor cell growth arrest,

differentiation and/or apoptosis (Richon, V.M., Webb, Y., Merger, R., et al. (1996) PNAS 93:5705-8). These compounds are targeted towards mechanisms inherent to the ability of a neoplastic cell to become malignant, as they do not appear to have toxicity in doses effective for inhibition of tumor growth in animals (Cohen, L.A., Amin, S., Marks, P.A., Rifkind, R.A., Desai, D., and Richon, V.M. (1999) Anticancer Research 19:4999-5006). The inhibition of HDAC by vorinostat is thought to occur through direct interaction with the catalytic site of the enzyme as demonstrated by X-ray crystallography studies (Finnin, M.S., Donigian, J.R., Cohen, A., et al. (1999) Nature 401:188-193).

U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511,990, disclose compounds useful for selectively inducing terminal differentiation of neoplastic cells, which compounds have two polar end groups separated by a flexible chain of methylene groups or a by a rigid phenyl group, wherein one or both of the polar end groups is a large hydrophobic group. Some of the compounds have an additional large hydrophobic group at the same end of the molecule as the first hydrophobic group which further increases differentiation activity about 100 fold in an enzymatic assay and about 50 fold in a cell differentiation assay. Methods of synthesizing the compounds used in the methods and pharmaceutical compositions of this invention are fully described in the aforementioned patents, the entire contents of which are incorporated herein by reference.

Cancer cells typically contain multiple genetic deficits that disrupt key cell pathways including regulation of cell division, cell migration, and cell death. Recent studies have shown that tumors such as pancreatic cancer and glioblastomas have a large number of genetic defects revealing an extremely complex pattern of abnormalities which suggest that therapeutic strategies that target biological pathways and in particular, multiple biological pathways are likely to be more effective therapeutics than drugs targeted at a single gene or protein. This concept supports the need for combinatorial treatment of patients with more man one anti-tumor therapeutic reagent.

While HDACi's have shown synergy when used in combination with chemotherapeutic agents, including, anthracyclines, carboplatinum, taxanes, topoisomerase inhibitors, the nucleoside analogs, gemcitabine and fludarabine, antiangiogenic agents, azacytidine, tyrosine kinase inhibitor, imatinib, proteasome inhibitor, boitezomib, the apoptosis inducer, TRAIL, the heat shock protein (HSP90) antagonist 17-allylamino-l 7-demethoxy-geldanamycin, rituximab, trastuzumab, and the EGFR inhibitor, erlotinib, the art is innocently silent with respect to combining an HDACi with an IGF-1R specific monoclonal antibody.

As well, tumor heterogeneity represents one potential limiting factor for the antitumor activity of inhibitors targeting a single-cellular pathway. For example, if multiple oncogenic processes are active in distinct tumor subpopulations, single-target therapies may provide limited effect. Furthermore, tumor cells are known to exhibit significant genetic instability, possibly enabling adaptation and resistance to specific molecular-targeted agents by switching to alternative growth and survival pathways.

In support of the above concept, accumulating evidence suggests that Vorinostat is not very effective in patients with NSCLC. See for example, Miyanaga et al., Mol Cancer Ther., 7(7): 1923-30. Epub 2008 Jul 7. See also Kakihana et al., J Thorac Oncol., 12:1455-65 (2009). For example, resistance to HDACi may reflect drug efflux, epigenetic alterations, stress response mechanisms and anti-apoptotic, and pro-survival mechanisms (Fantin et al., Clin Cancer Res 13: 7237-7242 (2007)). Resistance to HDACi can also be linked to factors, such as, poor pharmacokinetics and tumor cell micro-environment. Likewise, resistance to HDACi's can be associated with an increased capacity of cancer cells to resist oxidative stress. Investigators in the Oncology field have also suggested that HDACi's cause increased production of ROS, which may be a significant factor in the pro-apoptotic affects of HDACi. Increased activity of thioredoxins and or peroxiredoxins, redox proteins that play a role in protection of cells from ROS, may also cause resistance to HDACi. Norm L et al., Mol Cancer Res 1 : 682-689 (2003); Marks et al., J. Cellular Biochemistry, 107: 00-608 (2009).

Equally frustrating is the fact that to effectively identify a therapeutically effective combination therapeutic protocol which is more effective than the individual approaches alone, requires extensive pre-clinical and clinical testing, and it is not possible without such experimentation to predict which combinations show an additive or even synergistic effect.

What is clear from the above recitation is that while there are many compounds in ongoing or recently completed therapeutic trials, there remains an unmet need for additional therapeutic compounds capable of treating cancer, particularly early stage and advanced or metastasized lung cancer. Besides the aim to increase the therapeutic efficacy, another purpose of combination treatment detailed herein is the potential decrease of the doses of the individual components in the resulting combinations in order to decrease unwanted or harmful side effects caused by higher doses of the individual components.

The present invention attends to the above need by providing efficacious methods for the treatment of cancer, comprising a combination treatment protocol that result in decreased side effects and is effective at treating and controlling malignancies.

SUMMARY OF THE INVENTION

The invention provides improved combination therapeutics and methods for the treatment of cancer in a mammal, typically a human, by administering a combination of an HDACi, for example suberoylanilide hydroxamic acid (SAHA; vorinostat) and an antibody that specifically binds to human Insulin-Like Growth Factor receptor Type 1 (IGF-1R), for example

Dalotuzumab (MK06 6).

The term "antibodies" as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, and humanized or optimized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies to the IGF-1R proteins, including fragments thereof that express the same epitope as that bound by the antibodies of the invention.

It has been unexpectedly discovered that the combination of a first treatment procedure that includes administration of an IGF-1R inhibitor, as described herein, and a second treatment procedure using one or more HDACi's, as described herein, can provide therapeutically effective anticancer effects. Each of the treatments (administration of the IGf-lR inhibitor and

administration of an HDACi) is used in an amount or dose that in combination with the other provides a therapeutically effective treatment. The treatment protocol need not be restricted to the first treatment procedure being limited to an IGF-1R inhibitor and the second treatment procedure being limited to the administration of an HDACi. In a separate embodiment, the cancer is a vorinostat-resistance cancer.

The combination therapy can act through the induction of cancer cell differentiation, cell growth arrest and/or apoptosis. Furthermore, the effect of the IGF-1R inhibitor and the anticancer agent, e.g., HDACi may be additive or synergistic. The combination therapy is particularly advantageous, since the dosage of each agent in a combination therapy can be reduced as compared to monotherapy with the agent, while still achieving an overall anti-tumor effect.

As such, a broad aspect of the invention relies on the surprising discovery of a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an IGF-1R inhibitor, preferably an IGF-1R antibody in a first treatment procedure, and a second amount of an anti-cancer agent, e.g., suberoylanilide hydroxamic acid (SAHA) or a pharmaceutically acceptable salt or hydrate thereof, in a second treatment procedure, wherein the first and second amounts together comprise a therapeutically effective amount.

Treatment of cancer, as used herein, includes partially or totally inhibiting or delaying the progression of cancer including cancer metastasis in a mammal, for example a human.

While the examples detail the treatment of NSCLC, the methods of the present invention are useful in the treatment in a wide variety of cancers, including but not limited to solid tumors (e.g., tumors of the lung, breast, colon, prostate, bladder, rectum, brain or endometrium), hematological malignancies (e.g., leukemias, lymphomas, myelomas), carcinomas (e.g. bladder carcinoma, renal carcinoma, breast carcinoma, colorectal carcinoma), neuroblastoma, or melanoma. Non-limiting examples of these cancers include cutaneous T-cell lymphoma

(CTCL), noncutaneous peripheral T-cell lymphoma, lymphoma associated with human T-cell lymphotrophic virus (HTLV), adult T-cell leukemia lymphoma (ATLL), acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, mesothelioma, childhood solid tumors such as brain neuroblastoma, retinoblastoma, Wilms' tumor, bone cancer and soft-tissue sarcomas, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genito urinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular, rectal and colon), lung cancer, breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain cancer, liver cancer, adrenal cancer, kidney cancer, thyroid cancer, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, medullary carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, Kaposi's sarcoma, neuroblastoma and retinoblastoma.

In an embodiment of the invention, cancer that can be treated or prevented by

administering an anti-IGFl R antibody or antigen-binding fragment thereof and an HDACi is cancer that exhibits IGF1R expression and/or activation of IGF1R.

Thus, in one aspect, the method of the invention comprises administering to a patient in need thereof a first amount of an IGF-1R antibody, comprising light and heavy chains as described infra, in a first treatment procedure, and a second amount of an anti-cancer agent , preferably an HDACi, e.g., SAHA (vorinostat), in a second treatment procedure. The first and second treatments together comprise a therapeutically effective amount.

In another aspect of the invention, the IGF-1R antibody is Dalotuzumab (MK-0646). In yet a further aspect of the invention, administration of the combination results in enhanced therapeutic efficacy relative to administration of the HDACi alone.

In particular embodiments of this invention, the combination of the IGF-1R inhibitor and the anti-cancer agent (HDACi) are additive, i.e. the combination treatment regimen produces a result that is the additive effect of each constituent when it is administered alone. In accordance with this embodiment, the amount of the IGF-1R inhibitor and the HDACi together constitute an effective amount to treat cancer.

In another particular embodiment of this invention, the combination of the IGF-1R antibody and the HDACi is considered therapeutically synergistic when the combination treatment regimen produces a significantly better anticancer result (e.g., reduction in tumor volume, cell growth arrest, apoptosis, induction of differentiation, cell death etc.) than the additive effects of each constituent when it is administered alone at a therapeutic dose. Standard statistical analysis can be employed to determine when the results are significantly better. For example, a Mann-Whitney Test or some other generally accepted statistical analysis can be employed.

The treatment procedures can take place sequentially in any order, simultaneously or a combination thereof. For example, the first treatment procedure, administration of an IGF-1R antibody, can take place prior to the second treatment procedure, i.e. the HDACi, after the second treatment with the HDACi, at the same time as the second treatment procedure, or a combination thereof. For example, a total treatment period can be decided for the either one of IGF-1R or HDACi. The anti-cancer agent, e.g., HDACi can be administered prior to onset of treatment with the IGF-1R antibody or following treatment with the IGF-1R antibody. In addition, treatment with the anti-cancer agent can be administered during the period of IGF-1R antibody administration but does not need to occur over the entire IGF-1R antibody treatment period. Similarly, treatment with the IGF-1R antibody can be administered during the period of anti-cancer agent administration but does not need to occur over the entire anti-cancer agent treatment period. In another embodiment, the treatment regimen includes pre-treatment with one agent, either the IGF-1R antibody or the HDACi, followed by the addition of the second agent for the duration of the treatment period.

In one embodiment of the present invention, the IGF-1R inhibitor can be administered in combination with any one or more of an additional IGF-1R inhibitor or an HDACi, an anti- angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, a gene therapy agent or any combination thereof.

In one particular embodiment of the present invention, the HDACi is suberoylanilide hydroxamic acid (SAHA;vorinostat), which can be administered in combination with any one or more of another HDACi, an anti-angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, or any combination thereof. In yet another aspect of the invention. In another embodiment of the present invention, the IGF-1R is an IGF-1R antibody, preferably Dalotuzumab or one having the light and heavy chains as set forth herein, which can be administered in combination with any one or more of another IGF-1R inhibitor, an anti- angiogenic agent, a differentiation inducing agent, a cell growth arrest inducing agent, an apoptosis inducing agent, a cytotoxic agent, or any combination thereof.

The anti-IGF-1R antibody may be administered via parenteral, e.g., subcutaneous, intratumoral, intravenous, intradermal, oral, transmucosal, or rectal administration. While not intending to be bound to a particular theory of operation, it is believed that blockade of IGF-1R mediated signaling cascade through the administration of an anti-IGF-1R antibody potentiates anti-tumor immunity by negatively modulating the signaling cascade attendant the binding of a native IGF-1R ligand to the receptor.

IGF-1R inhibitors suitable for use in the present invention, include but are not limited to the IGF-1R antibody described and claimed in U.S patent No. 7,241,444. HDACis suitable for use in the present invention, include but are not limited to hydroxamic acid derivatives, Short Chain Fatty Acids (SCFAs), cyclic tetrapeptides, benzamide derivatives, or electrophilic ketone derivatives, as defined herein. Specific non-limiting HDACis suitable for use in the present invention, include but are not limited to hydroxamic acid derivatives, Short Chain Fatty Acids (SCFAs), cyclic tetrapeptides, benzamide derivatives, or electrophilic ketone derivatives, as defined herein. Specific non-limiting examples of HDACis suitable for use in the methods of the present invention are:

A) HYDROXAMIC ACID DERIVATIVES selected from SAHA (vorinostat), Pyroxamide, CBHA, Trichostatin A (TSA), Trichostatin C, Salicylbishydroxamic Acid, Azelaic Bishydroxamic Acid (ABHA), Azelaic-1-Hydroxamate-9-Anilide (AAHA), 6-(3- Chlorophenylureido) carpoic Hydroxamic Acid (3C1-UCHA), Oxamflatin, A-1 1906, Scriptaid, PXD-101, LAQ-824, CHAP, MW2796, and MW2996;

B) CYCLIC TETRAPEPTIDES selected from Trapoxin A, FR901228 (FK 228 or

Depsipeptide), FR225497, Apicidin, CHAP, HC-Toxin, WF27082, and Chlamydocin; C) SHORT CHAIN FATTY ACIDS (SCFAs) selected from Sodium Butyrate, Isovalerate, Valerate, 4 Phenylbutyrate (4-PBA), Phenylbutyrate (PB), Propionate, Butyramide, Isobutyramide, Phenylacetate, 3-Bromopropionate, Tributyrin, Valproic Acid and Valproate; D) BENZAMIDE DERIVATIVES selected from CI-994, MS-27-275 (MS-275) and a 3'- amino derivative of MS-27-275; E) ELECTROPHILIC KETONE DERIVATIVES selected from a trifluoromethyl ketone and an α-keto amide such as an N-methyl- α-ketoamide; and

F) Miscellaneous HDACis including natural products, psammaplins and Depudecin.

Specific HDACis include:

Suberoylanilide hydroxamic acid (SAHA; vorinostat), which is represented by the following structural formula:

Pyroxamide, which is represented by the following structural formula:

m-Carboxycinnamic acid bishydroxamate (CBHA), which is represented by the structural formula:

Other non-limiting examples of HDACis that are suitable for use in the methods of the present invention are:

A compound represented by the structure:

wherein R₃ and R₄ are independently a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, cycloalkyl, aryl, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydroxylamino group; and n is an integer from 5 to 8. A compound represented by the structure:

wherein R is a substituted or unsubstituted phenyl, piperidine, thiazole, 2-pyridine, 3- pyridine or 4-pyridine and n is an integer from 4 to 8.

A compound represented by the structure:

wherein A is an amide moiety, Rl and R2 are each selected from substituted or unsubstituted aryl, arylalkyl, naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; R4 is hydrogen, a halogen, a phenyl or a cycloalkyl moiety and n is an integer from 3 to 10.

The IGF-1R inhibitor (e.g. Dolutuzumab), and the anti-cancer agent (e.g., SAHA;

vorinostat) can be administered by any known administration method known to a person skilled in the art. Examples of routes of administration include but are not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, intraoccular, via local delivery by catheter or stent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in a slow release dosage form.

Of course, the route of administration of vorinostat or any one of the other HDACis is independent of the route of administration of the IGF-1R antibody. A currently preferred route of administration for vorinostat is oral administration. A currently preferred route of

administration for the IGF-1R is one of a parenteral, intraperitoneal, intravenous or

intramuscular injection.

The IGF-1R antibody can be administered in a total daily dose that may vary from patient to patient, and may be administered at varying dosage schedules. Furthermore, the compositions may be administered in cycles, with rest periods in between the cycles (e.g. treatment for two to eight weeks with a rest period of up to a week between treatments). The IGF-1R antibody can be administered in accordance with any dose and dosing schedule that, together with the effect of the HDACi, achieves a dose effective to treat cancer. For example, the IGF-1R antibody (MK- 0646/Dolutuzumab) is administered weekly, and is administered at a dose of 10 mg/kg i.v weekly . In certain embodiments, it is dosed twice a week at 15 mg Kg i.v. Alternative dosing regiment for the IGF-1R antibody is as follows:

(i) 15 mg kg loading, followed by 7.5 mg kg every week.

(ii) 20 mg/kg every other week

(iii) 30 mg kg every three weeks vorinostat or any one of the HDACis can be administered in accordance with any dose and dosing schedule that, together with the effect of the anti-cancer agent, achieves a dose effective to treat cancer. Suitable dosages are total daily dosage of between about 25-4000 mg/m2 administered orally once-daily, twice-daily or three times-daily, continuous (every day) or intermittently (e.g. 3-5 days a week). For example, vorinostat or any one of the HDACis can be administered in a total daily dose of up to 800 mg, preferably orally, once, twice or three times daily, continuously (every day) or intermittently (e.g., 3-5 days a week).

As such, the present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an IGF-1R at a dose of 10 mg/kg i.v weekly in a first treatment procedure, and a second amount of vorinostat at a daily does of up to 800 mg in a second treatment procedure, wherein the first and second amounts together comprise a therapeutically effective amount.

In one embodiment, the HDACi, e.g. vorinostat, is administered in a pharmaceutical composition, preferably suited for oral administration. For example, vorinostat is administered orally in a gelating capsule, which can comprise excipients such as microcrystalline cellulose, croscarmellose sodium and magnesium stearate.

In one embodiment, the composition is administered once daily at a dose of about 200- 600 mg. In another embodiment, the composition is administered twice daily at a dose of about 200-400 mg. In another embodiment, the composition is administered twice daily at a dose of about 200-400 mg intermittently, for example three, four or five days per week. In one embodiment, the daily dose is 200 mg which can be administered once-daily, twice-daily or three-times daily. In one embodiment, the daily dose is 300 mg which can be administered once- daily, twice-daily or three-times daily. In one embodiment, the daily dose is 400 mg which can be administered once-daily, twice-daily or three-times daily.

It is apparent to a person skilled in the art that any one or more of the specific dosages and dosage schedules of the IGF-1R antibody or the HDACi, is also applicable to any one or more of the anti-cancer agents to be used in the combination treatment. Moreover, the specific dosage and dosage schedule of the anti-cancer agent can further vary, and the optimal dose, dosing schedule and route of administration will be determined based upon the specific anti- cancer agent that is being used.

The present invention also provides in vitro methods for selectively inducing cell death, terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells, thereby inhibiting proliferation of such cells, by contacting the cells with a first amount of an IGF-1R antibody and a second amount of suberoylanilide hydroxamic acid (SAHA; vorinostat) or a pharmaceutically acceptable salt or hydrate thereof, wherein the first and second amounts together comprise an amount effective to induce terminal differentiation, cell growth arrest of apoptosis of the cells.

The combination therapy can provide a therapeutic advantage in view of the differential toxicity associated with the two treatment modalities. For example, treatment with an IOF-1R antibody can lead to a particular toxicity that is not seen with the anti-cancer agent, e.g., HDACi and vice versa. As such, this differential toxicity can permit each treatment to be administered at a dose at which said toxicities do not exist or are minimal, such that together the combination therapy provides a therapeutic dose while avoiding the toxicities of each of the constituents of the combination agents.

Furthermore, when the therapeutic effects achieved as a result of the combination treatment are enhanced or synergistic, for example, significantly better than additive therapeutic effects, the doses of each of the agents can be reduced even further, thus lowering the associated toxicities to an even greater extent.

The invention further provides compositions and kits comprising an HDACi and an anti-

IGF-1R antibody for use according to the description provided herein.

The invention further relates to the use of a first amount of an IGF-1R inhibitor and a second amount of an anti-cancer agent, e.g., HDACi and for the manufacture of a medicament for treating cancer.

Other characteristics and advantages of the invention appear in the continuation of the description with the examples and the figures whose legends are represented below.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

Figure 1A. - Graph detailing characterization of Vorinostat response in human NSCLC cell lines - The response of a panel of 14 NSCLC cells to vorinostat-based treatment was measured by MTT assay. IC₅₀ values were determined and arranged by decreasing order. Three independent experiments were performed with similar results; representative results of one experiment are presented. Mutational status and histological information were obtained from COSMIC (www.sanger.ac.uk/genetics/CGP/CellLines/).

Abbreviation: W: wt. M: wt, N:null, U: unknown S: squamous, AS: adenosquamous,. L: large cell, A: adenocarcinoma, B: Bronchioloalveolar carcinoma

Figure 1B shows a graph depicting the results of a soft agar colony forming assay.

Relative colony number in SAHA (vorinostat) 1uM - NSCLC cell lines were assessed for the ability to form colonies in soft agar. Sensitivities to vorinostat were evaluated. Results are expressed as percent colony number on 1uM of vorinostat relative to the number of DMSO- treated controls for each cell lines and are arranged by decreasing order. Three independent experiments were performed with similar results; representative results of one experiment are presented. Colonies formed under control conditions or in the presence of 1 micromolar SAHA (vorinostat) are shown in the micrograph for H1299, A549, H226Br and H358.

Figure 1C shows protein blots of sensitive and resistant cells wherein cleaved PARP (Poly-ADP-ribose polymerase) in each cell line in the presence or absence of vorinostat was determined.

Figure 1D shows a FACS analysis of sensitive and resistant cells wherein Annexin V induction was determined.

Figure 2A details an immunoblot showing that vorinostat induced IGFR expression and activation and p-AKT expression in resistant cells.

Figure 2B details an immunoblot showing that SAHA (vorinostat) downregulated IGFR expression and downstream p-AKT in sensitive cells (H358, A549 and 549M). Vorinostat induced IGFR expression and downstream p-AKT in resistant cells and downregulated IGFR expression and downstream p-AKT in sensitive cells (H358, A549 and 549M). Cells were cultured in complete medium for 48 hours and then in serum-free medium for 24 hours and stimulated with 10% FBS for 15 minutes. Expression of β-actin was used as a loading comparison.

Figure 2C shows an analysis of the spot intensity of receptor tyrosine kinase (RTK) array blots wherein H1299 cells were assayed for expression of various phosphorylated proteins in the presence or absence of vorinostat. Pixel densities in the graph are expressed as percent of the density of control after subtraction of an averaged background signal from each RTK spot. For each phosphoprotein analyzed, the bar on the right represented expression in the absence of vorinostat whereas the bar on the left represented expression in the presence of vorinostat.

Figure 2D shows an immunoblot of cell lines H226B, H460 and H1299, with or without SAHA (vorinostat), using antibodies that bind the indicated antigens.

Figure 2E shows quantitation of the anchorage-independent growth level observed in soft agar assays of the H596, H226Br, H226B, H460 and H1299 cell lines in the presence or absence of dalotuzumab, vorinostat or both. Relative colony numbers are shown (* - p<.05).

Figure 3A shows that IGF-1R antibody MK0646 induced IGF-1R downregulation in vorinostat-resistant cells. H596, H1299, H226Br and H226B cells were exposed to the indicated dose of MK0646 for 5 hours and stimulated with 50 ng/mL of IGF-1 before obtaining whole cell lysates and analyzing to determine the level of IGF1R expression.

Figure 3B depicts a graph that is representative of one experiment detailing minimal cytotoxicity of MK0646 on monolayer culture (MTT assay). H460, H596, H1299, H226Br and H226B cells were exposed to the indicated dose of MK0646 for 72 hours and the cell viability was measured by MTT assay. Three independent experiments were performed with similar results; representative results of one experiment are presented.

Error bars represent s.e.m.

Figure 3C is a graphical depiction detailing the effects of vorinostat and MK-0646 on cells cultured in soft agar. H460, H596, H1299 and H226B cell lines were assessed for the ability to form colonies in soft agar on the indicated concentration of vorinostat, M 0646 or the combination. Three independent experiments were performed with similar results;

representative results of one experiment are presented.

P-values were calculated by Mann-Whitney test

* p <.05 ** p <.01

Error bars represent s.e.m.

Fig 4A presents data relating to xenograft tumor growth in H226Br, H1299, H1944 and H1944/R cells. The combination treatment of vorinostat and dalotuzumab inhibited xenograft tumor growth in nude mice with two representative vorinostat-resistant cells (H226Br and H1299). Each treatment group had either 7 or 8 mice. Vorinostat 50mg/kg (V), dalotuzumab 15mgkg (D) or the combination were given i.p. twice a week.

Fig 4B presents data relating to the effect of vorinostat with or without dalotuzumab in the xenograft model for H1944 and H1944R cells. The tumor growth was inhibited by vorinostat treatment in tumors with H1944 cells, but only combination of vorinostat with dalotuzumab showed xenograft tumor inhibition in tumors with H1944R cells.

Figs 5A-5C present real time quantitative PCR data showing that IGF-2 increased in the resistant cells. (A) Transcription of IGF-1 was not significantly changed after vorinostat treatment in both representative resistant and sensitive cells, while (B) transcription of IGF-2 was significantly induced in the representative resistant cells, by real time quantitative PCR assay. (C) Compared to the parental H1944 cells, IGF-2 increased in H1944/R cells after vorinostat treatment.

DETAILED DESCRIPTION OF THE INVENTION

In its broadest aspect, the present invention relates to a method of treating cancer in a subject in need thereof, by administering to a subject in need thereof a first amount of an IGF-1R inhibitor in a first treatment procedure, and a second amount of an anti-cancer agent e.g., HDACi or a pharmaceutically acceptable salt or hydrate thereof, in a second treatment procedure, wherein the first and second amounts together comprise a therapeutically effective amount. The effect of the IGF-1R inhibitor and the HDACi may be additive or synergistic.

The term "treating" in its various grammatical forms in relation to the present invention refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causative agent (e.g., bacteria or viruses) or other abnormal condition. For example, treatment may involve alleviating a symptom (i.e., not necessary all symptoms) of a disease or attenuating the progression of a disease.

Because some of the inventive methods involve the physical removal of the etiological agent, the artisan will recognize that they are equally effective in situations where the inventive compound is administered prior to, or simultaneous with, exposure to the etiological agent (prophylactic treatment) and situations where the inventive compounds are administered after (even well after) exposure to the etiological agent.

Treatment of cancer, as used herein, refers to partially or totally inhibiting or delaying the progression of cancer including cancer metastasis; or inhibiting or delaying the recurrence of cancer including cancer metastasis; in a mammal, for example a human.

In addition, methods of the present invention include prevention of cancer or metastasis in mammalian (e.g., human) patients.

As used herein, the term "therapeutically effective amount" is intended to qualify the combined amount of the first and second treatments in the combination therapy. The combined amount will achieve a desired biological response. In the present invention, the desired biological response is partial or total inhibition or delay of the progression of cancer including cancer metastasis; inhibition or delay of the recurrence of cancer including cancer metastasis; or the prevention of the onset or development of cancer (chemoprevention) in a mammal, for example a human.

As used herein, the terms "combination treatment", "combination therapy", "combined treatment" or "combinatorial treatment", used interchangeably, refer to a treatment of an individual with at least two different therapeutic agents. According to the invention, the individual is treated with a first therapeutic agent, preferably an IGF-1R antibody. The second therapeutic agent may be another IGF-1R inhibitor, or may be any clinically established anti- cancer agent, preferably vorinostat. A combinatorial treatment may include a third or even further therapeutic agent.

The present invention includes compositions, combinations or kits comprising an anti- IGF1R antibody or antigen-binding fragment thereof in association with an HDACi and, optionally, in association with a further chemotherapeutic agent; as well as method of treating or preventing cancers with such compositions, combinations or kits. The term "in association with" indicates that the components of the compositions, combinations and kits of the invention 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 of a combination of the invention can be administered to a patient 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., orally, intravenously, subcutaneously). In particular embodiments of this invention, the combination of the HDACi and anticancer agent are additive, i.e. the combination treatment regimen produces a result that is the additive effect of each constituent when it is administered alone. In accordance with this embodiment, the amount of HDACi and the amount of the anti-cancer together constitute an effective amount to treat cancer.

In another particular embodiment of this invention, the combination of the IGF-1R inhibitor and the HDACi is considered therapeutically synergistic when the combination treatment regimen produces a significantly better anticancer result (e.g., cell growth arrest, apoptosis, induction of differentiation, cell death) than the additive effects of each constituent when it is administered alone at a therapeutic dose. Standard statistical analysis can be employed to determine when the results are significantly better. For example, a Mann-Whitney Test or some other generally accepted statistical analysis can be employed.

The terms "IGF1R", "IGFR1", "Insulin-like Growth Factor Receptor-I" and "Insulin-like Growth Factor Receptor, type I" are well known in the art. Although IGF-1R may be from any organism, it is preferably from an animal, more preferably from a mammal (e.g., mouse, rat, rabbit, sheep or dog) and most preferably from a human. The nucleotide and amino acid sequence of a typical human IGF-1R precursor is available at Genbank , e.g. Gene ID 3480 or NM000875. Cleavage of the precursor (e.g., between amino acids 710 and 711) produces an α- subunit and a β-subunit which associate to form a mature receptor.

An "immunoglobulin" is a tetrameric molecule. In a naturally-occuring

immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 5070 kDa). The amino- terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as .kappa, and .lambda, light chains. Heavy chains are classified as .mu., .DELTA., .gamma., .alpha., or .epsilon., and define the antibody's isotype as IgM, IgD, IgG (e.g., IgGl, IgG2, IgG3 or lgG4), IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites. The present invention includes compositions including antibodies and antigen-binding fragments thereof comprising any of the constant chains discussed herein; and methods of use thereof.

An "antibody" refers to an intact immunoglobulin or to an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

As used in the application, the term "anti-IGF-1R antibody" is collectively referred to as an anti-IGF-1R antibody disclosed in U.S. Patent No. 7,241,444, filed Dec. 16, 2003, the entire content of which is incorporated by reference herein in its entirety. The amino acid sequences of the various CDRs, light and heavy chain as well as the nucleotide sequences encoding the entire antibody claimed therein area also incorporated in their entirety by reference herein. Likewise, the disclosure of Serial No. 11/801,080 is also incorporated by reference herein in its entirety.

The term "patient" or "subject" or the like includes mammals such as humans.

Antibodies - IGF-1R (h7C10)

As detailed herein, an aspect of the present invention is directed to a method of improving the anti-tumor efficacy of an anti-cancer agent by co-administering an HDACi such as vorinostat, in association with an antibody or antigen-binding fragment thereof which

specifically binds to human Insulin-like growth factor-1 receptor (IGF-1R)-1 to a patient with cancer.

"h7C10" or "M -0646" or "dalotuzumab" is used interchangeably to describe a humanized antibody that is characterized as binding IGF-1R as well as binding the IR/IGF-1 hybrid receptor. The present invention includes compositions comprising such an antibody or an antigen-binding fragment thereof as well as methods of use thereof, e.g., as discussed herein. Such a antibody preferably includes the antibody described in the '444 patent, wherein the antibody or an antigen binding fragment thereof comprises a light chain and/or a heavy chain in which the skeleton segments FR1 to FR4 of said light chain and/or heavy chain are respectively derived from skeleton segments FR1 to FR4 of human antibody light chain and/or heavy chain.

The present invention includes antibodies and antigen-binding fragments thereof comprising the LCDRs and HCDRs set forth below. For example, the antibody (e.g., a humanized antibody) may comprise at least one light chain that comprises at least one or more (e.g., 3) complementary determining regions derived from a non-human source and having the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, or 3:

LCDR1: Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Gln

(SEQ ID NO: 1);

LCDR2: Lys Val Ser Asn Arg Leu Tyr

(SEQ ID NO: 2);

LCDR3: Phe Gln Gly Ser His Val Pro Trp Thr

(SEQ ID NO: 3);

And, at least one heavy chain comprising at least one or more (e.g., 3) complementary determining regions having an amino acid sequence selected from the group consisting of SEQ ID NOs 4, 5 or 6: HCDR1: Gly Gly yr Leu Trp Asn

(SEQ ID NO: 4);

HCDR2: Tyr Ile Ser Tyr Asp Gly Thr Asn Asn Tyr Lys Pro Ser Leu Lys Asp

(SEQ IDNO: 5);

HCDR3: Tyr Gly Arg Val Phe Phe Asp Tyr

(SEQ ID NO: 6).

The antibodies and fragments of the present invention can, in an embodiment of the invention, comprise a light chain comprising one or more of the amino acid sequences as set forth in one of SEQ ID NOs. 7 or 8, or a sequence having at least 80% identity (e.g., 90, 95, 98, or 99%) after optimum alignment with the sequence SEQ ID Nos: 7 or 8; and or a heavy chain that comprises one or more amino acid sequences as set forth in one of SEQ ID No.9, 10 or 11 , or a sequence having at least 80% identity (e.g., 90, 95, 98, or 99%) after optimum alignment with the sequence SEQ ID Nos 9, 10 or 11. Compositions comprising antibodies and antigen- binding fragments comprising the LCDR1, LCDR2 and LCDR3 in a light chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 7 or 8 and/or HCDR1, HCDR2, HCDR3 in a heavy chain immunoglobulin comprising the amino acid sequence set forth in SEQ ID NO: 9, 10 or 11 (e.g., as defined by Kabat or Chothia; see e.g., Kabat, "Sequences of Proteins of Immunological Interest" (National Institutes of Health, Bethesda, Md., 1987 and 1991);

Chothia et al. , J. Mol. Biol. 196:901 (1987); Nature 342:878 (1989); J. Mol. Biol. 186:651 (1989) or Al-Lazikani et al., J. Mol. Biol.273: 927-948 (1997)) are part of the present invention along with methods of use thereof. In certain embodiments, the methods of treatment include administering an antibody that binds the same epitope on IGF-1R as that bound by MK-0646.

As a consequence, the IGF-1R antibody for use in the proposed combination methods according to the invention is one that specifically binds insulin-like growth factor 1 receptor (IGF-1R). Preferably, the IGF-1R antibody as described and claimed in U.S Patent No.

7,241 ,444 ('444 patent) the content of which is incorporated by reference herein in its entirety.

Nucleic acid molecule for expressing the recombinant antibodies (IGF-1R specific mAbs) are described in the '444 patent, the content of which is incorporated by reference herein in its entirety.

"Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single- or double-stranded and, if single-stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. In some

embodiments of the invention, nucleic acids are "isolated." This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

Nucleic acids of the invention also include fragments of the nucleic acids of the invention. A "fragment" refers to a nucleic acid sequence that is preferably at least about 10 nucleic acids in length, more preferably about 40 nucleic acids, and most preferably about 100 nucleic acids in length. A "fragment" can also mean a stretch of at least about 100 consecutive nucleotides that contains one or more deletions, insertions, or substitutions. A "fragment" can also mean the whole coding sequence of a gene and may include 5' and 3' untranslated regions.

The antibodies for use in the present invention include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies (including bi- specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scfv) (including bi-specific scFvs), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and epitope-binding fragments of any of the above. In particular, antibodies for use in the present invention include immunoglobulin molecules and

immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an IGF-1R binding site that immunospecifically binds to IGF-1R. The immunoglobulin molecules for use in the invention can be of any type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Preferably, the antibodies for use in the invention are IgG, more preferably, IgGl .

The antibodies for use in the invention may be from any animal origin. Preferably, the antibodies are humanized monoclonal antibodies. Alternatively, te antibodies may be fully human so long as they bind the same epitope of the antibody claimed in the '444 patent. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice or other animals that express antibodies from human genes.

The antibodies for use in the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may immunospecifically bind to different epitopes of a polypeptide or may immunospecifically bind to both a polypeptide as well a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and ostelny et al., 1992, J. Immunol. 148:1547-1553.

The antibodies for use in the invention include derivatives of the antibodies. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody to be used with the methods for use in the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a preferred embodiment, the derivatives have conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

The antibodies for use in the present invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, synthesis in the presence of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The present invention also provides antibodies for use in the invention that comprise a framework region known to those of skill in the art. In certain embodiments, one or more framework regions, preferably, all of the framework regions, of an antibody to be used in the compositions and methods for use in the invention are human. In certain other embodiments for use in the invention, the fragment region of an antibody for use in the invention is humanized. In certain embodiments, the antibody to be used with the methods for use in the invention is a synthetic antibody, a monoclonal antibody, an intrabody, a chimeric antibody, a human antibody, a humanized chimeric antibody, a humanized antibody, a glycosylated antibody, a multispecific antibody, a human antibody, a single-chain antibody, or a bispecific antibody.

In certain embodiments, an antibody for use in the invention has a high binding affinity for IGF-1R.

In certain embodiments, an antibody for use in the invention has a half-life in a subject, preferably a human, of about 12 hours or more, about 1 day or more, about 3 days or more, about 6 days or more, about 10 days or more, about 15 days or more, about 20 days or more, about 25 days or more, about 30 days or more, about 35 days or more, about 40 days or more, about 45 days or more, about 2 months or more, about 3 months or more, about 4 months or more, or about 5 months or more. Antibodies with increased in vivo half-lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half- lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication No. WO 97/34631 and U.S. patent application Ser. No. 10/020,354, entitled "Molecules with Extended Half-Lives, Compositions and Uses Thereof, filed Dec. 12, 2001, by Johnson et al.; and U.S. Publication Nos.2005/003700 and 2005/0064514, which are incorporated herein by reference in their entireties). Such antibodies can be tested for binding activity to antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

Further, antibodies with increased in vivo half-lives can be generated by attaching to the antibodies polymer molecules such as high molecular weight polyethyleneglycol (PEG). PEG can be attached to the antibodies with or without a multifunctional linker either through site- specific conjugation of the PEG to the N- or C-terminus of the antibodies or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography. PEG-derivatized antibodies can be tested for binding activity to antigens as well as for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

In certain embodiments, an antibody for use in the present invention includes antigen- binding portions of an intact antibody that retain capacity to bind IGF-1R. Examples include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, ambivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chain antibodies are included by reference to the term "antibody."

Methods of Producing Antibodies to IGF-1R are well known. See for example, the '444 patent.

Screening for Antibody Specificity - Techniques for generating antibodies has been described above. One may further select antibodies with certain biological characteristics, as desired. Thus, once produced, the antibodies may be screened for their binding affinity for IGF- 1R. Screening for antibodies that specifically bind to IGF-1R may be accomplished using an enzyme-linked immunosorbent assay (ELISA) in which microtiter plates are coated with IGF- 1R. In some embodiments, antibodies that bind IGF-1R from positively reacting clones can be further screened for reactivity in an ELISA-based assay to other IGF-1R isoforms, for example, IGF-1R using microtiter plates coated with the other IGF-1R isoform(s). Clones that produce antibodies that are reactive to another isoform of IGF-1R are eliminated, and clones that produce antibodies that are reactive to IGF-1R only may be selected for further expansion and

development. Confirmation of reactivity of the antibodies to IGF-1R may be accomplished, for example, using a Western Blot assay in which protein from ovarian, breast, renal, colorectal, lung, endometrial, or brain cancer cells and purified IGF-1R and other IGF-1R isoforms are run on an SDS-PAGE gel, and subsequently are blotted onto a membrane. The membrane may then be probed with the putative anti-IGF-1R antibodies. Reactivity with IGF-1R and not another insulin-like receptor isoform confirms specificity of reactivity for IGF-1R.

General methods for detecting IGF-1R or its Derivatives - The assaying method for detecting IGF-1R using the antibodies of the invention or binding fragments thereof are not particularly limited. Any assaying method can be used, so long as the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen (e.g., the level of IGF-1R) in a fluid to be tested can be detected by chemical or physical means and the amount of the antigen can be calculated from a standard curve prepared from standard solutions containing known amounts of the antigen. Representative immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Pat. Nos.4,367,110 (double monoclonal antibody sandwich assay); Wide et al., Kirkham and Hunter, eds.

Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970); U.S. Pat No. 4,452,901 (western blot); Brown et al., J. Biol. Chem. 255: 4980-4983 (1980) (immunoprecipitation of labeled ligand); and Brooks et al., Clin. Exp. Immunol. 39:477 (1 80) (immunocytochemistry); immunofluorescence techniques employing a fluorescently labeled antibody, coupled with light microscopic, flow cytometric, or fluorometric detection etc. See also Immunoassays for the 80's, A. Voller et al., eds., University Park, 1981, Zola, Monoclonal Antibodies: A Manual of

Techniques, pp. 147-158 (CRC Press, Inc. 1 87).

A typical in vitro immunoassay for detecting IGF-1R comprises incubating a biological sample in the presence of a detectably labeled anti-IGF-1R antibody or antigen binding fragment of the present invention capable of selectively binding to IGF-1R, and detecting the labeled fragment or antibody which is bound in a sample. The antibody is bound to a label effective to permit detection of the cells or portions (e.g., IGF-1R or fragments thereof liberated from hyperplastic, dysplastic and/or cancerous cells) thereof upon binding of the antibody to the cells or portions thereof. The presence of any cells or portions thereof in the biological sample is detected by detection of the label.

The biological sample may be brought into contact with, and immobilized onto, a solid phase support or carrier, such as nitrocellulose, or other solid support or matrix, which is capable of immobilizing cells, cell particles, membranes, or soluble proteins. The support may then be washed with suitable buffers, followed by treatment with the detectably-labeled anti-IGF-1R antibody. The solid phase support may then be washed with buffer a second time to remove unbound antibody. The amount of bound label on the solid support may then be detected by conventional means. Accordingly, in another embodiment of the present invention,

compositions are provided comprising the monoclonal antibodies, or binding fragments thereof, bound to a solid phase support, such as described herein.

By "solid phase support" or "carrier" is intended any support capable of binding peptide, antigen or antibody. Well-known supports or carriers, include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to IGF- IR or an Anti-IGF-1R antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.

Alternatively, the surface can be flat, such as a sheet, culture dish, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody, peptide or antigen, or can ascertain the same by routine

experimentation.

In vitro assays in accordance with the present invention also include the use of isolated membranes from cells expressing a recombinant IGF-1R, soluble fragments comprising the ligand binding segments of IGF-1R, or fragments attached to solid phase substrates. These assays allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or ligand mutations and modifications, e.g., ligand analogues.

Assays For Efficacy of Combination Immunotherapy in in vivo Models - Tumor burden can be assessed at various time points after tumor challenge using techniques well known in the art Assays for monitoring anti-tumor response and determining the efficacy of combination immunotherapy are described below. While an improved or enhanced anti-tumor response may be most dramatically observed shortly following administration of the immunotherapy, e.g. within 5-10 days, the response may be delayed in some instances, depending on factors such as the expression level of IGF-1R, the dosage and dosing frequency of the anti-IGF-1R antibody, and the relative timing of administration of the IGF-1R inhibitor (IGF-1R antibody) relative to the timing of administration of the HDACi- vorinostat. Thus, any of the well known assays may be performed on biological samples harvested at various time points following treatment or administration of the combination therapeutic in order to fully assess the anti-tumor response following immunotherapy.

Monitoring Treatment · One skilled in the art is aware of means to monitor the therapeutic outcome and/or the systemic immune response upon administering a combination treatment of the present invention. In particular, the therapeutic outcome can be assessed by monitoring attenuation of tumor growth and/or tumor regression and or the level of tumor specific markers. The attenuation of tumor growth or tumor regression in response to treatment can be monitored using one or more of several end-points known to those skilled in the art including, for instance, number of tumors, tumor mass or size, or reduction/prevention of metastasis.

Histone Deacetylases and Histone Deacetylase Inhibitors

A broad aspect of the invention provides methods of effectively treating cancers without significant adverse effects to the human patient subject to treatment. The clinical outcomes of the treatment according to the invention are somewhat unexpected, in that the combination therapeutic comprising an anti-IGF-1R antibody and vorinostat are thought to be more effective in treating erlotinib resistant cancers. As well, the combination therapeutic is thought to be more effective in treating various cancers than erlotinib by itself. It is understood that other HDACis may be combined with the IGF-1R antibody. As well, the IGF-1R inhibitor need not be limited to an antibody. It may comprise any IGF-1R inhibiting moiety. Alternatively, the combination therapeutic may comprise more than one HDACi inhibitor thus comprising an anti-IGF-1R antibody combined with a chemotherapy cocktail comprising at least two or more

chemotherapeutic agents which do not significantly increase incident occurrences of adverse events, when compared with the chemotherapeutic alone.

Histone deacetylases (HDACs), as that term is used herein, are enzymes that catalyze the removal of acetyl groups from lysine residues in the amino terminal tails of the nucleosomal core histones. As such. HDACs together with histone acetyl transferases (HATs) regulate the acetylation status of histones. Histone acetylation affects gene expression and inhibitors of HDACs, such as the hydroxamic acid-based hybrid polar compound suberoylanilide

hydroxamic acid (SAHA; vorinostat) induce growth arrest, differentiation and or apoptosis of transformed cells in vitro and inhibit tumor growth in vivo. HDACs can be divided into three classes based on structural homology. Class I HDACs (HDACs 1 , 2, 3 and 8) bear similarity to the yeast RPD3 protein, are located in the nucleus and are found in complexes associated with transcriptional co-repressors. Class II HDACs (HDACs 4, 5, 6, 7 and 9) are similar to the yeast HDA1 protein, and have both nuclear and cytoplasmic subcellular localization. Both Class I and II HDACs are inhibited by hydroxamic acid-based HDACis, such as vorinostat. Class III

HDACs form a structurally distant class of NAD dependent enzymes that are related to the yeast SIR2 proteins and are not inhibited by hydroxamic acid-based HDACis.

Histone deacetylase inhibitors or HDACis, as that term is used herein are compounds that are capable of inhibiting the deacetylation of histones in vivo, in vitro or both. As such, HDACis inhibit the activity of at least one histone deacetylase. As a result of inhibiting the deacetylation of at least one histone, an increase in acetylated histone occurs and accumulation of acetylated histone is a suitable biological marker for assessing the activity of HDACis. Therefore, procedures that can assay for the accumulation of acetylated histones can be used to determine the HDACi activity of compounds of interest. It is understood that compounds that can inhibit histone deacetylase activity can also bind to other substrates and as such can inhibit other biologically active molecules such as enzymes. It is also to be understood that the compounds of the present invention are capable of inhibiting any of the histone deacetylases set forth above, or any other histone deacetylases.

For example, in patients receiving HDACis, the accumulation of acetylated histones in peripheral mononuclear cells as well as in tissue treated with an HDACi can be determined against a suitable control.

HDACi activity of a particular compound can be determined in vitro using, for example, an enzymatic assay which shows inhibition of at least one histone deacetylase. Further, determination of the accumulation of acetylated histones in cells treated with a particular composition can be determinative of the HDACi activity of a compound.

Assays for the accumulation of acetylated histones are well known in the literature. See, for example, Marks, P.A. et al, J. Natl. Cancer Inst., 92:1210-1215, 2000, Butler, L.M. et l., Cancer Res. 60:5165-5170 (2000), Richon, V. M. et al., Proc. Natl. Acad. Sci., USA, 95:3003- 3007, 1998, and Yoshida, M. et l., J. Biol. Chem., 265:17174-17179, 1990.

For example, an enzymatic assay to determine the activity of an HDACi compound can be conducted as follows. Briefly, the effect of an HDACi compound on affinity purified human epitope-tagged (Flag) HDAC1 can be assayed by incubating the enzyme preparation in the absence of substrate on ice for about 20 minutes with the indicated amount of inhibitor compound. Substrate ([³H]acetyl-labeled murine erythroleukemia cell-derived histone) can be added and the sample can be incubated for 20 minutes at 37°C in a total volume of 30 μL· The reaction can then be stopped and released acetate can be extracted and the amount of radioactivity release determined by scintillation counting. An alternative assay useful for determining the activity of an HDACi compound is the "HDAC Fluorescent Activity Assay; Drug Discovery Kit-AK-500" available from BIOMOL® Research Laboratories, Inc., Plymouth Meeting, PA.

In vivo studies can be conducted as follows. Animals, for example, mice, can be injected intraperitoneally with an HDACi compound. Selected tissues, for example, brain, spleen, liver etc, can be isolated at predetermined times, post administration. Histones can be isolated from tissues essentially as described by Yoshida et al., J. Biol. Chem. 265:17174-17179, 1990. Equal amounts of histones (about 1 g) can be electrophoresed on 15% SDS-polyacrylamide gels and can be transferred to Hybond-P filters (available from Amersham). Filters can be blocked with 3% milk and can be probed with a rabbit purified polyclonal anti-acetylated histone H4 antibody (αAc-H4) and anti-acetylated histone H3 antibody (αAc-H3) (Upstate Biotechnology, Inc.). Levels of acetylated histone can be visualized using a horseradish peroxidase-conjugated goat anti-rabbit antibody (1:5000) and the SuperSignal chemiluminescent substrate (Pierce). As a loading control for the histone protein, parallel gels can be run and stained with Coomassie Blue (CB).

In addition, hydroxamic acid-based HDACis have been shown to up regulate the expression of the p21^WAF1 gene. The p21^WAF1 protein is induced within 2 hours of culture with HDACis in a variety of transformed cells using standard methods. The induction of the p21^WAF1 gene is associated with accumulation of acetylated histones in the chromatin region of this gene. Induction of p21^WAF1 can therefore be recognized as involved in the G1 cell cycle arrest caused by HDACis in transformed cells.

HDACis are effective at treating a broader range of diseases characterized by the proliferation of neoplastic diseases, such as any one of the cancers described herein. However, the therapeutic utility of HDACis is not limited to the treatment of cancer. Rather, there is a wide range of diseases for which HDACis have been found useful.

Typically, HDACis fall into five general classes: 1 ) hydroxamic acid derivatives; 2)

Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; and 5) electrophilic ketones.

Thus, the present invention includes within its broad scope compositions comprising HDACis which are 1) hydroxamic acid derivatives; 2) Short-Chain Fatty Acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting histone deacetylases, for use in inhibiting histone deacetylase, inducing terminal differentiation, cell growth arrest and/or apoptosis in neoplastic cells, and/or inducing differentiation, cell growth arrest and/or apoptosis of tumor cells in a tumor. Non-limiting examples of such HDACis are set forth below. It is understood that the present invention includes any salts, crystal structures, amorphous structures, hydrates, derivatives, metabolites, stereoisomers, structural isomers and prodrugs of the HDACis described herein.

A. Hydroxamic Acid Derivatives such as suberoylanilide hydroxamic acid (SAHA;

vorinostat) (Richon et al., Proc. Natl. Acad. Sci. USA 95,3003-3007 (1 98)); m- carboxycinnamic acid bishydroxamide (CBHA) (Richon et al., supra); pyroxamide; trichostatin analogues such as trichostatin A (TSA) and trichostatin C ( oghe et al. 1998. Biochem.

Pharmacol. 56: 1359-1364); salicylbishydroxamic acid (Andrews et al., International J.

Parasitology 30,761-768 (2000)); suberoyl bishydroxamic acid (SBHA) (U.S. Patent No.

5,608,108); azelaic bishydroxamic acid (ABHA) (Andrews et al., supra); azelaic-1-hydroxamate- 9-anilide (AAHA) (Qiu et al., Mol. Biol. Cell 11, 2069-2083 (2000)); 6-(3-chlorophenylureido) carpoic hydroxamic acid (3C1-UCHA); oxam latin [(2E)-5-[3-[(phenylsufonyl) aminol phenyl]- pent-2-en-4-ynohydroxamic acid] (Kim et al. Oncogene, 18: 2461 2470 (1999)); A-161906, Scriptaid (Su et al. 2000 Cancer Research, 60: 3137-3142); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews et al., supra); MW2996 (Andrews et al., supra); or any of the hydroxamic acids disclosed in U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, ,087,367 and 6,511, 990.

B. Cyclic Tetrapeptides such as trapoxin A (TPX)-cyclic tetrapeptide (cyclo-(L- phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amino-8-oxo-9,10-epoxy decanoyl)) (Kijima et al, J Biol. Chem. 268,22429-22435 (1993)); FR901228 (FK 228, depsipeptide) (Nakajima et al., Ex. Cell Res. 241,126-133 (1998)); FR225497 cyclic tetrapeptide (H. Mori et al., PCT

Application WO 00/08048 (17 February 2000)); apicidin cyclic tetrapeptide [cyclo(N-O-methyl- L-tryptophanyl-L -isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)] (Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93,1314313147 (1996)); apicidin Ia, apicidin Ib, apicidin Ic, apicidin IIa, and apicidin IIb (P. Dulski et al., PCT Application WO 97/11366); CHAP, HC-toxin cyclic tetrapeptide (Bosch et al , Plant Cell 7, 1941-1950 (1995)); WF27082 cyclic tetrapeptide (PCT Application WO 98/48825); and chlamydocin (Bosch et al., supra).

C. Short chain fatty acid (SCFA) derivatives such as: sodium

butyrate (Cousens et al , J. Biol. Chem. 254,1716-1723 (1979)); isovalerate (McBain et al , Biochem. Pharm. 53: 1357-1368 (1997)); valerate (McBain et al., supra) ; 4-phenylbutyrate (4- PBA) (Lea and Tulsyan, Anticancer Research, 15,879-873 (1995)); phenylbutyrate (PB) (Wang et al, Cancer Research, 59, 2766-2799 (1999)); propionate (McBain et al., supra); butyramide (Lea and Tulsyan, supra); isobutyramide (Lea and Tulsyan, supra); phenylacetate (Lea and Tulsyan, supra); 3-bromopropionate (Lea and Tulsyan, supra); tributyrin (Guan et al., Cancer Research, 60,749-755 (2000)); valproic acid, valproate and Pivanex™.

D. Benzamide derivatives such as CI-994; MS-275 [N- (2-aminophenyl)-4- [N- (pyridin-3- yl methoxycarbonyl) aminomethyl] benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA 96, 4592-4597 (1999)); and 3'-amino derivative of MS-275 (Saito etal., supra).

E. Electrophilic ketone derivatives such as trifluororaethyl ketones (Frey et al, Bioorganic & Med. Chem. Lett. (2002), 12, 3443-3447; U.S. 6,511,990) and α-keto amides such as N-methyl- α-ketoamides

F. Other HDACis such as natural products, psammaplins and Depudecin ( won et al. 1 98. PNAS 95: 3356-3361).

Preferred hydroxamic acid based HDACis are suberoylanilide hydroxamic acid (SAHA; vorinostat), m-carboxycinnamic acid bishydroxamate (CBH A) and pyroxamide. vorinostat has been shown to bind directly in the catalytic pocket of the histone deacetylase enzyme, vorinostat induces cell cycle arrest, differentiation and/or apoptosis of transformed cells in culture and inhibits tumor growth in rodents, vorinostat is effective at inducing these effects in both solid tumors and hematological cancers. It has been shown that vorinostat is effective at inhibiting tumor growth in animals with no toxicity to the animal. The vorinostat -induced inhibition of tumor growth is associated with an accumulation of acetylated histones in the tumor, vorinostat is effective at inhibiting the development and continued growth of carcinogen-induced (N- methylnitrosourea) mammary tumors in rats, vorinostat was administered to the rats in their diet over the 130 days of the study. Thus, vorinostat is a nontoxic, orally active antitumor agent whose mechanism of action involves the inhibition of histone deacetylase activity.

Preferred HDACis are those disclosed in U.S. Patent Numbers 5,369,108, 5,932,616, 5,700,811, 6,087,367 and 6,511, 990, issued to some of the present inventors disclose compounds, the entire contents of which are incorporated herein by reference, non-limiting examples of which are set forth below:

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 1, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R₁ and R₂ can be the same or different; when R₁ and R₂ are the same, each is a substituted or unsubstituted arylamino, cycloalkylamino, pyridineamino, piperidino, 9- purine-6- amine or thiazoleamino group; when R₁ and R₂ are different R₁=R₃-N-R₄, wherein each of R₃ and R₄ are independently the same as or different from each other and are a hydrogen atom, a hydroxy, group, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl alkyloxy, aryloxy, arylalkyloxy or pyridine group, or R₃ and R₄ are bonded together to form a piperidine group, R₂ is a hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino or alkyloxy group and n is an integer from about 4 to about 8.

In a particular embodiment of formula 1, R₁ and R₂ are the same and are a substituted or unsubstituted thiazoleamino group; and n is an integer from about 4 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 2, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₃ and R₄ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, arylalkyloxy, aryloxy, arylalkyloxy or pyridine group, or R₃ and R₄ are bonded together to form a piperidine group, R₂ is a hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino or alkyloxy group and n is an integer from about 4 to about 8.

In a particular embodiment of formula 2, each of R₃ and R₄ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydroxylamino, hydroxyl, amino, alkylamino, or alkyloxy group; n is an integer from 5 to 7; and R₃-N-R₄ and R₂ are different.

In another particular embodiment of formula 2, n is 6. In yet another embodiment of formula 2, R₄ is a hydrogen atom, R₃ is a substituted or unsubstituted phenyl and n is 6. In yet another embodiment of formula 2, R₄ is a hydrogen atom, R₃ is a substituted phenyl and n is 6, wherein the phenyl substituent is selected from the group consisting of a methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro, 2,4-difluoro, 2,5-difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro, 2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methoxy, phenyloxy, benzyloxy, phenylaminooxy,

phenylaminocarbonyl, methoxycarbonyl, methylaminocarbonyl, dimethylamino, dimethylamino carbonyl, or hydroxylaminocarbonyl group.

In another embodiment of formula 2, n is 6, R₄ is a hydrogen atom and R₃ is a cyclohexyl group. In another embodiment of formula 2, n is 6, R₄ is a hydrogen atom and R₃ is a methoxy group. In another embodiment of formula 2 n is 6 and R₃ and R₄ bond together to form a piperidine group. In another embodiment of formula 2, n is 6, R₄ is a hydrogen atom and R₃ is a benzyloxy group. In another embodiment of formula 2, R₄ is a hydrogen atom and R₃ is a γ- pyridine group. In another embodiment of formula 2, R₄ is a hydrogen atom and R₃ is a β- pyridine group. In another embodiment of formula 2, R₄ is a hydrogen atom and R₃ is an a- pyridine group. In another embodiment of formula 2, n is 6, and R₃ and R₄ are both methyl groups. In another embodiment of formula 2, n is 6, R₄ is a methyl group and R₃ is a phenyl group.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 3, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein n is an integer from 5 to about 8.

In a preferred embodiment of formula 3, n is 6. In accordance with this embodiment, the HDACi is vorinostat (4), or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient. vorinostat can be represented by the following structural formula:

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 5, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 6 (pyroxamide), or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 7, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 8, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 9, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

(9)

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 10, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R₃ is hydrogen and R₄ cycloalkyl, aryl, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydroxylamino group; and n is an integer from 5 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 11 , or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R₃ and R₄ are independently a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, cycloalkyl, aryl, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydroxylamino group; and n is an integer from 5 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 12, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group; R is a hydrogen atom, a hydroxyl, group, a substituted or unsubstituted alkyl, arylalkyloxy, or aryloxy group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8.

In a particular embodiment, the HDACi is a compound of formula 12 wherein X, Y and

R are each hydroxyl and both m and n are 5.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 13, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

0 whe)rein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino or aryloxyalkylamino group; each of R₁ and R₂ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, aryl, alkyloxy, or aryloxy group; and each of m, n and o are independently the same as or different from each other and are each an integer from about 0 to about 8.

In one particular embodiment of formula 13, each of X and Y is a hydroxyl group and each of R₁ and R₂ is a methyl group. In another particular embodiment of formula 1 , each of X and Y is a hydroxyl group, each of R₁ and R₂ is a methyl group, each of n and o is 6, and m is 2.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 14, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino or aryloxyalkylamino group; each of R₁ and R₂ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, aryl, alkyloxy, or aryloxy group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 15, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino or aryloxyalkylamino group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8.

In one particular embodiment of formula 15, each of X and Y is a hydroxyl group and each of m and n is 5.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 16, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino or aryloxyalkylamino group; R₁ and R₂ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, arylalkyloxy or aryloxy group; and each of m and n are independently the same as or different from each other and are each an integer from about 0 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 17, or a pharmaceutically acceptable salt or hydrate thereof and a pharmaceutically acceptable carrier or excipient

wherein each of X an Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylarylamino, or aryloxyalkylamino group; and n is an integer from about 0 to about 8.

In one particular embodiment of formula 17, each of X and Y is a hydroxylamino group; R₁ is a methyl group, R₂ is a hydrogen atom; and each of m and n is 2. In another particular embodiment of formula 17, each of X and Y is a hydroxylamino group; R₁ is a

carbonylhydroxylamino group, R₂ is a hydrogen atom; and each of m and n is 5. In another particular embodiment of formula 17, each of X and Y is a hydroxylamino group; each of R₁ and R₂ is a fluoro group; and each of m and n is 2.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 18, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of X and Y are independently the same as or different from each other and are a hydroxyl, amino or hydroxylamino group, a substituted or unsubstituted alkyloxy, alkylamino, dialkylamino, arylamino, alkylaryl amino, alkyloxyamino, aryloxyamino, alkyloxyalkyamino or aryloxyalkylamino group; each of R₁ and R₂ are independently the same as or different from each other and are a hydrogen atom, a hydroxyl group, a substituted or unsubstituted alkyl, aryl, alkyloxy, aryloxy, carbonylhydroxylamino or fluoro group; and each of m and n are

independently the same as or different from each other and are each an integer from about 0 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 19, or a pharmaceutically acceptable salt

thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₁ and R₂ are independently the same as or different from each other and are a hydroxyl, alkyloxy, amino, hydroxylamino, alkylamino, dialkylamino, arylamino,

alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group. In a particular embodiment, the HDACi is a compound of structural formula 19 wherein R₁ and R₂ are both hydroxylamino.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 20, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₁ and R₂ are independently the same as or different from each other and are a hydroxyl, alkyloxy, amino, hydroxylamino, alkylamino, dialkylamino, arylamino,

alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group. In a particular embodiment, the H ACi is a compound of structural formula 20 wherein R₁ and R₂ are both hydroxylamino.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 21, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₁ and R₂ are independently the same as or different from each other and are a hydroxyl, alkyloxy, amino, hydroxylamino, alkylamino, dialkylamino, arylamino,

alkylarylamino, alkyloxyamino, aryloxyamino, alkyloxyalkylamino, or aryloxyalkylamino group.

In a particular embodiment, the HDACi is a compound of structural formula 21 wherein R₁ and R₂ are both hydroxylamino

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 22, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R is a phenylamino group substituted with a cyano, methylcyano, nitro, carboxyl, aminocarbonyl, methylaminocarbonyl, dimethylaminocarbonyl, trifluoromethyl,

hydroxylaminocarbonyl, N-hydroxylaminocarbonyl, methoxycarbonyl, chloro, fluoro, methyl, methoxy, 2,3-difluoro, 2,4-difluoro, 2,5-difluoro, 2,6-difuloro, 3,5-difluoro, 2,3,6-trifluoro, 2,4,6- trifluoro, 1,2,3-trifluoro, 3,4,5-trifluoro, 2,3,4,5-tetrafluoro, or 2,3,4,5,6-pentafluoro group; and n is an integer from 4 to 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 23 (m-carboxycinnamic acid bishydroxamide - CBHA), or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 24, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 25, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R is a substituted or unsubstituted phenyl, piperidine, thiazole, 2-pyridine, 3- pyridine or 4-pyridine and n is an integer from about 4 to about 8.

In one particular embodiment of formula 25, R is a substituted phenyl group. In another particular embodiment of formula 25, R is a substituted phenyl group, where the substituent is selected from the group consisting of methyl, cyano, nitro, thio, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro, 2,4-difluoro, 2,5- difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro, 2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methyloxy, phenyloxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methyloxycarbonyl, methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl, or hydroxylaminocarbonyl group.

In another particular embodiment of formula 25, R is a substituted or unsubstituted 2- pyridine, 3-pyridine or 4-pyridine and n is an integer from about 4 to about 8.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 26, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein R is a substituted or unsubstituted phenyl, pyridine, piperidine or thiazole group and n is an integer from about 4 to about 8 or a pharmaceutically acceptable salt thereof.

In a particular embodiment of formula 26, R is a substituted phenyl group. In another particular embodiment of formula 26, R is a substituted phenyl group, where the substituent is selected from the group consisting of methyl, cyano, nitro, thio, trifluoromethyl, amino, aminocarbonyl, methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro, 2,4-difluoro, 2,5- difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro, 2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro, 2,3,4,5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl, hydroxyl, methyloxy, phenyloxy, benzyloxy, phenylaminooxy, phenylaminocarbonyl, methyloxycarbonyl, methylaminocarbonyl, dimethylamino, dimethylaminocarbonyl, or hydroxylaminocarbonyl group.

In another particular embodiment of formula 26, R is phenyl and n is 5. In another embodiment, n is 5 and R is 3-chlorophenyl.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 27, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₁ and R₂ is directly attached or through a linker and is substituted or unsubstituted, aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridineamino, piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, pyridyl, or quinolinyl or isoquinolinyl; n is an integer from about 3 to about 10 and R₃ is a hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group. The linker can be an amide moiety, e.g., O-, -S-, -NH-, NR₅, - CH₂-, - CH₂)_m- , -(CH=CH)-, phenylene, cycloalkylene, or any combination thereof, wherein R₅ is a substitute or unsubstituted C₁-C₅ alkyl.

In certain embodiments of formula 27, R₁ is -NH-R₄ wherein R₄ is substituted or unsubstituted, aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridineamino, piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 28, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

wherein each of R₁ and R₂ is, substituted or unsubstituted, aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, cycloalkyl, cycloalkylamino, pyridineamino, piperidino, 9-purine-6-amino, thiazoleamino, hydroxyl, branched or unbranched alkyl, alkenyl, alkyloxy, aryloxy,

arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; R₃is hydroxamic acid, hydroxylamino, hydroxyl, amino, alkylamino or alkyloxy group; R₄ is hydrogen, halogen, phenyl or a cycloalkyl moiety; and A can be the same or different and represents an amide moiety, O-, -S-, -NH-, NR₅, - CH₂-, - CH₂)_m-, -(CH=CH)-, phenylene, cycloalkylene, or any combination thereof wherein R₅ is a substitute or unsubstituted C₁-C₅ alkyl; and n and m are each an integer from 3 to 10.

In further particular embodiment compounds having a more specific structure within the scope of compounds 27 or 28 are:

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 29:

wherein A is an amide moiety, R₁ and R₂ are each selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino,

thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; and n is an integer from 3 to 10.

For example, the compound of formula 29 can have the structure 30 or 31 :

wherein R₁, R₂ and n have the meanings of formula 29.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 32:

wherein R₇ is selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; n is an integer from 3 to 10 and Y is selected from:

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 33:

wherein n is an integer from 3 to 10, Y is selected from

and R_7' is selected from

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 34:

aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; n is an integer from 3 to 10 and R_7' is selected from

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 35:

wherein A is an amide moiety, R₁ and R₂ are each selected from substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl; R₄ is hydrogen, a halogen, a phenyl or a cycloalkyl moiety and n is an integer from 3 to 10.

For example, the compound of formula 35 can have the structure 36 or 37:

wherein R₁, R₂, R₄ and n have the meanings of formula 35.

In one embodiment, the HDACi useful in the methods of the present invention is represented by the structure of formula 38 :

wherein L is a linker selected from the group consisting of an amide moiety, 0-, -S-, -NH-, NR₅, -CH₂-, - CH₂)_m-, -(CH=CH)-, phenylene, cycloalkylene, or any combination thereof wherein R₅ is a substitute or unsubstituted C₁-C₅ alkyl; and wherein each of R₇ and R₈ are independently a substituted or unsubstituted aryl (e.g., phenyl), arylalkyl (e.g., benzyl), naphthyl, pyridineamino, 9-purine-6-amino, thiazoleamino, aryloxy, arylalkyloxy, pyridyl, quinolinyl or isoquinolinyl;, n is an integer from 3 to 10 and m is an integer from 0-10.

For example, a compound of formula 38 can be represented by the structure of formula

(39):

Other HDAC is suitable for use in the methods of the present invention include those shown in the following more specific formulas:

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 40, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 41, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10 or an enantiomer thereof. In one particular embodiment of formula 42, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 43, n=5.

A compound represented by the structure:

(44)

wherein n is an integer from 3 to 1 ,0 or an enantiomer thereof. In one particular embodiment of formula 44, n=5. A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 45, n=5.

wherein n is an integer from 3 to 10 or an enantiomer thereof. In one particular embodiment of formula 46, n-5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof In one particular embodiment of formula 47, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 48, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 49, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 50, n=5.

A compound represented by the structure:

wherein n is an integer from 3 to 10, or an enantiomer thereof. In one particular embodiment of formula 51, n=5.

Other examples of such compounds and other HDACis can be found in U.S. Patent No.

5,369,108, issued on November 29, 1994, U.S. Patent No. 5,700,811, issued on December 23, 1997, U.S. Patent No. 5,773,474, issued on June 30, 1998, U.S. Patent No. 5,932,616, issued on August 3, 1999 and U.S. Patent No. 6,511,990, issued January 28, 2003, all to Breslow et al.,' U.S. Patent No. 5,055,608, issued on October 8, 1991, U.S. Patent No. 5,175,191, issued on December 29, 1992 and U.S. Patent No. 5,608, 108, issued on March 4, 1997, all to Marks et al ; as well as Yoshida, M., et al, Bioassays 17, 423-430 (1995); Saito, A., et al., PNAS USA 96, 4592-4597, (1999); Furamai R. et αί, PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et al., Cancer Res. 61(11), 4459-4466 (2001); Su, G.H., et al, Cancer Res. 60, 3137-3142 (2000); Lee, B.I. et al, Cancer Res. 61(3), 931-934; Suzuki, T., et al, J. Med. Chem.42(15), 3001-3003 (1999); published PCT Application WO 01/18171 published on March 15, 2001 to Sloan- ettering Institute for Cancer Research and The Trustees of Columbia University; published PCT Application WO02/246144 to Hoffmann-La Roche; published PCT Application WO02/22577 to Novartis; published PCT Application WO02/30879 to Prolifix; published PCT Applications WO 01/38322 (published May 31, 2001), WO 01/70675 (published on September 27, 2001) and WO 00/71703 (published on November 30, 2000) all to Methylgene, Inc.;

published PCT Application WO 00/21979 published on October 8, 1999 to Fujisawa

Pharmaceutical Co., Ltd. ; published PCT Application WO 98/40080 published on March 11 , 1998 to Beacon Laboratories, L.L.C.; and Curtin M. (Current patent status of HDACis Expert Opin. Ther. Patents (2002) 12(9): 1375-1384 and references cited therein).

vorinostat or any of the other HDACs can be synthesized according to the methods outlined in the Experimental Details Section, or according to the method set forth in U.S. Patent Nos. 5,369,108, 5,700,811, 5,932,616 and 6,511,990, the contents of which are incorporated by reference in their entirety, or according to any other method known to a person skilled in the art.

Specific non-limiting examples of HDACis are provided in the Table below. It should be noted that the present invention encompasses any compounds which are structurally similar to the compounds represented below, and which are capable of inhibiting histone deacetylases.

Chemical Definitions

An "aliphatic group" is non-aromatic, consists solely of carbon and hydrogen and can optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group can be straight chained, branched or cyclic. When straight chained or branched, an aliphatic group typically contains between about 1 and about 12 carbon atoms, more typically between about 1 and about 6 carbon atoms. When cyclic, an aliphatic group typically contains between about 3 and about 10 carbon atoms, more typically between about 3 and about 7 carbon atoms. Aliphatic groups are preferably C₁-C₁₂ straight chained or branched alkyl groups (i.e., completely saturated aliphatic groups), more preferably C|-C« straight chained or branched alkyl groups. Examples include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl and tert-butyl.

An "aromatic group" (also referred to as an "aryl group") as used herein includes carbocyclic aromatic groups, heterocyclic aromatic groups (also referred to as "heteroaryl") and fused polycyclic aromatic ring system as defined herein.

A "carbocyclic aromatic group" is an aromatic ring of 5 to 14 carbons atoms, and includes a carbocyclic aromatic group fused with a 5 -or 6-membered cycloalkyl group such as indan. Examples of carbocyclic aromatic groups include, but are not limited to, phenyl, naphthyl, e.g., 1 -naphthyl and 2-naphthyl; anthracenyl, e.g., 1-anthracenyl, 2-anthracenyl; phenanthrenyl; fluorenonyl, e.g., 9-fluorenonyl, indanyl and the like. A carbocyclic aromatic group is optionally substituted with a designated number of substituents, described below.

A "heterocyclic aromatic group" (or "heteroaryl") is a monocyclic, bicyclic or tricyclic aromatic ring of 5- to 14-ring atoms of carbon and from one to four heteroatoms selected from O, N, or S. Examples of heteroaryl include, but are not limited to pyridyl, e.g., 2-pyridyl (also referred to as "α-pyridyl), 3-pyridyl (also referred to as β-pyridyl) and 4-pyridyl (also referred to as (γ-pyridyl); thienyl, e.g., 2-thienyl and 3-thienyl; furanyl, e.g., 2-furanyl and 3-furanyl;

pyrimidyl, e.g., 2-pyrimidyl and 4-pyrimidyl; imidazolyl, e.g., 2-imidazolyl; pyranyl, e.g., 2- pyranyl and 3-pyranyl; pyrazolyl, e.g., 4-pyrazolyl and 5-pyrazolyl; thiazolyl, e.g., 2-thiazolyl, 4- thiazolyl and 5 -thiazolyl; thiadiazolyl; isothiazolyl; oxazolyl, e.g., 2-oxazoyl, 4-oxazoyl and 5- oxazoyl; isoxazoyl; pyrrolyl; pyridazinyl; pyrazinyl and the like. Heterocyclic aromatic (or heteroaryl) as defined above may be optionally substituted with a designated number of substituents, as described below for aromatic groups.

A "fused polycyclic aromatic" ring system is a carbocyclic aromatic group or heteroaryl fused with one or more other heteroaryl or nonaromatic heterocyclic ring. Examples include, quinolinyl and isoquinolinyl, e.g., 2-quinolinyl, 3-quinolinyl, 4- quinolinyl, 5-quinolinyl, 6- quinolinyl, 7-quinolinyl and 8-quinolinyl, 1 -isoquinolinyl, 3-quinolinyl, 4-isoquinolinyl, 5- isoquinolinyl, 6-isoquinolinyl, 7-isoquinolinyl and 8-isoquinolinyl; benzofuranyl e.g., 2- benzofuranyl and 3-benzo furanyl; dibenzofuranyl.e.g., 2,3-dihydrobenzofuranyl;

dibenzothiophenyl; benzothienyl, e.g., 2-benzothienyl and 3-benzothienyl; indolyl, e.g., 2- indolyl and 3-indolyl; benzothiazolyl, e.g., 2-benzothiazolyl; benzooxazolyl, e.g., 2- benzooxazolyl; benzimidazolyl, e.g., 2-benzoimidazolyl; isoindolyl, e.g., 1-isoindolyl and 3- isoindolyl; benzotriazolyl; purinyl; thianaphthenyl and the like. Fused polycyclic aromatic ring systems may optionally be substituted with a designated number of substituents, as described herein.

An "aralkyl group" (arylalkyl) is an alkyl group substituted with an aromatic group, preferably a phenyl group. A preferred aralkyl group is a benzyl group. Suitable aromatic groups are described herein and suitable alkyl groups are described herein. Suitable substituents for an aralkyl group are described herein. An "aryloxy group" is an aryl group that is attached to a compound via an oxygen (e.g., phenoxy).

An "alkoxy group"(alkyloxy), as used herein, is a straight chain or branched C₁-C₁₂ or cyclic C₃-C₁₂ alkyl group that is connected to a compound via an oxygen atom. Examples of alkoxy groups include but are not limited to methoxy, ethoxy and propoxy.

An "arylalkoxy group" (arylalkyloxy) is an arylalkyl group that is attached to a compound via an oxygen on the alkyl portion of the arylalkyl (e.g., phenylmethoxy).

An "arylamino group" as used herein, is an aryl group that is attached to a compound via a nitrogen.

As used herein, an "arylalkylamino group" is an arylalkyl group that is attached to a compound via a nitrogen on the alkyl portion of the arylalkyl.

As used herein, many moieties or groups are referred to as being either "substituted or unsubstituted". When a moiety is referred to as substituted, it denotes that any portion of the moiety that is known to one skilled in the art as being available for substitution can be substituted. For example, the substitutable group can be a hydrogen atom which is replaced with a group other than hydrogen (i.e., a substituent group). Multiple substituent groups can be present. When multiple substituents are present, the substituents can be the same or different and substitution can be at any of the substitutable sites. Such means for substitution are well-known in the art. For purposes of exemplification, which should not be construed as limiting the scope of this invention, some examples of groups that are substituents are: alkyl groups (which can also be substituted, with one or more substituents, such as CF₃), alkoxy groups (which can be substituted, such as OCF₃), a halogen or halo group (F, CI, Br, I), hydroxy, nitro, oxo, -CN, - COH, -COOH, amino, azido, N-alkylamino or Ν,Ν-dialkylamino (in which the alkyl groups can also be substituted), esters (-C(O)-OR, where R can be a group such as alkyl, aryl, etc., which can be substituted), aryl (most preferred is phenyl, which can be substituted), arylalkyl (which can be substituted) and aryloxy.

Stereochemistry

Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral centers). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the formulas of the invention, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

When the HDACis of the present invention contain one chiral center, the compounds exist in two enantiomeric forms and the present invention includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixtures. The enantiomers can be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David ozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

Designation of a specific absolute configuration at a chiral carbon of the compounds of the invention is understood to mean that the designated enantiomeric form of the compounds is in enantiomeric excess (ee) or in other words is substantially free from the other enantiomer. For example, the "R" forms of the compounds are substantially free from the "S" forms of the compounds and are, thus, in enantiomeric excess of the "S" forms. Conversely, "S" forms of the compounds are substantially free of "R" forms of the compounds and are, thus, in enantiomeric excess of the "R" forms. Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%. For example, the enantiomeric excess can be about 60% or more, such as about 70% or more, for example about 80% or more, such as about 90% or more. In a particular embodiment when a specific absolute configuration is designated, the

enantiomeric excess of depicted compounds is at least about 90%. In a more particular embodiment, the enantiomeric excess of the compounds is at least about 95%, such as at least about 97.5%, for example, at least 99% enantiomeric excess. When a compound of the present invention has two or more chiral carbons it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to 4 optical isomers and 2 pairs of enantiomers ((S,S) (R,R) and (R,S) (S,R)). The pairs of enantiomers (e.g., (S,S) (R,R)) are mirror image stereoisomers of one another. The stereoisomers which are not mirror-images (e.g., (S,S) and (R,S)) are diastereomers. The diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. The present invention includes each diastereoisomer of such compounds and mixtures thereof.

As used herein, "a," an" and "the" include singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an active agent" or "a

pharmacologically active agent" includes a single active agent as well a two or more different active agents in combination, reference to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

This invention is also intended to encompass pro-drugs of the HDACis disclosed herein.

A prodrug of any of the compounds can be made using well known pharmacological techniques.

This invention, in addition to the above listed compounds, is intended to encompass the use of homologs and analogs of such compounds. In this context, homologs are molecules having substantial structural similarities to the above-described compounds and analogs are molecules having substantial biological similarities regardless of structural similarities.

The present invention includes compositions, combinations or kits comprising an anti- IGF1R antibody or antigen-binding fragment thereof in association with an HDACi optionally in association with a further chemotherapeutic agent as well as method of treating or preventing various cancers using such compositions, combinations and kits. Further chemotherapeutic agents include, e.g., alkylating agents, antibiotics, antimetabolic agents, hormonal agents, plant- derived agents and various other agents, e.g., as discussed herein.

Alkylating Agents

Alkylating agents react with nucleophilic residues, such as the chemical entities on the nucleotide precursors for DNA production. They affect the process of cell division by alkylating these nucleotides and preventing their assembly into DNA.

Examples of alkylating agents include, but are not limited to, bischloroethylamines

(nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridines (e.g., thiotepa), alkyl alkone sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, streptozocin), nonclassic alkylating agents

(altretamine, dacarbazine, and procarbazine), platinum compounds (carboplastin and cisplatin).

These compounds react with phosphate, amino, hydroxyl, sulfihydryl, carboxyl, and imidazole groups. Under physiological conditions, these drugs ionize and produce positively charged ion that attach to susceptible nucleic acids and proteins, leading to cell cycle arrest and/or cell death. The alkylating agents are cell cycle phasenonspecific agents because they exert their activity independently of the specific phase of the cell cycle. The nitrogen mustards and alkyl alkone sulfonates are most effective against cells in the G1 or M phase. Nitrosoureas, nitrogen mustards, and aziridines impair progression rom the G1 and S phases to the M phases. Chabner and Collins eds. (1990) "Cancer Chemotherapy: Principles and Practice", Philadelphia: JB

Lippincott.

The alkylating agents are active against wide variety of neoplastic diseases, with significant activity in the treatment of leukemias and lymphomas as well as solid tumors.

Clinically this group of drugs is routinely used in the treatment of acute and chronic leukemias; Hodgkin's disease; non-Hodgkin's lymphoma; multiple myeloma; primary brain tumors;

carcinomas of the breast, ovaries, testes, lungs, bladder, cervix, head and neck, and malignant melanoma.

The major toxicity common to all of the alkylating agents is myelosuppression.

Additionally, Gastrointestinal adverse effects of variable severity occur commonly and various organ toxicities are associated with specific compounds. Black and Livingston (1 90) Drugs 39: 489-501; and 39: 652-673. Antibiotics

Antibiotics (e.g., cytotoxic antibiotics) act by directly inhibiting DNA or RNA synthesis and are effective throughout the cell cycle. Examples of antibiotic agents include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin and anthracenedione), mitomycin C, bleomycin, dactinomycin, plicatomycin. These antibiotic agents interfere with cell growth by targeting different cellular components. For example, anthracyclines are generally believed to interfere with the action of DNA topoisomerase II in the regions of transcriptionally active DNA, which leads to DNA strand scissions.

Bleomycin is generally believed to chelate iron and forms an activated complex, which then binds to bases of DNA, causing strand scissions and cell death.

The antibiotic agents have been used as therapeutics across a range of neoplastic diseases, including carcinomas of the breast, lung, stomach and thyroids, lymphomas, myelogenous leukemias, myelomas, and sarcomas. The primary toxicity of the anthracyclines within this group is myelosuppression, especially granulocytopenia. Mucositis often accompanies the granulocytopenia and the severity correlates with the degree of myelosuppression. There is also significant cardiac toxicity associated with high dosage administration of the anthracyclines.

Antimetabolic Agents Antimetabolic agents (i.e., antimetabolites) are a group of drugs that interfere with metabolic processes vital to the physiology and proliferation of cancer cells. Actively proliferating cancer cells require continuous synthesis of large quantities of nucleic acids, proteins, lipids, and other vital cellular constituents.

Many of the antimetabolites inhibit the synthesis of purine or pyrimidine nucleosides or inhibit the enzymes of DNA replication. Some antimetabolites also interfere with the synthesis of ribonucleosides and RNA and/or amino acid metabolism and protein synthesis as well. By interfering with the synthesis of vital cellular constituents, antimetabolites can delay or arrest the growth of cancer cells. Examples of antimetabolic agents include, but are not limited to, fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin, hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin, fludarabine phosphate, cladribine (2- CDA), asparaginase, and gemcitabine.

Antimetabolic agents have widely used to treat several common forms of cancer including carcinomas of colon, rectum, breast, liver, stomach and pancreas, malignant melanoma, acute and chronic leukemia and hair cell leukemia. Many of the adverse effects of antimetabolite treatment result from suppression of cellular proliferation in mitotically active tissues, such as the bone marrow or gastrointestinal mucosa. Patients treated with these agents commonly experience bone marrow suppression, stomatitis, diarrhea, and hair loss. Chen and Grem (19 2) C«rr. Opin. Oncol 4: 1089-1098.

Hormonal Agents

The hormonal agents are a group of drug that regulate the growth and development of their target organs. Most of the hormonal agents are sex steroids and their derivatives and analogs thereof, such as estrogens, progestogens, anti-estrogens, androgens, anti-androgens and progestins. These hormonal agents may serve as antagonists of receptors for the sex steroids to down regulate receptor expression and transcription of vital genes. Examples of such hormonal agents are synthetic estrogens (e.g., diethylstibestrol), antiestrogens (e.g., tamoxifen, toremifene, fluoxymesterol and raloxifene), anti androgens (bicalutamide, nilutamide, flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole and tetrazole), luteinizing hormone release hormone (LHRH) analogues, ketoconazole, goserelin acetate, leuprolide, megestrol acetate and mifepristone.

Hormonal agents are used to treat breast cancer, prostate cancer, melanoma and meningioma. Because the major action of hormones is mediated through steroid receptors, 60% receptor-positive breast cancer responded to first-line hormonal therapy, and less than 10% of receptor-negative tumors responded. The main side effect associated with hormonal agents is flare. The frequent manifestations are an abrupt increase of bony pain, erythema around skin lesions, and induced hypercalcemia. Specifically, progestogens are used to treat endometrial cancers, since these cancers occur in women that are exposed to high levels of oestrogen unopposed by progestogen.

Antiandrogens are used primarily for the treatment of prostate cancer, which is hormone dependent. They are used to decrease levels of testosterone, and thereby inhibit growth of the tumor.

Hormonal treatment of breast cancer involves reducing the level of oestrogen-dependent activation of oestrogen receptors in neoplastic breast cells. Anti-oestrogens act by binding to oestrogen receptors and prevent the recruitment of coactivators, thus inhibiting the oestrogen signal.

LHRH analogues are used in the treatment of prostate cancer to decrease levels of testosterone and so decrease the growth of the tumor.

Aromatase inhibitors act by inhibiting the enzyme required for hormone synthesis. In post-menopausal women, the main source of oestrogen is through the conversion of

androstenedione by aromatase.

Plant-derived Agents

Plant-derived agents are a group of drugs that are derived from plants or modified based on the molecular structure of the agents. They inhibit cell replication by preventing the assembly of the cell's components that are essential to cell division.

Examples of plant derived agents include vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine and vinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide (VM-26)), taxanes (e.g., paclitaxel and docetaxel). These plant-derived agents generally act as antimitotic agents that bind to tubulin and inhibit mitosis. Podophyllotoxins such as etoposide are believed to interfere with DNA synthesis by interacting with topoisomerase II, leading to DNA strand scission.

Plant-derived agents are used to treat many forms of cancer. For example, vincristine is used in the treatment of the leukemias, Hodgkin's and non-Hodgkin's lymphoma, and the childhood tumors neuroblastoma, rhabdomyosarcoma, and Wilms' tumor. Vinblastine is used against the lymphomas, testicular cancer, renal cell carcinoma, mycosis fungoides, and Kaposi's sarcoma. Doxetaxel has shown promising activity against advanced breast cancer, non-small cell lung cancer (NSCLC), and ovarian cancer.

Etoposide is active against a wide range of neoplasms, of which small cell lung cancer, testicular cancer, and NSCLC are most responsive.

The plant-derived agents cause significant side effects on patients being treated. The vinca alkaloids display different spectrum of clinical toxicity. Side effects of vinca alkaloids include neurotoxicity, altered platelet function, myelosuppression, and leukopenia. Paclitaxel causes dose-limiting neutropenia with relative sparing of the other hematopoietic cell lines. The major toxicity of the epipophyllotoxins is hematologic (neutropenia and thrombocytopenia). Other side effects include transient hepatic enzyme abnormalities, alopecia, allergic reactions, and peripheral neuropathy.

Other Therapies

Recent developments have introduced, in addition to the traditional cytotoxic and hormonal therapies used to treat cancer, additional therapies for the treatment of cancer.

For example, many forms of gene therapy are undergoing preclinical or clinical trials.

In addition, approaches are currently under development that are based on the inhibition of tumor vascularization (angiogenesis). The aim of mis concept is to cut off the tumor from nutrition and oxygen supply provided by a newly built tumor vascular system.

In addition, cancer therapy is also being attempted by the induction of terminal differentiation of the neoplastic cells. Suitable differentiation agents include the compounds disclosed in any one or more of the following references, the contents of which are incorporated by reference herein.

a) Polar compounds (Marks et al (1 87), Friend, C, Scher, W., Holland, J. W., and Sato,

T. (1971) Proc. Natl. Acad. Sci. (USA) 68: 378-382; Tanaka, M., Levy, J., Terada, M., Breslow, R., Rifkind, R. A., and Marks, P. A. (1975) Proc. Natl. Acad. Sci. (USA) 72: 1003-1006;

Reuben, R. C, Wife, R. L., Breslow, R., Rifkind, R. A., and Marks, P. A. (1976) Proc. Natl. Acad. Sci. (USA) 73: 862-866);

b) Derivatives of vitamin D and retinoic acid (Abe, E., Miyaura, C, Sakagami, H.,

Takeda, M., Konno, K., Yamazaki, T., Yoshika, S., and Suda, T. (1981) Proc. Natl. Acad. Sci. (USA) 78: 4990-4994; Schwartz, E. L., Snoddy, J. R., Kreutter, D., Rasmussen, H., and

Sartorelli, A. C. (1983) Proc. Am. Assoc. Cancer Res. 24: 18; Tanenaga, ., Hozumi, M., and Sakagami, Y. (1980) Cancer Res. 40: 914-919);

c) Steroid hormones (Lotem, J. and Sachs, L. (1975) Int. J. Cancer 15: 731-740);

d) Growth factors (Sachs, L. (1978) Nature (bond.) 274: 535, Metcalf, D. (1985) Science, 229: 16-22);

e) Proteases (Scher, W., Scher, B. M., and Waxman, S. (1983) Exp. Hematol. 11 : 490- 498; Scher, W., Scher, B. M., and Waxman, S. (1982) Biochem. & Biophys. Res. Comm. 109: 348-354);

f) Tumor promoters (Huberman, E. and Callaham, M. F. (1979) Proc. Natl. Acad. Sci. (USA) 76: 1293-1297; Lottem, J. and Sachs, L. (1979) Proc. Natl. Acad. Sci. (USA) 76: 5158- 5162); and

g) inhibitors of DNA or RNA synthesis (Schwartz, E. L. and Sartorelli, A. C. (1982) Cancer Res. 42: 2651 -2655, Terada, M., Epner, E., Nudel, U., Salmon, J., Fibach, E., Rifkind, R. A., and Marks, P. A. (1978) Proc. Natl. Acad. Sci. (USA) 75: 2795-2799; Morin, M. J. and Sartorelli, A. C. (1984) Cancer Res. 44: 2807-2812; Schwartz, E. L., Brown, B. J., Nierenberg, M., Marsh, J. C, and Sartorelli, A. C. (1983) Cancer Res. 43: 2725-2730; Sugano, H., Furusawa, M., Kawaguchi, T., and Ikawa, Y. (1973) Bibl. Hematol. 39: 943-954; Ebert, P. S., Wars, L, and Buell, D. N. (1976) Cancer Res. 36: 1809-1813; Hayashi, M., Okabe, J., and Hozumi, M. (1979) Gann 70: 235-238).

In an embodiment of the invention, a kit, composition or combination including an anti- IGF1R antibody or antigen-binding fragment thereof in association with an HDACi (e.g., vorinostat) is further in association with one or more of the following chemotherapeutic agents: ALT-110; AMN-107 (Nilotinib); amrubicin; ARQ-197; atrasentan; AV-299; AZD 1152; AZD 2171 ; batabulin; BIO-111; BIO-140; calcitriol; CC 8490; cilengitide; dasatinib; decatanib; DN- 101; edotecarin; enzastaurin; erlotinib; everolimus; gimatecan; gossypol (e.g.; gossypol acetate); GSK461364; GSK690693; IL13-PE38QQR; INO 1001; IPdR; ipilimumab; KRX-0402; Lep-etu; lonafarnib; lucanthone; LY 317615; MK-0457; MLN8054; neuradiab; nolatrexed; oblimersen; ofatumumab; ON 0910.Na; oregovomab; panitumumab; pazopanib; PHA-739358; R-763; RTA 744; rubitecan; Sdx 102; talampanel; temsirolimus; tesmilifene; tetrandrine; ticilimumab; TKI- 258; TLK 286; trabectedin; vandetanib; vitespan; Xr 311; zanolimumab; 131-I-TM-601;

zolendronate; histrelin; azacitidine; dexrazoxane; alemtuzumab; lenalidomide; gemtuzumab; ketoconazole; nitrogen mustard; ibritumomab tiuxetan; decitabine; hexamethylmelamine;

bexarotene; tositumomab; arsenic trioxide; editronate; cyclosporine; Edwina-asparaginase, strontium 89, romidepsin; ADS-100380; CG-781; CG-1521; SB-556629; chlamydocin; JNJ- 16241199;

;etoposide; gemcitabine;doxorubicin;

5'-deoxy-5-fluorouridine; vincristine; a CDK inhibitor, such as ZK-304709 or Seliciclib; a MEK inhibitor, such as PD0325901 or AZD-6244 (ARRY-142886); capecitabine; camptothecin;

irinotecan; a combination of irinotecan, 5-fluorouracil, and leucovorin; or PEG-labeled irinotecan; the FOLFOX regimen, which consists of oxaliplatin together with infusional fluorouracil and folinic acid; Oxaliplatin; an antiestrogen such as tamoxifen or toremifene citrate; an aromatase inhibitor such as anastrazole, exemestane, or letrozole; anastrazole; exemestane; letrozole; an estrogen such as diethylstilbestrol (DES), estradiol, or conjugated estrogens; an anti-angiogenic agent such as Bevacizumab, VEGFR-2 antibody IMC-1C11, other VEGF-R inhibitors such as CHIR-258, Vatalanib (PTK/ZK; CGP-79787; ZK-222584), AG-013736, 3-[5- (methylsulfonylpiperadinemethyl)-indolyl]-quinolone, or the VEGF trap (AVE-0005), a soluble decoy receptor comprising portions of VEGF receptors 1 and 2; a luteinizing hormone-releasing hormone (LHRH) or gonadotropin releasing hormone (GnRH) agonist such as an goserelin acetate; leuprolide acetate; or triptorelin pamoate; sunitinib; sunitinib malate; a progestational agent such as medroxyprogesterone acetate; hydroxyprogesterone caproate; megestrol acetate, or progestins; a selective estrogen receptor modulator (SERM) such as raloxifene; an anti-androgen such as bicalutamide, flutamide, nilutamide, or megestrol acetate. Inhibitors of the EGF

Receptor or HER2, such as CP-724714, TAK-165 (mubritinib), HKI-272, OSI-774 (erlotinib), lapatinib (GW2016), canertinib (CI-1033), EKB-569, PKI-166 (CGP-75166), ABX-EGF antibody, cetuximab, GW-S72016, or any anti-EGFR or anti-HER2 antibody; a farnesyl protein transferase inhibitor (FTI) such as lonafamib; BMS-214662; tipifamib or R155777; amifostine;

NVP-LAQ824; suberoyl analide hydroxamic acid; valproic acid; trichostatin A; FK-228;

SU11248; sorafenib, KRN951; aminoglutethimide; amsacrine; anagrelide; anastrozole;

asparaginase, bacillus Calmette-Guerin (BCG) vaccine; bleomycin; buserelin; busulfan;

satraplatin; carboplatin; carmustine; chlorambucil; cisplatin; cladribine; clodronate;

cyclophosphamide; cyproterone; cytarabine; dacarbazine; dactinomycin; daunorubicin;

diethylstilbestrol; epirubicin; fludarabine; fludrocortisones; fluoxymesterone; flutamide;

hydroxyurea; idarubicin; ifosfamide; imatinib; leucovorin; leuprolide; levamisole; lomustine; mechlorethamine; melphalan; mercaptopurine; mesna; methotrexate; mitomycin; mitotane; mitoxantrone; mlutamide; octreotide; edotreotide (yttrium-90 labeled or unlabeled); oxaliplatin; pamidronate; Pentostatin; plicamycin; porfimer; procarbazine; raltitrexed; rituximab;

streptozocin; teniposide; testosterone; thalidomide; thalidomide combined with dexamethasone; thioguanine; thiotepa; tretinoin; vindesine; all trans-retinoic acid; or 13-cis-retinoic acid;

abraxane (an injectable suspension of paclitaxel protein-bound particles comprising an albumin- bound form of paclitaxel with a mean particle size of approximately 130 nanometers, free of solvents and cremophor (polyoxyethylated castor oil); phenylalanine mustard; uracil mustard; estramustine; altretamine; floxuridine; 5-deooxyuridine; cytosine arabinoside; 6-mecaptopurine; 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; 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; LY294002; LY292223; LY292696; LY293684; LY293646; wortmannin; BAY-43-

9006; ZM336372; L-779;450; a Raf inhibitor, flavopiridol; UCN-01; any mTOR inhibitor; rapamycin; everolimus; temsirolimus; AP-23573; RAD001; ABT-578; BC-210.

In an embodiment of the invention, a kit, composition or combination including an anti-

IGF1R antibody or antigen-binding fragment thereof in association with an HDACi is further 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, NJ), diphenhydramine (sold as Benadryl® by Pfizer, New York, NY), hydroxyzine

(sold as Atarax® by Pfizer; New York, NY), metoclopramide (sold as Reglan® by AH Robins Co,; Richmond, VA), lorazepam (sold as Ativan® by Wyeth; Madison, NJ), alprazolam (sold as Xanax® by Pfizer, New York, NY), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, NJ), droperidol (Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, GA), dexamethasone (sold as Decadron® by Merck and Co.; Rahway, NJ), methylprednisolone (sold as Medrol® by Pfizer; New York, NY), prochlorperazine (sold as Compazine® by Glaxosmithkline; Research Triangle Park, NC), granisetron (sold as Kytril® by Hoffmann-La Roche Inc.; Nutley, NJ), ondansetron ( sold as Zofran® by by Glaxosmithkline; Research Triangle Park, NC), dolasetron (sold as Anzemet® by Sanofi-Aventis; New York, NY), tropisetron (sold as Navoban® by Novartis; East Hanover, NJ); and/or pegfilgrastim, erythropoietin, epoetin alfa or darbepoetin alfa.

The use of all of these approaches in the combination methods of the invention are within the scope of the present invention.

Modes and Doses of Administration

The methods of the present invention comprise administering to a patient in need thereof a first amount of an IGF-1R inhibitor, e.g., preferably an IGF-1R antibody exemplified by Dolutuzumab, in a first treatment procedure, and a second amount of an anti-cancer agent, e.g., HDACi, more preferably vorinostat in a second treatment procedure. The first and second treatments together comprise a therapeutically effective amount. It is understood that administration of HDACi and the IGF-1R inhibitor are interchangeable in that the first treatment protocol may comprise an HDACi followed by the second treatment protocol comprising an IGF-1R inhibitor.

"Patient" as that term is used herein, refers to the recipient of the treatment. Mammalian and non-mammalian patients are included. In a specific embodiment, the patient is a mammal, such as a human, canine, murine, feline, bovine, ovine, swine or caprine. In a particular embodiment, the patient is a human.

Administration of the HDACi

Dose and Route of Administration

The combination therapeutic comprising IGF-1R specific antibodies and

chemotherapeutic agents of the invention are administered to a human patient, in accord with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred. Three distinct delivery approaches are expected to be useful for delivery of the antibodies in accordance with the invention.

Conventional intravenous delivery will presumably be the standard delivery technique for the majority of tumours. However, in connection with some tumours, such as those in the peritoneal cavity exemplified by tumours of the ovaries, biliary duct, other ducts, and the like,

intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumour and to minimize antibody clearance. In a similar manner certain solid tumours possess vasculature that is appropriate for regional perfusion. Regional perfusion will allow the obtention of a high dose of the antibody at the site of a tumour and will minimize short term clearance of the antibody.

As with any protein or antibody infusion based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills, (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAH A or H ACA response), and (iii) toxicity to normal cells that express the EGF receptor, e.g., hepatocytes which express EGFR and/or IGF- 1R. Standard tests and follow up will be utilized to monitor each of these safety concern In particular, liver function will be monitored frequently during clinical trails in order to assess damage to the liver, if any.

For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. In a combination therapy regimen, the compositions of the present invention are administered in a therapeutically effective or synergistic amount. As used herein, a therapeutically effective amount is such that co-administration of anti-IGF-1R antibody and one or more other therapeutic agents, or administration of a composition of the present invention, results in reduction or inhibition of the targeting disease or condition. A therapeutically synergistic amount is that amount of anti-IGF-1R antibody and one or more other therapeutic agents necessary to synergistically or significantly reduce or eliminate conditions or symptoms associated with a particular disease.

In a broad embodiment, the treatment of the present invention involves the combined administration of an anti-IGF-1R antibody and one or more chemotherapeutic agents. The combined administration includes co administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeutic agent may precede, or follow administration of the antibody or may be given simultaneously therewith. The clinical dosing of therapeutic combination of the present invention are likely to be limited by the extent of adverse reactions skin rash as observed with monoclonal anti-IGF-1R antibodies and a tyrosine kinase inhibitor (TKI) (Erlotinib and Gefitinib ) used in the clinic today.

Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved. Further information about suitable dosages is provided in the Example below.

Depending on the type and severity of the disease, about 1 μgkg to 50 mg/kg (e.g.0.1-20 mg/kg) of antibody is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 .mu.g/kg to about 100 mg kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful.

In one aspect, the antibody of the invention is administered bi-weekly, weekly or may be administered every two to three weeks, at a dose ranged from about 5 mg/kg to about 15 mg/kg. More preferably, such dosing regimen is used in combination with a chemotherapy regimen for treating erlotinib resistant cancers such as NSCLC. In some aspects, the chemotherapy regimen involves the traditional high-dose intermittent administration. In some other aspects, the chemotherapeutic agents are administered using smaller and more frequent doses without scheduled breaks ("metronomic chemotherapy"). The progress of the therapy of the invention is easily monitored by conventional techniques and assays.

In one embodiment, the dosing sequence comprises administering vorinostat concurrently with the IGF-1R antibody - vorinostat is administered twice a week at a dose of 50 mg/kg together with the IGF-1R antibody, wherein each is administered via IP injection. In alternative embodiments, vorinostat may be administered orally while the antibody is administered at the same time via injection. In other alternatives, the IGF-1R antibody is administered at a dose of 10 mg/kg i.v weekly while vorinostat may be administered at 50/kg mg twice a week.

Alternative dosing regiment for the IGF-1R antibody is as follows:

(i) 15 mg kg loading, followed by 7.5 mg/kg every week.

(ii) 20 mg/kg every other week

(iii) 30 mg/kg every three weeks

For parenteral administration, the antibody can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by known or suitable techniques. The

administration of the combination therapeutic may continue until disease progression.

Routes of Administration

The HDACi (e.g. vorinostat), can be administered by any known administration method known to a person skilled in the art. Examples of routes of administration include but are not limited to oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, intraoccular, via local delivery by catheter or stent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in a slow release dosage form.

For example, the HDACis of the invention can be administered in such oral forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. Likewise, the HDACi can be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. A currently preferred administration of the HDACi is oral administration.

The HDACi can also be administered in the form of a depot injection or implant preparation, which may be formulated in such a manner as to permit a sustained release of the active ingredient. The active ingredient can be compressed into pellets or small cylinders and implanted subcutaneously or intramuscularly as depot injections or implants. Implants may employ inert materials such as biodegradable polymers or synthetic silicones, for example, Silastic, silicone rubber or other polymers manufactured by the Dow-Corning Corporation.

The HDACi can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or

phosphatidylcholines.

The HDACi can also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.

The HDACi can also be prepared with soluble polymers as targetable drug carriers. Such polymers can include polyvinlypyrrolidone, pyran copolymer, polyhydroxy-propyl- methacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, an HDACi can be prepared with

biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels.

In a currently preferred embodiment, the HDACi, e.g. vorinostat, is administered orally in a gelatin capsule, which can comprise excipients such as microcrystalline cellulose, croscarmellose sodium and magnesium stearate. A further preferred embodiment is 200 mg of solid vorinostat with S9.5 mg of microcrystalline cellulose, 9 mg of sodium croscarmellose and 1.5 mg of magnesium stearate contained in a gelatin capsule.

Dosages and Dosage Schedules

The dosage regimen utilizing the HDACi can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the cancer to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease.

For example, vorinostat or any one of the HDACis can be administered in a total daily dose of up to 800 mg, the HDACi can be administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). The HDACi can be administered at a total daily dosage of up to 800 mg, e.g., 200 mg, 300 mg, 400 mg, 600 mg or

800 mg, which can be administered in one daily dose or can be divided into multiple daily doses as described above. Preferably, the administration is oral.

In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration of an HDACi may be administration one to six days per week or it may mean administration in cycles (e.g. daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.

vorinostat or any of the HDACis are administered to the patient at a total daily dosage of between 25-4000 mg m². A particular treatment protocol comprises continuous administration

(i.e., every day), once, twice or three times daily at a total daily dose in the range of about 200 mg to about 600 mg.

Another currently preferred treatment protocol comprises intermittent administration of vorinostat once daily at a dose of 50 mg/kg twice a week via IP injection together with or concurrently with the IGF-1R antibody.

In addition, the HDACi may be administered according to any of the schedules described above, consecutively for a few weeks, followed by a rest period. For example, the HDACi may be administered according to any one of the schedules described above from two to eight weeks, followed by a rest period of one week, or twice daily at a dose of from 50 to 300 mg for one to three, or three to five days a week. In another particular embodiment, the HDACi is

administered once twice weekly together with the IGF-1R antibody.

Intravenously or subcutaneously, the patient would receive the HDACi in quantities sufficient to deliver between about 3-1500 mg/m² per day , for example, about 3, 30, 0, 90, 180, 300, 600, 900, 1200 or 1500 mg m² per day. Such quantities may be administered in a number of suitable ways, e.g. large volumes of low concentrations of HDACi during one extended period of time or several times a day. The quantities can be administered for one or more consecutive days, intermittent days or a combination thereof per week (7 day period). Alternatively, low volumes of high concentrations of HDACi during a short period of time, e.g. once a day for one or more days either consecutively, intermittently or a combination thereof per week (7 day period). For example, a dose of 300 mgm² per day can be administered for 5 consecutive days for a total of 1500 mg m² per treatment. In another dosing regimen, the number of consecutive days can also be 5, with treatment lasting for 2 or 3 consecutive weeks for a total of 3000 mg/m² and 4500 mg/m² total treatment.

Typically, an intravenous formulation may be prepared which contains a concentration of HDACi of between about 1.0 mg mL to about 10 mg/mL, e.g. 2.0 mgmL, 3.0 mg mL, 4.0 mg/mL, 5.0 mg mL, 6.0 mg/mL, 7.0 mgmL, 8.0 mgmL, 9.0 mgmL and 10 mg/mL and administered in amounts to achieve the doses described above. In one example, a sufficient volume of intravenous formulation can be administered to a patient in a day such that the total dose for the day is between about 300 and about 1500 mg/m².

Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, also include suitable buffers and isotonicity agents, as described below. They can be formulated to deliver a daily dose of HDACi in one or more daily subcutaneous administrations, e.g., one, two or three times each day.

The HDACis can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.

It should be apparent to a person skilled in the art that the various modes of

administration, dosages and dosing schedules described herein merely set forth specific embodiments and should not be construed as limiting the broad scope of the invention. Any permutations, variations and combinations of the dosages and dosing schedules are included within the scope of the present invention.

Combination Administration The first treatment procedure, administration of an IGF-1R inhibitor, can take place prior to the second treatment procedure, i.e., HDACi, after the treatment with the HDACi, at the same time as the treatment with the HDACi, or a combination thereof.

Vorinostat or any one of the HDACis can be administered in accordance with any dose and dosing schedule that, together with the effect of the anti-cancer agent, achieves a dose effective to treat cancer.

In certain embodiments, the IGF-1R (MK-0646) antibody is administered twice weekly concurrently with vorinostat, each administered via injection, with vorinostat being administered at a dose of 50 mg kg.

Pharmaceutical compositions

As described above, the compositions comprising the HDACi and/or the anti-cancer agent can be formulated in any dosage form suitable for oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal, via inhalation, vaginal, or intraocular administration, for administration via local delivery by catheter or stent, or for subcutaneous, intraadiposal, intraarticular, intrathecal administration, or for administration in a slow release dosage form.

The IGF-1R inhibitor and the HDACi can be formulated in the same formulation for simultaneous administration, or they can be in two separate dosage forms, which may be administered simultaneously or sequentially as described above.

The invention also encompasses pharmaceutical compositions comprising

pharmaceutically acceptable salts of the HDACis and or the anti-cancer agents. Suitable pharmaceutically acceptable salts of the compounds described herein and suitable for use in the method of the invention, are conventional non-toxic salts and can include a salt with a base or an acid addition salt such as a salt with an inorganic base, for example, an alkali metal salt (e.g., lithium salt, sodium salt, potassium salt, etc.), an alkaline earth metal salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt a salt with an organic base, for example, an organic amine salt (e.g., triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, Ν,Ν'-dibenzylethylenediamine salt, etc.) etc.; an inorganic acid addition salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate, etc.); an organic carboxylic or sulfonic acid addition salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate,

methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.); a salt with a basic or acidic amino acid (e.g., arginine, aspartic acid, glutamic acid, etc.) and the like.

The invention also encompasses pharmaceutical compositions comprising hydrates of the HDACis and/or the anti-cancer agents. The term "hydrate" includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.

In addition, this invention also encompasses pharmaceutical compositions comprising any solid or liquid physical form of vorinostat or any of the other HDACis. For example, the HDACis can be in a crystalline form, in amorphous form, and have any particle size. The HDACi particles may be micronized, or may be agglomerated, particulate granules, powders, oils, oily suspensions or any other form of solid or liquid physical form.

For oral administration, the pharmaceutical compositions can be liquid or solid. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.

Any inert excipient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. The compositions may further comprise a

disintegrating agent and a lubricant, and in addition may comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof. Furthermore, the compositions of the present invention may be in the form of controlled release or immediate release formulations.

The HDACis can be administered as active ingredients in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials or "pharmaceutically acceptable carriers") suitably selected with respect to the intended form of administration. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.

For liquid formulations, pharmaceutically acceptable carriers may be aqueous or non- aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.

Aqueous carriers include water, alcoholic aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil. Solutions or suspensions can also include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.

Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In addition, the compositions may further comprise binders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCl, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween S0, Pluronic F6S, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), a glidant (e.g., colloidal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

It is especially advantageous to formulate oral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

The preparation of pharmaceutical compositions that contain an active component is well understood in the art, for example, by mixing, granulating, or tablet-forming processes. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient For oral administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions and the like as detailed above.

The amount of the compound administered to the patient is less than an amount that would cause toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound.

Preferably, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. The optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the particular compound used and the type of cancer being treated.

The percentage of the active ingredient and various excipients in the formulations may vary. For example, the composition may comprise between 20 and 90%, preferably between 50- 70% by weight of the active agent.

For IV administration, Glucuronic acid, L-lactic acid, acetic acid, citric acid or any pharmaceutically acceptable acid/conjugate base with reasonable buffering capacity in the pH range acceptable for intravenous administration can be used as buffers. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed. Typically, a pH range for the intravenous formulation can be in the range of from about 5 to about 12. A preferred pH range for intravenous formulation comprising an HDACi, wherein the HDACi has a hydroxamic acid moiety, can be about 9 to about 12.

Subcutaneous formulations, preferably prepared according to procedures well known in the art at a pH in the range between about 5 and about 12, also include suitable buffers and isotonicity agents. They can be formulated to deliver a daily dose of the active agent in one or more daily subcutaneous administrations. The choice of appropriate buffer and pH of a formulation, depending on solubility of the HDACi to be administered, is readily made by a person having ordinary skill in the art. Sodium chloride solution wherein the pH has been adjusted to the desired range with either acid or base, for example, hydrochloric acid or sodium hydroxide, can also be employed in the subcutaneous formulation. Typically, a pH range for the subcutaneous formulation can be in the range of from about 5 to about 12. A preferred pH range for subcutaneous formulation of an HDACi a hydroxamic acid moiety, can be about 9 to about 12.

Although the methods of the present invention can be practiced in vitro, it is

contemplated that the preferred embodiment for the methods of selectively inducing cell death, terminal differentiation, cell growth arrest and/or apoptosis of neoplastic cells will comprise contacting the cells in vivo, i.e., by administering the compounds to a subject harboring neoplastic cells or tumor cells in need of treatment.

The invention is illustrated in the examples in the Experimental Details Section that follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS SECTION

EXAMPLE 1: Synthesis of SAHA (vorinostat)

SAHA can be synthesized according to the method outlined below, or according to the method set forth in US Patent 5,369,108, the contents of which are incorporated by reference in their entirety, or according to any other method.

Synthesis of SAHA

Step 1 - Synthesis of Suberanilic acid

In a 22 L flask was placed 3,500 g (20.09 moles) of suberic acid, and the acid melted with heat. The temperature was raised to 175°C, and then 2,040 g (21.92 moles) of aniline was added. The temperature was raised to 190°C and held at that temperature for 20 minutes. The melt was poured into a Nalgene tank that contained 4,017 g of potassium hydroxide dissolved in 50 L of water. The mixture was stirred for 20 minutes following the addition of the melt. The reaction was repeated at the same scale, and the second melt was poured into the same solution of potassium hydroxide. After the mixture was thoroughly stirred, the stirrer was turned off, and the mixture was allowed to settle. The mixture was then filtered through a pad of Celite (4,200 g) (the product was filtered to remove the neutral by-product (from attack by aniline on both ends of suberic acid). The filtrate contained the salt of the product, and also the salt of unreacted suberic acid. The mixture was allowed to settle because the filtration was very slow, taking several days.). The filtrate was acidified using 5 L of concentrated hydrochloric acid; the mixture was stirred for one hour, and then allowed to settle overnight. The product was collected by filtration, and washed on the funnel with deionized water (4 x 5 L). The wet filter cake was placed in a 72 L flask with 44 L of deionized water, the mixture heated to 50°C, and the solid isolated by a hot filtration (the desired product was contaminated with suberic acid which is has a much greater solubility in hot water. Several hot triturations were done to remove suberic acid. The product was checked by NMR [D₆DMSO] to monitor the removal of suberic acid). The hot trituration was repeated with 44 L of water at 50°C. The product was again isolated by filtration, and rinsed with 4 L of hot water. It was dried over the weekend in a vacuum oven at 65°C using a Nash pump as the vacuum source (the Nash pump is a liquid ring pump (water) and pulls a vacuum of about 29 inch of mercury. An intermittent argon purge was used to help carry off water); 4, 182.8 g of suberanilic acid was obtained.

The product still contained a small amount of suberic acid; therefore the hot trituration was done portionwise at 65°C, using about 300 g of product at a time. Each portion was filtered, and rinsed thoroughly with additional hot water (a total of about 6 L). This was repeated to purify the entire batch. This completely removed suberic acid from the product. The solid product was combined in a flask and stirred with 6 L of methanol water (1 :2), and then isolated by filtration and air dried on the filter over the week end. It was placed in trays and dried in a vacuum oven at 65°C for 45 hours using the Nash pump and an argon bleed. The final product has a weight of 3,278.4 g (32.7% yield).

Step 2 -Synthesis of Methyl Suberanilate

To a 50 L flask fitted with a mechanical stirrer, and condenser was placed 3,229 g of suberanilic acid from the previous step, 20 L of methanol, and 398.7 g of Dowex 50WX2-400 resin. The mixture was heated to reflux and held at reflux for 18 hours. The mixture was filtered to remove the resin beads, and the filtrate was taken to a residue on a rotary evaporator.

The residue from the rotary evaporator was transferred into a 50 L flask fitted with a condenser and mechanical stirrer. To the flask was added 6 L of methanol, and the mixture heated to give a solution. Then 2 L of deionized water was added, and the heat turned off. The stirred mixture was allowed to cool, and then the flask was placed in an ice bath, and the mixture cooled. The solid product was isolated by filtration, and the filter cake was rinsed with 4 L of cold methanol/water (1:1). The product was dried at 45°C in a vacuum oven using a Nash pump for a total of 64 hours to give 2,850.2 g (84% yield) of methyl suberanilate, CSL Lot # 98-794-92-3 1.

To a 50 L flask with a mechanical stirrer, thermocouple, and inlet for inert atmosphere was added 1 ,451.9 g of hydroxylamine hydrochloride, 19 L of anhydrous

Step 3 - Synthesis of Crude SAHA

methanol, and a 3.93 L of a 30% sodium methoxide solution in methanol. The flask was then charged with 2,748.0 g of methyl suberanilate, followed by 1.9 L of a 30% sodium methoxide solution in methanol. The mixture was allowed to stir for 16 hr and 10 minutes. Approximately one half of the reaction mixture was transferred from the reaction flask (flask 1 ) to a 50 L flask (flask 2) fitted with a mechanical stirrer. Then 27 L of deionized water was added to flask 1 and the mixture was stirrer for 10 minutes. The pH was taken using a pH meter; the pH was 11.56. The pH of the mixture was adjusted to 12.02 by the addition of 100 ml of the 30% sodium methoxide solution in methanol; this gave a clear solution (the reaction mixture at this time contained a small amount of solid. The pH was adjusted to give a clear solution from which the precipitation the product would be precipitated). The reaction mixture in flask 2 was diluted in the same manner; 27 L of deionized water was added, and the pH adjusted by the addition of 100 ml of a 30 % sodium methoxide solution to the mixture, to give a pH of 12.01 (clear solution).

The reaction mixture in each flask was acidified by the addition of glacial acetic acid to precipitate the product. Flask 1 had a final pH of 8.98, and Flask 2 had a final pH of 8.70. The product from both flasks was isolated by filtration using a Buchner funnel and filter cloth. The filter cake was washed with 15 L of deionized water, and the funnel was covered and the product was partially dried on the funnel under vacuum for 15.5 hr. The product was removed and placed into five glass trays. The trays were placed in a vacuum oven and the product was dried to constant weight. The first drying period was for 22 hours at 60°C using a Nash pump as the vacuum source with an argon bleed. The trays were removed from the vacuum oven and weighed. The trays were returned to the oven and the product dried for an additional 4 hr and 10 minutes using an oil pump as the vacuum source and with no argon bleed. The material was packaged in double 4-mill polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2633.4 g (95.6%).

Step 4 - Recrystallization of Crude SAHA (vorinostat)

The crude SAHA was recrystallized from methanol/water. A 50 L flask with a mechanical stirrer, thermocouple, condenser, and inlet for inert atmosphere was charged with the crude SAHA to be crystallized (2,525.7 g), followed by 2,625 ml of deionized water and 15,755 ml of methanol. The material was heated to reflux to give a solution. Then 5,250 ml of deionized water was added to the reaction mixture. The heat was turned off, and the mixture was allowed to cool. When the mixture had cooled sufficiently so that the flask could be safely handled (28°C), the flask was removed from the heating mantle, and placed in a tub for use as a cooling bath. Ice/water was added to the tub to cool the mixture to -5°C. The mixture was held below that temperature for 2 hours. The product was isolated by filtration, and the filter cake washed with 1.5 L of cold

methanol/water (2:1). The funnel was covered, and the product was partially dried under vacuum for 1.75 hr. The product was removed from the funnel and placed in 6 glass trays. The trays were placed in a vacuum oven, and the product was dried for 64.75 hr at 60°C using a Nash pump as the vacuum source, and using an argon bleed. The trays were removed for weighing, and then returned to the oven and dried for an additional 4 hours at 60°C to give a constant weight. The vacuum source for the second drying period was a oil pump, and no argon bleed was used. The material was packaged in double 4-mill polyethylene bags, and placed in a plastic outer container. The final weight after sampling was 2,540.9 g (92.5%).

EXAMPLE 2: Treatment of lung cancer with anti-IGF1R and vorinostat

METHODS Cells and reagents. Human non-small cell lung cancer cell lines H226B, H226Br, H292, H322, H358, H427, H460, H596, H1299, H1944, H1993, H2126, A549 and A549M were obtained from the American Tissue Culture Collection. While cells other than H427 were cultured in RPMI 1640, H427 cells were cultured in DME /F12, supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified environment with 5% CO2. Clinical-grade vorinostat and MK0646 (IGF-1R specific) were provided by Merck & Co. Stock solutions of vorinostat were prepared as 20 raM stock solutions in dimethyl sulfoxide (DMSO), stored at -20 °C and diluted to appropriate concentrations in culture medium before addition to the cells. MK0646 were kept in the original container at 4°C.

Western blot analysis. Whole-cell lysates were prepared in lysis buffer as described elsewhere_{Ref 1}. Complete protease inhibitor cocktail (Roche, Alameda, CA) with 1M NaF, 1M β-glycerophosphate and 0.2M Sodium orthovanadate was added to lysis buffer before use. Protein concentrations were measured using the Bio-Rad protein assay (Bio-Rad Laboratories). Equivalent amounts of protein (25-50 μg) were resolved by

SDS-polyacrylamide electrophoresis in 6%-12% gels (80 V for 20 minutes and 100 V for 1 hour) and transferred by electroblotting overnight at 20 V to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). After nonspecific binding to the blot was blocked in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and 5% nonfat powdered milk, the blot was incubated with primary antibody at the appropriate dilution in TBS-5% bovine serum albumin at 4° C for 16 hours. The membrane was then washed multiple times with TBST and incubated with the appropriate horseradish peroxidase- conjugated secondary antibody for 1 hour at room temperature. The protein-antibody complexes were detected by using the enhanced chemiluminescence kit ( Amersham, Arlington Heights, IL), according to the manufacturer's recommended protocol. Loading and transferring control was confirmed by probing the membranes with anti-β-actin antibody.

The following antibodies were used for Western blotting. Phospho-IGF-1R antibody (no. 3021), EGFR antibody (no. 2232), poly ADP-ribose polymerase (PARP) antibody (no. 9542), mTOR antibody (no.2983), phospho-mTOR (no. 2971), Akt antibody (no. 9272), phospho-Akt (Ser473) antibody (no. 9271), and phospho- ERK1/2 antibody (no. 9106) were purchased from Cell-Signaling Biotechnology. IGF-1R (no. 713), ERK antibody (no. 93-G) and β-actin (no. 1675) were purchased from Santa Cruz Biotechnology.

Cell proliferation assay. Cells were seeded into 96-well microculture plates at

2500 to 10,000 cells per well and allowed to attach for 24 hours. Cells were treated with vehicle (DMSO 0.1%) or indicated concentration of vorinostat or MK0646 in the standard culture media for 72 hours. Cell proliferation was measured with the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentrations required to inhibit cell growth by 50% were determined by interpolation from the dose-response curves.

Anchorage-independent clonogenic growth assay. For the anchorage- independent clonogenic growth assay, 2.5 to 7.5 x 103 cells were suspended in 0.5 mL of 0.4% soft agar that was layered on top of 1 mL of 1 % solidified agar in each well of 12- well plates. The plates were then incubated for 10 to 15 days in complete medium containing 0.5, 1 and 5 μmοl L concentrations of vorinostat, 10μg/mL of MK0646, or combination. The medium was changed twice a week during this period, at the end of which tumor cell colonies measuring at least 80 μm were stained in 0.001 % crystal violet and counted using ImageJ software (Rasband, W.S., Image), U. S. National Institutes of Health, Bethesda, Maryland, USA, rsb.info.nih.gov/ij/, 1997-2009). Mean growth inhibition was calculated by dividing the colony number of each treatment group by the colony number of control group. Expected MGI is calculated by multiplying MGIs by single treatment. Index was calculated by dividing the expected colony number by the observed colony number. An index of > 1 indicates synergistic effect and an index of < 1 indicates less than additive effect.

In Vivo Tumor Model. After irradiation with 350 rad from a cesium-137 source, xenograft tumors were generated by subcutaneous injection of the H1299 and H226B cells into the flanks of athymic nude mice (Charles River Laboratories) as described

elsewhere _{ef 2}. Briefly, nude mice were injected at a single dorsal flank site with 5 x 107 H1299 and H226Br cells in 100 μL of phosphate-buffered saline (PBS). Injection of these cells into nude mice induced exponentially growing tumors. When tumors reached a volume of 50-75cm3 (termed day 0 for our experiments), mice were treated with intraperitoneal (IP) injection of vehicle (50% polyethylenglycol 400 in distilled water), vorinostat at 50 mg/kg , M 0646 15mg kg, or combination twice a day for 15-28 days. Tumor growth was quantified by measuring the tumors in two dimensions with calipers twice a week. Volumes were calculated by the formula 0.5 x a x b2, where a and b are the longer and shorter diameters, respectively. Tumor volumes were expressed as the mean and standard error. Mice with necrotic tumors or with tumors that had a diameter of more than 1.5 cm were humanely killed by exposure to CO₂. All animal procedures were performed in accordance with a protocol approved by the M.D. Anderson Cancer Center Institutional Animal Care and Usage Committee.

Statistical Analysis Data are expressed as the mean and 95% confidence interval from at least triplicate samples. Data were calculated either with the Microsoft Excel software program (version 2003; Microsoft Corporation, Redmond, WA) or in the SPSS statistical program (SPSS version 17; SPSS, Chicago, IL). The statistical significance of differences between groups was analyzed with Mann- Whitney test. All statistical tests were two-sided. A P value of less than .05 was considered to be statistically significant. RESULTS

Characterization of vorinostat response in human NSCLC cell lines. Treatment with vorinostat at doses ranging from 0.5 to 50 μΜ exhibited a range of sensitivities after

3-day treatment (Fig. 1 A). IC₅₀ values were not correlated with a specific mutational status or histological subtypes. Five cell lines were classified as relatively resistant based on this result. To validate the resistance, soft agar colony forming assay was done. When the relative number of colonies were calculated by dividing the colony number of control (DMSO 0.1 %) and arranged by the decreasing order, the 5 resistant cells were again clustered in the resistant group (Fig 1B).

Apoptosis induction in the relatively resistant and sensitive cells. Vorinostat induced cleavage of PARP dose-dependently in the representative sensitive cell lines

(H1944 and A549M). Also 5uM of SAHA (vorinostat) could induce apoptosis in sensitive cell (358), but could not induce apoptosis in resistance cells (226Br, 460, 596 and 1299).

See Fig 1C. In addition, vorinostat was demonstrated, by FACS analysis, to induce apoptosis marker Annexin V in sensitive lines 358 and A549M but not in resistant lines

226Br and 1299. See Fig 1D.

Because IGF-1R pathway may be involved in development, maintenance and progression of lung cancer and IGF-1R/IGF pathway display extensive crosstalk with the estrogen receptor, EGFR and HER2 signaling, the IGF-1R pathway may be associated with resistance to cytotoxic agents or targeted agents. When the f ve relatively resistant cells were treated with increasing concentrations of vorinostat, IGF-1R expression remained unchanged or increased by vorinostat treatment in relatively resistant cells while relatively sensitive cells showed decreased expression of IGF-1R after the drug treatment (Fig 2A and 2B). Down stream phospho-A T was also induced or suppressed by vorinostat in H226Br and A549 cells.

A human phospho-RTK array of the H1299 cell demonstrated that vorinostat induced activation of the IGF-1R/IR pathway in these cells. Expression of phosphorylated insulin receptor and IGF1R was observed in the presence of vorinostat relative to expression in the absence of vorinostat. H1299 cells were treated with 5μΜ vorinostat or DMSO for 72 hours in serum deprived condition. Whole-cell lysates were incubated on RTK antibody arrays comprising antibodies that bind to various phosphorylated proteins (e.g., including anti-phospho IGF1R and anti-phospho insulin receptor). Each RTK antibody was spotted in duplicate and developed. The relative spot densities of each blot and their ratio was determined and the pixel density corresponding to each spot was graphed. See Fig. 2C.

An immunoblot of cell lines H226B, H460 and H1299, with or without SAHA (vorinostat), using the indicated antibodies is shown in Fig 2D. The effect of

dalotuzumab, vorinostat or a combination of both, on anchorage-independent growth of representative vorinostat-resistant cells, H596, H1299, H226B, H460 and H226Br, in a soft agar assay, is shown in Fig 2E. Relative colony numbers are shown (* = p<.05).

Effects of MK0646 on the relatively resistant cells and synergy with vorinostat. In the relatively resistant cells, the IGF-1R antibody (MK-0646) effectively downregulated IGF-1R expression at a very low dose (Fig 3A). But, when the response to MK0646 was evaluated by MTT assay, these IGF-1R inhibitions did not translate into cell growth inhibition. Even though at the dose of MK0646 100ug/ml, there was a minimal growth inhibition in the relatively resistant cell lines (Fig 3B). Because cells growing in monolayer on standard tissue culture plates could have different response to drug treatment compared with cells growing in vivo in three-dimensional environment, the investigators also tested the effects of vorinostat and MK0646 on the cells cultured in soft agar. As a single treatment, each drug showed some level of growth inhibition in the relatively resistant cells. When combined with MK0646, the growth inhibition was more prominent compared to vorinostat single treatment (Fig 3C and Table 1). Taken together, this result suggests that vorinostat and MK0646 exhibit an additive effect in these vorinostat resistant cells.

Table 1. Combinatioa effect on soft agar colony forming assays

The antitumor potency of the combination was evaluated using xenograft tumor models established in nude mice. Nude mouse xenograft tumor growth of H226Br, H1299, H1 44 and H1944 R cells was assayed. A combination treatment of vorinostat and dalotuzumab inhibited xenograft tumor growth in nude mice in two representative vorinostat-resistant cells (H226Br and H1299). Each treatment group had either 7 or 8 mice. Vorinostat 50mg/kg (V), dalotuzumab 15mg/kg (D) or the combination were given i.p. twice a week. See Fig 4A. The effect of vorinostat or vorinostat with or without dalotuzumab in the xenograft model for H1944 and H1944R cells was also determined. The tumor growth was inhibited by vorinostat treatment in tumors with H1944 cells, but only combination of vorinostat with dalotuzumab showed xenograft tumor inhibition in tumors with H1944R cells. See Fig 4B.

Real time quantitative PCR showed an IGF-2 increase in the resistant cells. Fig 5A includes data demonstrating that transcription of IGF-1 was not significantly changed after vorinostat treatment in both representative resistant and sensitive cells, while the real time quantitative PCR assay data in Fig 5B demonstrated that transcription of IGF-2 was significantly induced in the representative resistant cells. Fig 5C contains data demonstrating that, compared to the parental H1944 cells, IGF-2 increased in H1944/R cells after vorinostat treatment.

CONCLUSIONS

The results of all the combination studies described hereinabove indicate that combination treatment with IGF-1R inhibitor and an HDAC inhibitor, e.g., SAHA

(vorinostat) may be useful for cancer therapy, particularly for patients with vorinostat- resistant NSCLC. The data suggest that (a) resistance to vorinostat is related to induction expression and subsequent activation of IGF-1R and (b) integration of IGF-IR-targeted agents e.g., IGF-1R inhibitor would provide an added benefit to the treatment regimens with vorinostat for patients with lung cancer. Results from the study provide further support for clinical trials of MK0646 and vorinostat combination in patients presenting with lung cancer.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the meaning of the invention described. Rather, the scope of the invention is defined by the claims that follow.

REFERENCE 1. Han JY, Oh SH, Morgillo F, Myers JN, Kim E, Hong WK, et al. Hypoxia- inducible factor 1 alpha and antiangiogenic activity of farnesyltransferase inhibitor SCH66336 in human aerodigestive tract cancer. J Natl Cancer Inst 2005 Sep

7;97(17): 1272-86.

2. Lee HY, Moon H, Chun H, Chang YS, Hassan K, Ji L, et al. Effects of insulin-like growth factor binding protein-3 and farnesyl transferase inhibitor SCH66336 on Akt expression and apoptosis in non-small-cell lung cancer cells. J Natl Cancer Inst. 2004 Oct 20;96(20): 1536-48.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a histone deacetylase inhibitor in association with an isolated antibody or antigen-binding fragment thereof that comprises LCDR1, LCDR2 and LCDR3 from a light chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9; and that comprises HCDR1 , HCDR2 and HCDR3 from a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13.

2. The composition according to claim 1 , wherein said antibody comprises a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13; and, a light chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.

3. The composition of claim 1 wherein the antibody or antigen-binding fragment thereof comprises a light chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9; and a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

11, 12 and 13.

4. The composition of claim 1 wherein the histone deacetylase inhibitor is suberoylanilide hydroxamic acid or a pharmaceutically acceptable salt or hydrate thereof; a hydroxamic acid derivative, a Short Chain Fatty Acid, a cyclic tetrapepude, a benzamide derivative, an electrophilic ketone derivative; CBHA, Trichostatin A (TSA), Trichostatin C,

Salicylbishydroxamic Acid, Azelaic Bishydroxamic Acid (ABHA), Azelaic-1-

Hydroxamate-9-Anilide (AAHA), 6-(3-Chlorophenylureido) carpoic Hydroxamic Acid (3Cl-UCHA), Oxamflatin, A-161906, Scriptaid, PXD-101, LAQ-824, CHAP, MW2796, MW2996; or a compound that is represented by the structure:

wherein R₃ and R₄ are independently a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, cycloalkyl, aryl, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydrox lamino group; and n is an integer from 5 to 8.

5. A pharmaceutical composition comprising the composition of claim 1 and a

pharmaceutically acceptable carrier.

6. A method of treating a vorinostat-resistant cancer comprising: administering a therapeutically effective amount of a histone deacetylase inhibitor in association with an isolated antibody or antigen-binding fragment thereof that specifically binds to human Insulin-like growth factor-1 receptor, wherein the antibody or fragment comprises

LCDR1, LCDR2 and LCDR3 from a light chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9; and wherein the antibody or fragment comprises HCDR1, HCDR2 and HCDR3 from a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13.

7. The method according to claim 6, wherein said antibody or fragment is an antibody that comprises a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13; and, a light chain

immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.

8. A method of treating a non-small cell lung cancer comprising: administering a therapeutically effective amount of a histone deacetylase inhibitor in association with an antibody or antigen-binding fragment thereof that specifically binds to human Insulin-like growth factor-1 receptor, wherein the antibody or fragment comprises LCDR1 , LCDR2 and LCDR3 from a light chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9; and wherein said antibody or fragment comprises HCDR1, HCDR2 and HCDR3 from a heavy chain immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13.

9. The method according to claim 8, in which said non-small cell lung cancer is vorinostat-resistant.

10. The composition according to claim 1, wherein said light chain comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequence set forth in SEQ ID NOs. 1, 2 and 3; and wherein said heavy chain comprises HCDR1, HCDR2 and HCDR3 comprising the amino acid sequence set forth in SEQ ID NOs. 4, 5 and 6.

11. The composition of claim 10 wherein the antibody or fragment is an antibody which is a humanized antibody.

12. The composition of claim 11 wherein the inhibitor is vorinostat.

13. The composition according to claim 1 , wherein said histone deacetylase inhibitor is suberoylanilide hydroxamic acid or a pharmaceutically acceptable salt or hydrate thereof.

14. The composition of claim 1 , wherein said histone deacetylase inhibitor is a hydroxamic acid derivative, a Short Chain Fatty Acid, a cyclic tetrapeptide, a benzamide derivative, or an electrophilic ketone derivative.

15. The composition of claim 14, wherein said histone deacetylase inhibitor is a hydroxamic acid derivative selected from the group consisting of vorinostat, Pyroxamide, CBHA, Trichostatin A (TSA), Trichostatin C, Salicylbishydroxamic Acid, Azelaic Bishydroxamic Acid (ABHA), Azelaic-1-Hydroxamate-9-Anilide (AAHA), 6-(3- Chlorophenylureido) carpoic Hydroxamic Acid (3C1-UCHA), Oxamflatin, A-161906, Scriptaid, PXD-101, LAQ-824, CHAP, MW2796, and MW2996.

16. The composition according to claim 14, wherein said HDACi is pyroxamide or a pharmaceutically acceptable salt thereof.

17. The composition of claim 1 , wherein said histone deacetylase inhibitor is represented by the structure:

wherein R₃ and R₄ are independently a substituted or unsubstituted, branched or unbranched alkyl, alkenyl, cycloalkyl, aryl, alkyloxy, aryloxy, arylalkyloxy, or pyridine group, cycloalkyl, aryl, aryloxy, arylalkyloxy, or pyridine group, or R₃ and R₄ bond together to form a piperidine group; R₂ is a hydroxylamino group; and n is an integer from 5 to 8.

18. The method of claim 6, wherein said antibody or antigen-binding fragment thereof and said histone deacetylase inhibitor are administered simultaneously.

19. The method of claim 6, wherein said antibody or antigen-binding fragment thereof and said histone deacetylase inhibitor are administered sequentially.

20. The method of claim 6, wherein said histone deacetylase inhibitor is administered orally, parenterally, intraperitoneally, intravenously, intraaiterially, transdermal!/, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery by catheter or stent, subcutaneously, intraadiposally, intraarticularly, intrathecally, or in a slow release dosage form.

21. A method of treating a vorinostat-resistant cancer, in a subject, comprising the step of administering, to the subject, a first amount comprising a total daily dose of up to 10 mg/kg i.v weekly of an isolated antibody that binds specifically with human insulin-like growth factor-1 receptor and that comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 or 9 and a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12 and 13; in a first treatment procedure; and a second amount of an histone deacetylase inhibitor in a second treatment procedure administered at about 50-2400 mg vorinostat or a pharmaceutically acceptable salt or hydrate thereof; wherein the first and second amounts, together, comprise a therapeutically effective amount.