WO2009089186A2

WO2009089186A2 - Peptide-conjugated oligonucleotide therapeutic and method of making and using same

Info

Publication number: WO2009089186A2
Application number: PCT/US2009/030164
Authority: WO
Inventors: Robert Benezra; Erik Henke
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 2008-01-05
Filing date: 2009-01-05
Publication date: 2009-07-16
Also published as: WO2009089186A3

Abstract

Conjugates for the efficient delivery of sequence-specific antisense to cells of a selected type for the inhibition of a target protein have the general formula: peptide-HBL-antisense in which the peptide is a homing peptide which directs the conjugate to cells of a particular type, antisense is an antisense oligonucleotide having a sequence selected to provide sequence-specific inhibition of the target protein, and HBL is a heterobifunctional linker having reactivity towards amino and sulfhydryl groups.

Description

PEPTIDE-CONJUGATED OLIGONUCLEOTIDE THERAPEUTIC AND METHOD OF MAKING AND USING SAME

Related Applications

This application claims priority from US Provisional Applications 61/019,244 filed January 5, 2008 and 61/027,100 filed February 8, 2008, both of which are incorporated herein by reference.

Background of the Invention

This application relates to a peptide-conjugated oligonucleotide therapeutic agent and to methods of using same in therapy.

Sequence-specific antisense oligonucleotides are well known both as a concept for providing targeted therapy, and as myriad specific agents directed to specific targets. Sequence-specific antisense oligonucleotides interact with mRNA having a complementary sequence to interfere with expression of the protein encoded by the mRNA. Crooke, S.T. "Basic Principles of Antisense Therapeutics" in Antisense Research and Application, Pages 1- 52, Springer (1998). Because the sequence of the antisense can be selected to complement the mRNA for just one protein, sequence-specific antisense oligonucleotides have been seen as offering vast potential for the treatment of many types of disease characterized by protein over-expression. To date, however, this potential has not been realized, owing in part to the inability to reliably deliver effective amounts of the oligonucleotide into the cells containing the target mRNA in vivo.

Various strategies have been proposed to overcome these difficulties so that antisense therapy can realize its potential. In some proposals, vectors are introduced which lead to the in situ production of antisense species (either full length or as oligonucleotides) using cellular mechanisms for the production of polynucleotides. See Weiss et al., Cell. MoI. Life Sci. 1999, 55:334-358. Others have suggested coupling the oligonucleotide to various moieties to enhance delivery of the oligonucleotide to cells. See Manoharan, M., Antisense and Nucleic Acid Drug Development. 2002, 12(2): 103-128

Peptides that specifically home to tumor endothelial cells in various tissues are known. For example, Porkka et al. Proc. Nat'lAcad. Sci. (USA) 99: 7444-7449 (2002), which is incorporated herein by reference, describe peptides that home to the nuclei of tumor cells and tumor endothelial cells in vivo. Other cell-type specific homing peptides are described in US Patent Publication Nos. US2003/0152578, US2003/0149235, and US2002/0041898, which are incorporated herein by reference. US Patent Publication No. 2003/0152578 relates to conjugates of these homing peptides with drugs or with detectable labels. US Patent Publication No. 2002/0041898 mentions conjugates of homing peptides with various types of molecules including antisense oligonucleotides, but does not disclose any specific method for this conjugation. US Patent No. 5,670,617 discloses conjugation of nucleic acids with the HIV tat protein as a transport protein to enhance intracellular delivery.

Harrison et al., Nucleic Acids Res. (1998) 13: 3136-3145 discloses synthesis of peptide-oligonucleotide conjugates using 4-(maleimidomethyl)-l-cyclohexane carboxylic acid N-hydroxysuccinimde ester (SMCC) as a linker. This paper reports only the use of short peptides, however, to allow assessment of the affect of peptide sequence on antisense activity.

These approaches have not provided a general answer to the issue delivery of antisense to cells of interest for efficient reduction of expression of a target protein. When a targeting moiety is added to an oligonucleotide, it may limit the ability of that oligonucleotide to interact with target mRNA for example as a consequence of steric or chemical (hydrophilic/hydrophobic) interactions, alter the ability of the molecule to pass through the cell membrane and/or change the intracellular trafficking pattern for the molecule such that it sequestered in or excreted from the cell, or otherwise made less available for interaction with target nucleotides in the cell.

Summary of the Invention

The present invention provides therapeutic conjugates for the efficient delivery of sequence-specific antisense to cells of a selected type for the inhibition of a target protein. The therapeutic conjugates of the invention have the general formula:

peptide-HBL-antisense

in which the peptide is a homing peptide which directs the conjugate to cells of a particular type, antisense is an antisense oligonucleotide having a sequence selected to provide sequence-specific inhibition of the target protein, and HBL is a heterobifαnctional linker having reactivity towards amino and sulfliydryl groups. Exemplary linkers useful as HBL in this formula include SMCC, GMBS (4-maleimidobutyric acid N-hydroxysuccinimide ester) and EMCS ([N-e-Maleimidocaproyloxy]succinimide ester). SMCC, GMBS and EMCS are known linkers that have been used to form conjugates of peptides, for example with larger proteins or polysaccharides, and are commercially available as a cross-linking agent for proteins (www.piercenet.com).

In specific embodiments of the invention, the peptide is a peptide that specifically homes to endothelial cells in the tumor. In preferred embodiments, the peptide portion of the conjugate is the F3 peptide of Seq. ID No. 3.

In specific embodiments, the antisense oligonucleotide that inhibits expression of inhibitor of DNA-binding protein 1 (IdI). In preferred embodiment, the antisense oligonucleotide targeting IdI has the sequence of SEQ ID Nos. 1 or 2.

The invention further provides a method of using the conjugates of the invention to treat diseases associated with over expression of a target protein. In accordance with this method, a patient, particularly a human patient diagnosed with the disease is treated by administration of a peptide-conjugate antisense oligonucleotide conjugate, in which the peptide portion is selected to direct the conjugate to cells relevant to the disease, and the antisense portion is selected to be a sequence-specific inhibitor of expression of the target protein. In specific embodiments, the method is used to treat cancer, particularly tumors whose growth is very dependent on de novo blood vessel formation.

Brief Description of the Drawings

Fig. 1 shows a synthetic procedure for making the peptide conjugated oligonucleotide of the invention.

Fig. 2 shows a tumor growth curve for mice treated with 12 nmol/d IV of IdI -PCAO starting on day 4 after tumor implantation (vertical line).

Fig. 3 shows increased haemorrhage in treated tumors.

Fig. 4 shows reduction in cells staining positive for IdI in treated tumors.

Fig. 5 shows tumor growth in Lewis Lung cell model with various treatments.

Fig. 6 shows percent of tumor free mice in Lewis Lung cell model after removal of primary tumors with various treatments. - A -

Fig. 7 shows tumor volume following treatment with IdI-PCAO, with and without 17-AAG treatment.

Fig. 8 shows accumulation of conjugates in 4Tl breast carcinoma using different peptide portions.

Detailed Description of the Invention

In the present application the term "peptide-conjugated antisense oligonucleotide" or PCAO refers to a compound of the structure peptide-HBL-antisense in which the peptide is a sequence providing cell-type specific targeting. HBL is a heterobifunctional linker and antisense is an antisense oligonucleotide (including DNA, RNA, morpholino oligonucleotides, peptide nucleic acids, LNA or chemically modified derivatives thereof) with sequence-based specificity for a therapeutic target gene found in the targeted cell type. As will be apparent, the peptide and the antisense are selected in compatible pairs, and the general formula should not be construed as covering meaningless combinations of peptides and antisense that would not be expected to have any therapeutic value.

The antisense may be an RNA molecule that forms a double-stranded hairpin (shRNA) or it may be double stranded. In the case where the antisense is part of a double stranded molecule, for example when using siRNA, the peptide may be conjugated with either the sense or the antisense strand, the other strand being associated by hybridization. Thus, in a more general sense, the structure can be described as peptide-HBL-sequence-specific oligonucleotide with the sequence specific oligonucleotide portion comprising a sequence-specific antisense oligonucleotide, either as a direct conjugate or via hybridization to a conjugated strand.

Peptides useful in the compounds may be diverse based on the desired target cell-type. The length of the peptide portion is variable, and need only be sufficient to provide the cell- type specificity desired. In some embodiments, the peptide has a length of from 6 to 50 amino acids. Specific peptides include those disclosed in Porkka et al. Proc. Nat'l Acad. Sd. (USA) 99: 7444-7449 (2002) and US Patent Publication Nos. US2003/0152578, US2003/0149235, and US2002/0041898. In a specific embodiments of the invention, the peptide portion of compound comprises peptide of the sequence:

CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (Seq. ID No. 3)

also referred to herein as HMGN2-F3 or simply F3 which homes to tumor endothelial cells. In other specific embodiments of the peptide portion of the composition is a Grp78 homing peptide such as one comprising the sequence:

SGRP78- 1 WIFPWIQL (Seq. Id No. 4) , or

SGRP78-2 WDLAWMFRLPVG (Seq. ID No. 5)

described in Arap et al. Cancer Cell, 2004, 6(3):275-84 which is incorporated herein by reference. Grp78 homing peptides are useful in targeting the therapeutic agent of the invention to cells for the treatment of glioma. In other specific embodiments, the peptide portion of the compound comprises a tumor endothelial marker as disclosed in St Croix et al. Science 289: 119701202 (2000) which is incorporated herein by reference. In still further specific embodiments, the peptide is a tumor endothelial marker as listed in the following table:

TEM Accession Number

Vscp DQ832275

ETSvg4 (Pea3) DQ832277

Apelin DQ832282 as described in Seaman et al, Cancer Cell, 2007 PMID: 17560335, which is incorporated herein by reference.

The oligonucleotide portion of the compound of the invention may likewise be quite variable depending on the specific gene being targeted. In general, it may be any oligonucleotide which has been shown to have sequence specific activity for inhibition of the target gene. In specific embodiments of the invention, the antisense oligonucleotide is targeted to a transcription regulating factor. Examples include antisense targeting inhibitors of DNA bindings (IdI, Id2, Id3 and Id4) which are known for various sources including US Patent No. 6,372,433, Kleef et al. Cancer Res. 1998 Sep l;58(17):3769-72, and Chaudhary et al., Endocrinology Vol. 142, No. 5 1727-1736 which are incorporated herein by reference. Other transcription regulating factors include Olig2 (Accession # NP_005797) (Lingon et al, Neuron 2007 PMID: 17296553, Marie et al, Lancet 2001 PMID: 1 1498220). Antisense to Olig2 with the sequence (5'-TCATCTGCTTCTTGTCCT-S', Seq. Id No. 6) is disclosed in Fu et al., Development 129, 681-693 (2002). Still other targets for which antisense oligonucleotides are known include, without limitation bcl-2, BCR-ABL, C-raf-1, Ha-ras, c-myc, PKC, PKA, p53 and MDM2, insulin-dependent growth factor (IGF) and its binding proteins, particularly IGFBP-2 and IGFBP-5. Antisense to kinase suppressor of ras inactivation for therapy of ras mediated tumori genesis is known from commonly assigned US Patent Publication No. 2006025203 which is incorporated herein by reference. Antisense that specifically hybridizes to a nucleic acid encoding a DNA dependent protein kinase subunit so as to prevent expression of the DNA dependent protein kinase subunit and increase the sensitivity of the cell to heat, chemical, or radiation-induced DNA damage is disclosed in commonly assigned US Patent Publication No. 20050032726 which is incorporated herein by reference.

Sequences useful in siRNA modulation of protein expression are described in numerous sources, including without limitation US Patent Publications 20040077574, 20040127450, 20050042646, 20050124569, 20050137155, 20050171039, 20050196767, 20050209179, 20060094676 which are incorporated herein by reference.

In specific embodiments of the invention, the compound comprises the IDl targeting antisense sequence gcaccagctccttgaggcgtgag (Seq. ID No. 1) or an RNA counter-part thereof. In preferred embodiments, this sequence is provided as a 5-13-5 gapmer, with the first and last 5 bases in the sequence being 2'-O-methyl RNA bases, and the intervening 13 bases being phosphorothioate linked DNA bases. This preferred oligonucleotide has the sequence: GCACCagctccttgaggcGUGAG (Seq. ID No. 2), with the RNA bases shown in upper case. In other embodiments, the antisense portion has the sequence CAGCCGTTCATGTCGT (Seq. ID No. 31) which targets human-Id 1, human-Id3, mouse-Id 1, and mouse- Id3, or CAGTGGTTCATGTCGA (Seq ID No. 32) which targets hld3 and mld3. The peptide portions and the oligonucleotide portions of the compositions of the invention are covalently coupled to one another via the heterobifunctional linker (HBL) that has reactivity with an amino and sulfhydryl groups. In certain embodiments, the heterobifunctional linker is a compound with a maleimide and a succinimide group. The oligonucleotide is connected to the bifunctional linker using the maleimide activity of the linker and an amino functionality on the oligonucleotide. The peptide is reacted with the bifunctional linker via succinimide portion and a sulfhydryl functionality on the peptide. Known linkers of this type with varying spacers are listed in the following table:

Specific examples of compounds within the scope of the present invention include those listed in table 1.

Table 1

Methods for formation of peptide-oligonucleotide conjugates such as these are known. In particular, the use of a small linker (4-(maleirnidomethyl)-l-cyclohexane- carboxylic acid N-hydroxysuccinimide ether, SMCC) for this purpose is described in the Harrison et al., Nucleic Acids Res. 26: 3136-3145 (1998), which is incorporated herein by reference, and this method can be used in connection with making the therapeutic agents of the invention. Another specific method which was used to make the compositions described in the examples of this application and which is illustrated in Fig. 1 uses chemistry similar to that of Harris et al., but uses 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) in place of SMCC. GMBS and SMCC have the same reactive groups, the maleimido group and the hydroxysuccinimide group, but differ in the intervening structure or spacer. The same is true of EMCS. Both synthetic methods results in a compound with an HBL, with the oligonucleotide connected directly or indirectly through the nitrogen atom and the peptide connected through a sulfur to a carbon atom of the succinimide ring.

The therapeutic agent of the invention can be administered in various ways, including topically, intra-nasally, and via subcutaneous, retro-orbital, intramuscular, intravenous, and intraperitoneal injection. The amount of the therapeutic agent administered is sufficient to provide a therapeutic affect without inappropriate levels of toxicity. It will be appreciated that the extent of tolerable side effects is dependent on the seriousness of the condition being treated. In general, therapeutic levels of 10%, 25%, 50%, 75% or up to 100% of the maximum tolerated dosage as determined through standard toxicity testing are appropriate.

The therapeutic agent can be formulated in sterile injectable solutions, in lipid carriers, in topical creams and ointments or other carriers dependent on the condition, hi addition, in the case of cancer treatments, the therapeutic agent can be formulated or administered with a chemotherapy agent or agents that are effective against the particular cancer. Examples of chemotherapy agents include, without limitation, vinca alkaloids such as vinblastine, navelbine and vindesine; probenicid, nucleotide analogs such as 5-fluorouracil, cytarabine and gemcitabine; alkylating agents such as cyclophosphamide or ifosfamide; cisplatin or carboplatin; leucovorin; taxanes such a paclitaxel or docetaxel; anti-CD20 monoclonal antibodies, with or without radioisotopes, antifolates such as methotrexate, edatrexate and 10-progargyl-lO-deazaaminopterin; and antibiotics such as doxorubicin and mitomycin.

The therapeutic agents of the invention can, through the appropriate selection of the peptide and the antisense portions of the compound, be used to treat a wide variety of cancers, including without limitation, breast cancer, prostate cancer, lung cancer, glioma and other brain cancers, colorectal cancer, pancreatic cancer, liver cancer, and sarcoma.

Therapeutic agents with Idl-targeted antisense

In one embodiment of the invention, the therapeutic agents of the invention are effective to inhibit expression of IdI, and as a result are useful in the treatment of conditions where angiogenesis is significant, including tumors and diabetic retinopathy. In this embodiment, the homing peptide F3 selectively directs the therapeutic agent to tumor endothelial cells, and as a result helps minimize the side effects of the treatment and elevate local concentration of the active agent (the oligonucleotide). hi order to determine if the IdI PCAO retained the homing specificity of the F3 peptide and could be taken up by cells in the absence of lipid or other specialized carrier, fluorescence labelled IdI-PCAOs were supplied to different cell lines at a concentration of 200 nM. Confocal fluorescence microscopy showed uptake only by endothelial cells (HUVECs and the murine endothelioma cell line EOMA) whereas all other tested tumor cell lines as well as normal murine embryonic fibroblasts (MEFs) and human dermal fibroblasts (NHDFs) were negative. While the IdI-PCAOs were readily taken up into endothelial cells, no internalization was observed using non-conjugated fluorescence labelled IdI-AOs presumably due to the absence of lipid carrier and the absence of conjugation with the homing peptide. Fluorescein and terra methyl-rhodamine red labelled PCAOs were also tested to control for the effects of the fluorophore and similar results were obtained. To rule out effects from the fixation process we performed epifluorescence live imaging on viable HUVECs and HeLa cells and similar results were observed. The fluorescent IdI-PCAO derivatives co-localized with nucleolin in the nucleus of HUVECs. This is in accordance with published data suggesting that nucleolin is the cell surface binding partner for F3, and that F3 is transported with nucleolin into the cytoplasm and subsequently into the nucleus.

Homing studies were also performed in animals bearing spontaneous rumors in MMTV-HER2/neu (YD) and PTEN+/- backgrounds. Accumulation of IdI-PCAOs in the rumor endothelium was observed in these models, showing that the homing properties are maintained in spontaneous rumor models. To test if the PCAOs down-regulate IdI protein levels in vivo, rumor bearing transgenic MMTV-HER2/neu (YD) IdI+/- mice were injected intravenously with IdI-PCAOs. Since repeated application of the drug was necessary to yield significant down-regulation in vitro, the animals were treated with 15 nmol/d of IdI-PCAO or ldl-AO for three consecutive days. Over 80 % of rumor vessels in animals treated with IdI-PCAO were completely negative for IdI expression by IHC. Vessels from animals that received the IdI-AO alone showed no detectable down-regulation of IdI.

The effect of the IdI-PCAOs on a developing allograft tumor was examined. The benefit of this model versus a spontaneous model is that tumor onset and progression time can be controlled. This allows for narrowing of total treatment time as much as possible. IdI negative cells from a spontaneous tumor formed in a MMTV-HER2/neu (YD) Id-/- animal were employed to ensure that observed effects were caused by IdI inhibition in the microenvironment, presumably the endothelium, and not the tumor cells. While fluorophore-labelled PCAOs did not accumulate in the tumor cells in short term experiments, we wanted to exclude effects resulting from accumulation of the drug in the tumor when given over a prolonged period. Animals were implanted with MMTVHER2/neu (YD) IdI-/- cells and treated with 10 nmol/d (app. 5.7 mg/kgBW*d, equivalent to 3.7 mg/kg*d pure AO) IdI-PCAO for eight consecutive days by i.v. injection, starting at day 4 when tumors became palpable. Tumor-growth was only modestly, but statistically significantly (P = 0.0078) inhibited by treatment with IdI-PCAOs (Fig. 2). Tumor specimens dissected after completion of the treatment however showed a dramatic increase in haemorrhage (Fig. 3, P = 0.022, an effect also observed previously in spontaneous tumors arising in Id-deficient mice. IdI-PCAO treated tumors also showed significantly lower counts of IdI -positive endothelial cells, indicating the effectiveness of the drug (Fig. 4, P = 0.015 for IdI-PCAO treated versus both, saline and F3 + IdI-AO receiving tumors). IdI-PCAOs induced hypoxia as shown by increased staining for Hifl -alpha-expression relative to saline or F3 plus IdI-AO controls. This result was consistent with previously observed analysis of genetic IdI loss in spontaneous MMTV-HER2/neu (YD) tumors.

Id proteins are attractive targets for anti-angiogenic tumor therapy because (1) they are essential for the mobilization of endothelial progenitors from the bone marrow to the tumor, and (2) are not expressed in normal adult vasculature and lead to severe perturbations in tumor vascular integrity when partially inhibited genetically. However, the hurdles for hitting these targets are significant since Ids are intranuclear proteins which work by direct physical association with other proteins. The results summarized here show that the therapeutic agents of the invention provide the ability to practically realize the potential of the Id proteins (particularly IdI) as targets for use in anti-angiogeneic tumor therapy, through the use of the antisense targeting strategy using a homing peptide. The resulting peptide conjugated antisense oligonucleotide (or PCAO) retains its homing specificity and ability to inhibit IdI protein expression both in vitro and in vivo.

While it has been previously shown that F3 can be used to transport a payload like fluorophores or nanoparticles into the tumor vasculature, it was anticipated that the homing potential of the highly basic F3 peptide might be affected by conjugation to the anionic oligonucleotide. However, surprisingly the PCAO seems to show higher selectivity for endothelial cells than F3 itself which is also taken up by tumor cells in vitro. Furthermore, the tumor vessel phenotype observed after intervention with IdI-PCAOs and after genetic IdI loss is different than that found after treatment with anti- VEGF agents like the monoclonal antibody Avastin.

In vivo, PCAOs closely recapitulate the effects of IdI loss observed in genetically manipulated mice which strongly supports the idea that we have effectively hit the intended target. As a single agent IdI-PCAO was able to reduce the growth rate of Lewis Lung cancer (LLC) tumors significantly similar to what has been observed after genetic reduction of IdI levels. Male C57/B6 were engrafted with GFP/fluc expressing Lewis lung carcinoma cells. After tumor establishment osmotic pumps were implanted to deliver IdI-PCAO or control substances. Fig. 5 shows Tumor growth was followed for 14 days post implantation. Treatment started on day 8 after the pumps implanted on day 7 started working (grey field: working period of pumps during primary tumor growth). As shown, tumor volume was substantially lower with IdI-PCAO treatment (bottom line).

Moreover, after treatment with IdI-PCAOs and removal of primary tumors, metastatic growth of LLC was substantially delayed as was observed after genetic reduction of Idl/3 levels. Fig. 6 shows a Kaplan-Meyer plot of tumor free survival after primary tumors were surgically removed 14 days after injection. Metastatic growth was monitored by intravital bioluminescence imaging (grey field: residual working period of pumps after removal of primary tumors). As shown, the percentage of tumor free mice was substantially greater with the IdI-PCAO treatment (top line).

Increased haemorrhage in treated tumors was also observed in mice treated with IdI- PCAO as compared to the controls. Importantly, we now have shown that inhibition of IdI by PCAO in established tumors can also substantially inhibit tumor growth in the LLC model, thus establishing the clinical relevance of the method of the invention. Since we observed increased haemorrhage and vascular permeability in treated tumors which is in general attributed with a higher rate of tumor cell embolization, it is probable that IdI-PCAO interferes with metastasis by blocking angiogenesis in the new distal bed rather than inhibiting escape of cells into the circulation from the primary tumor. Indeed metastatic cells were observed in the lungs of IdI-PCAO treated animals, but these cells failed to colonize as long as treatment was applied. This is in accordance with earlier findings that genetic IdI loss prevents the establishment of metastasis in the lung after i.v. injection of LLC cells.

In a particularly preferred embodiment, the therapeutic peptide-conjugated oligonucleotide is administered in combination with an inhibitor of Hsp90 such as 17-AAG (17-(allylamino)-17-demethoxygeldanamycin). Other inhibitors of Hsp90 are known, for example from US patent Publications 2004/0102458, 2005/0049263, 2005/0107343, 2005/0113339, 2006/0205705 and 2007/0072855, which are incorporated herein by reference.

It has been observed previously that genetic Id loss in combination with the Hsp90-inhibitor 17- AAG) was more effective than either alone in reducing tumor burden (de Candia, P. et al. Angiogenesis impairment in Id-deficient mice cooperates with an Hsp90 inhibitor to completely suppress HER2/neu-dependent breast tumors. Proc Natl Acad Sci U S A 100, 12337-42 (2003)), perhaps due to the requirement of Hsp90 for maintaining Hifl- alpha or HER2/neu stability. IdI-PCAOs were therefore tested in combination with 17-AAG in the MMTV-HER2/neu (YD) IdI-/- allograft model. The IdI-PCAO was delivered in this prolonged experiment via subcutaneously implanted osmotic pumps (7 nmol/d). 17-AAG was given by intraperitoneal injection according to an established protocol, on 3 consecutive days per week after tumor establishment. As controls, saline or the un-conjugated components of the IdI-PCAO (F3-peptide and IdI-AO each at 20 nmol/d) were administered. IdI-PCAO alone delayed tumor growth to a similar degree as 17-AAG. Combination of both drugs however yielded virtually complete inhibition of tumor growth over the treatment period (Fig. 7, bottom line, P = 0.0001, P < 0.0001, P - 0.0002 versus control, 17-AAG and F3 plus IdI-AO plus 17-AAG respectively). In contrast administration of the antisense oligonucleotide and peptide in un-conjugated form did not inhibit tumor growth and showed no enhancement of the 17- AAG alone effect. To further control for non-specific effects we repeated the experiment using the same tumor model and added this time rcIdl-PCAO which was delivered at the same rate as the IdI-PCAO (7 nmol/d) and the F3 -peptide without addition of IdI-AO (20 nmol/d) as control substances. In this second experiment IdI-PCAO administration in combination with 17- AAG again had a significant effect on growth (P = 0.0079) while neither rcIdl-PCAO nor F3 enhanced 17- AAG efficacy (P = 0.97 and 1.00 respectively). Injection of Evans blue in selected animals showed massively increased leakage from the IdI-PCAO treated tumor blood vessels likely accounting for the hypoxic stress and sensitivity to 17- AAG. Treatment with IdI-PCAO as well as with 17- AAG alone resulted in a decrease of IdI -positive endothelial cells in the tumor. While IdI-PCAO down-regulated IdI expression in the cells, 17- AAG lead to a decreased vascular density, which resulted in the lower count for IdI -positive cells. Combination of both drugs further diminished IdI -positive cells. The decrease in Idl-positive cells after administration of IdI-PCAO alone was less dramatic than in the intravenous protocol described above. This might be due to the slight decrease in dosage and the changed route of administration (s.c. vs. i.v.). While IdI-PCAO administration caused up-regulation of Hifl -alpha, 17- AAG injections counteracted this response. The hypoxic regions characteristically surrounded necrotic areas that also displayed signs of cystification.

Adverse effects of IdI-PCAO treatment were not observed. Animals did not suffer from weight loss and wound healing (e.g. after replacement of the subcutaneous implanted pumps) was not grossly impaired. Also, kidneys of all animals were removed after the 3 week treatment and examined and no signs of renal toxicity were observed. hi a further specific example, the therapeutic agent combines a Grp78 targeting peptide with an IdI targeting antisense oligonucleotide. A therapeutic agent of this type was synthesized using the Grp-78 targeting peptide of Seq. ID No. 4, GMBS as the HBL, and IdI antisense of Seq. ID No. 2, labeled with FITC and injected retro-orbitally into a mouse model with a high-grade malignant glioma. Four hours post injection the mouse was sacrificed, and tissue was processed for imaging. Grp78-GMBS-ASIdl was observed to accumulate around tumor blood vessels in the imaged tissue, indicating that it crossed the blood brain barrier and selectively accumulated in tumor endothelium. Examples

Example 1: Synthesis of F3-ASO conjugates

Coupling was done according to Harrison and Balasubramanian, Nucleic Acids Res 26, 3136-45 (1998), with some modifications as described below. Fully modified oligonucleotides were obtained directly from Operon Biotechnologies with a C6-amino linker attached to the 5 '-end of a gap-mer with the sequence GCACCagctccttgaggcGUGAG (SEQ ID No: 2, upper case: 2'0-methyl RNA bases; lower case phosphorothioate linked DNA bases). The reverse complimentary sequence with the same modification was used as a control oligonucleotide (rcIdl-AO and in conjugated form as rcldl -PCAO). For uptake and homing studies, oligos were obtained from the supplier with a 3 '-end fluorescein or rhodamin red label. The cysteine modified F3-peptide sequence is CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (SEQ ID NO: 3).

The oligos were dissolved in 200 mM TrisHCl pH 8.4 to a final concentration of 1 mM and stored at -20⁰C. 2.8 mg GMBS (4-maleimidobutyric acid N-hydroxysuccinimide ester, 10 μmol, 100 eq.) in 40 μL acetonitrile were added to 100 μL (100 nmol) of the oligo solution. The reaction vessel was wrapped in aluminum foil and incubated with shaking at 25 ⁰C for 90 min. The oligonucleotide was precipitated with 1 mL acetonitril and remaining GMBS was removed by vigorous washing with acetonitril (9 x 1 mL). After drying in vacuo the activated oligo was dissolved in 50 μL buffer (100 mM Na-phosphate, 400 mM NaCl at pH 7.0) to which 400 nmol F3-N-Cys (4 eq.) in 50 μL buffer (40 mM Na-phosphate, 20 mM EDTA, pH 7.0) were slowly added. The reaction mixture was incubated with shaking for 24 h at 25 C. The coupling-product was purified via reverse phase high performance liquid chromatography (Akta Purifier System, GE Healthcare; Column: OligoDNA RP 150x7.8 mm, Tosoh Bioscience, Montgomeryville, PA). To first eluate the non-conjugated peptide a 0.05% TFA in water/acetonitrile gradient (5-20% acetonitrile) was used. To separate the non-conjugated AO from the PCAO a second step was done using an ammonium acetate (20 mM, pH 6.8) / acetonitrile gradient (5-25% acetonitrile). Fractions containing the PCAO were combined, concentrated in a vacuum concentrator system (Eppendorf, Westbury, NY), and precipitated with ethanol. Yields in a multitude of experiments (>10, scales up to 600 nmol) varied between 40 and 78 % as calculated from absorbance measurements at 260 nm. Identity of the conjugate was verified by mass spectrometry. Negative ion electrospray ionization mass spectronomy was performed on a Sciex Q-star/Pulsar instrument (MDS Sciex, Concorde, ON). IdI-PCAO: calculated mass: 11441.6 Da, found: 11441.8 Da.

Example 2: Effect of F3-ASO conjugates on IdI expression

HUVEC (4th passage) were seeded 150,000 cells into the wells of six- well dishes (BD Bioscience, San Jose, CA). Medium was exchanged after 18h with standard medium containing either F3-ASO conjugates or ASO at a concentration of 200 nM. The treatment was repeated every 24 h for two more days. Samples were drawn every 24 h by lysis with 150 μL/well M-PER lysis buffer (Pierce). Samples were stored in liquid nitrogen. Protein concentration was determined with the BCA protein assay kit (Pierce), and an equal amount (30 μg) was loaded into the wells of a 4-20% gradient SDS polyacrylamide gel. After electrophoresis the proteins were transferred onto a PVDF membrane and IdI amounts were detected with an anti-Idl antibody (C-20, SantaCruz Biotechnology, Santa Cruz, CA) and an anti-rabbit-HRP conjugate as secondary antibody (Amersham). Significantly greater reduction in IdI expression was observed using the F3-Idl-ASO conjugate than using the ASO conjugate alone.

Example 3: Tube Formation Assay

HUVECs (Cambrex, East Rutherford, NJ) in 4th passage were seeded at 500,000 cells into 25 mL culture flasks in EGM-2 medium. Medium was exchanged after 18h with standard medium containing either F3-ASO conjugates or ASO at a concentration of 200 nM. The treatment was repeated every 24 h for two more days. 72 h after starting the treatment wells of a 24 well culture dish were covert with 150 μL matrigel and the matrigel was gelatinized for 30 min at 37 ⁰C. Pretreated HUVEC were trypsinized, the detached cells were collected by centrifugation and resuspended in EGM-2 media containing the F3-ASO conjugates or ASO at 200 nM. Cells were seeded at 25,000 or 50,000 cells on the gelatinized Matrigel. The plates were incubated at 37 ⁰C with 5 % CO2 for 16 - 18 h. Media was removed, and the matrigel surface was washed twice with 500 μL PBS before cells were labeled with 250 μL 8 μM Calcein AM (Pierce, Rockford, IL) in PBS for 30 min at 37 ⁰C. After two additional washing steps (500 μL PBS) cells were visualized under an inverted microscope. Fluorescence was excited at 488 nm and recorded at 538 nm. Well-defined tube formation did not occur in HUVEC treated with F3-Idl -ASO (IdI -PCAO). See Table 2. This shows that treatment with the F3-Idl-ASO conjugate interferes with the ability of HUVEC to form capillary-like structures, which is necessary in angiogenic processes. The tube formation assay is a standard assay for anti-angiogenic drugs as reviewed in Auerbach et al. Clin. Chem. 49 (2003), 32-40.

Table 2

Example 4: Wound healing assay

HUVEC (4th passage) were seeded 100,000 cells into the wells of a fibronectin coated two-well chamber slides (BD Bioscience). Medium was exchanged after 18h with standard medium containing either F3-ASO conjugates or ASO at a concentration of 200 nM. The treatment was repeated every 24 h for two more days. 72h after starting the treatment a scratch was applied with a micropipette-tip. Media was removed the cell-layer once washed with PBS and media containing the F3-conjugate or the control ASO was added. The chamber slides were incubated at 37 ⁰C with 5 % CO2 for 22 h. The cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 20 min at r.t, and washed twice with PBS. The slides were mounted and images were taken under a light microscope to access the grade of closure of the applied scratch.

While the applied scratch closed completely in the non-treated and ASO treated cell cultures, a significant inhibition of the wound closing process was observed in the F3-Idl-ASO conjugate treated culture. This indicates that treatment with the F3-Idl-ASO conjugates inhibits the motility of HUVEC. The movement of endothelial cells towards the progressing tumor is an important part of the angiogenic process. Carmeliet P. and Jain R.K., Nature, 407 (2000), 249-257.

Example 5: In vivo distribution of F3-Idl-ASO-FITC conjugates in a PTEN +/- transgenic tumor model.

18 nmol of F3-Idl- ASO-FITC conjugate were i.v. injected into a mouse with advanced lymph hyperplasia. After 3h the animal was sacrificed and organs were removed. The organs were paraformaldehyde-fixed, embedded in OCT-medium and sectioned. Tissue sections were counterstained with Hoechst 33342 and mounted with fluorescence mounting media. Experiments were repeated in the Pten +/- model and twice in YD neu mice.

A strong accumulation of the FITC-signal was observed in the vessel walls of the tumor and in the kidneys. No significant accumulation was observed in other organs (heart, brain, liver, spleen, intestines). The strong signal in the tumor endothelium is consistent with targeted delivery of the active agent (IdI-ASO) by conjugation to F3. The accumulation in the kidneys indicates rapid renal clearance, also observed with other peptide drugs after i.v. application.

Example 6; Down regulation of IdI expression in the tumor endothelium of YD neu breast carcinoma.

YD neu transgenic animals bearing breast carcinoma were treated for 3 subsequent days with 25 nmol of either IdI-ASO or F3-W1-ASO conjugate in PBS by i.v. injection. 24 h after the last injection animals were sacrificed, the tumor removed, fixed in paraformaldehyde and embedded in parafine. IHC staining was performed for IdI (antibody C-20, SantaCruz Biotechnology, Santa Cruz, CA) on sections. While treatment with antisense alone showed no effect, IdI expression was no longer observed in the endothelium of F3-W1-ASO treated tumor.

Example 7; Transfection of endothelial cells (ECs) with antisense oligonucleotides (AOs): ECs (HUVEC-2 or MS-I) were seeded 16 h prior to transfection at 10⁵ cells/well in six-well Multi well dishes (MWD) in standard growth media without antibiotics. AOs were re-precipitated with ethanol from sodium acetate (10 mM, ph 4.8) buffer prior to use. Cells were transfected in 1 mL OptiMEM I media (Invitrogen, Carlsbad, CA) with Lipofectin (Invitrogen) or Cytofectin (Gene Therapy Systems, San Diego, CA) according to the manufactures' recommendations. The transfection was repeated at 24 and 48 hours.

Example 8 Plasma stability assay: Female balb/c mice were bled by submandibular punctuation using a 4.5 mm lancet (Medipoint, Mineola, NY). Plasma was separated from heparinized whole blood by centrifugation (18000 g, 5 min at 4° C). Antisense oligonucleotides and PCAOs were dissolved in plasma at 25 μM and 10 μL aliquots in microfuge tubes and were incubated at 37⁰C for the indicated time. After incubation, 5 μL quencher solution (1.6 M NaCl, 100 mM EDTA pH 8.0) were added and the samples were stored at - 80° C. For analysis samples were diluted to 60 μL with agarose sample buffer, incubated 5 min at 95⁰C and separated in a 1.5 % low melting point agarose gel. AOs/PCAOs were visualized with ethidium bromide under UV-light.

Example 9: Transfectϊon of cells with IdI-PCAOs: HUVEC were seeded 24 h prior to transfection at 10⁵ cells/well in six- well MWDs in standard growth media (EGM-2) without antibiotics. At the day of transfection the AO-conjugates and control oligos were diluted in EGM-2 to the indicated concentration. Media was replaced with the supplemented EGM-2. The procedure was repeated every 24 h for two more days, samples were drawn at the indicated time points and analyzed via western blot. Other cell lines were treated similarly, with the exception that IdI-PCAO was added to standard growth media, DME or RPMI 1640 in accordance to cell type. Uptake studies in different cell lines with fluorescence labelled IdI-PCAO were performed in both the standard growth media (DME, RPMI 1640 or EGM-2) or in OptiMEM I (supplemented with 2% FBS) to control for media effects.

Example 10: Scratch assay: HUVEC were plated in fibronectin coated two-well chamber slides (BD Bioscience) at 2.5 xlO4 cells/well. Growth media was supplemented with 200 nM IdI-PCAO (or IdI-AO) and renewed every 24 h. 72 h after plating a scratch was applied using a 20 μl pipette tip. Chambers were washed with media and supplemented media was added. 18 h after the scratch was applied, cells were fixed and imaged.

Example 11: Transduction of LLC cells with an eGFP/Fluc dual-modality reporter:

Ecotropic retrovirus based on the SFG vector5, expressing an Aequorea Victoria eGFP/firefly luciferase (eGFP/FLuc) fusion protein was produced in PhoenixE cells and was used with at least 1 x 106 infectious particles/ml against NIH3T3cells. The in vitro transduction of early passage LLC cells with the retroviral vector was accomplished by exposing the cell monolayer to a filtered (0.45 μm) culture medium obtained from the vector producer cells for 8 h in the presence of 8 μg/ml polybrene (Sigma, MO). Stably transduced cells were enriched by fluorescence assisted cell sorting (FACS) using eGFP-expression as an marker for successful transduction.

Example 12; Proliferation assav: HUVEC were plated at 2x10⁴ cells in the wells of 24 well MWDs. After 18 h media was exchanged with standard growth media (EGM-2) supplemented with IdI-PCAO or IdI-AO plus F3 (200 nM or 1 mM). Supplemented media was renewed every 24 h. Media was removed from sample plates at different time points, and the plates were stored at -80 C. All samples were analyzed in parallel using the CyQuant system (Invitrogen, Carlsbad, CA) according to the manufacture's instructions.

Example 13: Delivery of IdI-PCAOs in vivo. 12 nmol fluorescence-labelled IdI -PCAOs (app 6.8 mg/kg BW) or IdI-AOs were dissolved in TBS and injected into the tail vein or subcutaneously of tumor-bearing mice. Mice were sacrificed, organs and tumors were dissected, fixed overnight in 4% PFA and finally immersed in 20% sucrose for 24 h. After embedding in OCT (Miles Inc., Elkhart, IN) and sectioning, samples were probed for CD31 using a biotinylated secondary antibody and a streptavidin-Alexa488 conjugate as a tertiary agent.

Example 14: Allograft model of Her2-overexpressing breast cancer: Female nude NCR mice (Taconic) were engrafted with 5 x 106 MMTV-HER2/neu (YD) IdI-/- tumor cells in the left flank. The animals were randomly divided to three cohorts of four animals and treatment was started 96h later when tumors became palpable. The first cohort received 10 nmol IdI-PCAO conjugate in 200 μL TBS. The other two cohorts served as negative controls and received either TBS or 10 nmol F3-peptide plus 10 nmol IdI-AO in TBS (app. 5.7 mg/kg BW). Application of the conjugate and control solutions was performed by intravenous injection into the tail vein and was repeated every 24 h for 7 consecutive days. 24 h after the last injection animals were sacrificed. Tumors, kidneys, livers and femurs were collected, fixed with paraformaldehyde, and embedded in paraffin. Altematively, animals were implanted s.c. with osmotic pumps (Durect Corp., Cupertino, CA) that delivered 7 nmol/d IdI -PCAO (3.5 mg/kg BW) in TBS over a 14 day period. Controls received 20 nmol/d F3-peptide plus 20 nmol/d IdI-AO (in TBS) or TBS. 2 x 106 MMTV-HER2/neu (YD) IdI-/- tumor cells were injected into the left flank 24 h after implantation of the pumps. After the 14 days treatment period, pumps were replaced using a model with a work period of 7 days. Treatment with 17- AAG (75 mg/kg, i.p. on 3 consecutive days/week) was started when tumors reached a size of 20 mm3. 17- AAG was dissolved at 50 mg/kg in DMSO and diluted with EPL 1 :1 before injection. Control animals received DMSO:EPL 1 :1 i.p at the same schedule. Tumor size was measured using a calliper. Volume was calculated as V= ( /6 x longest diameter x perpendicular diameter2).

Example 16: Allograft model of metastatic Lewis lung carcinoma (LLCV 7.5xlO⁵ Dual reporter labelled LLC cells were implanted in the right dorsal flank of male C57J/B6 mice (Jackson laboratories, Bar Harbour). After seven days, animals were implanted with osmotic pumps (lOOμL volume, work period 14 days). The pumps were filled with saline solution of either IdI-PCAO (3.5 mM), rcIdl-PCAO (3.5 mM) or F3-peptide plus IdI-AO (12.5 mM each). Concentration and release rate of the pumps resulted in a delivery rate of 229 μg/d (IdI-PCAO and rcIdlPCAO) or 265 μg/d and 580 μg (F3 and IdI-AO). 14 days after tumor implantation animals were anaesthetised and primary tumors were surgically removed. Complete removal of the tumor tissue was checked 3 days post operation by in vivo luciferase imaging and re-growing primary tumors were removed. For in vivo luciferase imaging, 100 μL of D-luciferin (Gold Bio Technology, St. Louis, MO; 15 mg/mL potassium-salt in PBS) were injected retro-orbitally to animals anaesthetised by isofluorane inhalation. Photographic and luminescence images were acquired using an IVIS 100 system (Xenogen, Hopkinton, MA). Animals were sacrificed when disstressed. Tumor burden and metastasis data acquired by in vivo luminescence was confirmed by histology.

Image acquisition and analysis: Epifluorescence, bright field and phase contrast images were acquired using Zeiss Axiostar 200 microscopes. Leica laser confocal microscopes were used for co-localisation studies. For quantification, large fields of the tissue sections were acquired using an automated image acquisition and montaging system (Zeiss Axiostar 200M microscope with MetaMorph Software, Molecular Devices, Sunnyvale, CA). For evaluation of single cell staining (IdI, CD31, Hifl -alpha) an average of 30 adjacent, single images were acquired from the centre of the section using a 2Ox objective. Images were montaged to yield one large image covering an average area of 0.82 mm2. Three or 4 large field images were used to quantify each section. Stained areas were quantified using MetaMorph or ImageJ software (http://rsb.info.nih.gov/ij/). Images were threshholded and stained area (CD31) was calculated or particles per field were counted (IdI or Hifl positive cells). To quantify extent of haemorrhage, H&E whole tumor sections were imaged with a 5x objective and evaluated using the colour threshold function of the Metamorph software.

Example 15: Targeting of Murine Mammary Carcinoma

A conjugate was prepared using peptide sGRP78 (CWIFPWIQL, Seq ID No. 7), a peptide that binds to GRP78, a stress response chaperone expressed on the surface of various tumors was tested for its ability to transport oligonucleotides into tumor cells. The peptide was conjugated to an anti-Id 1 oligonucleotide using GMBS via a 5 '-amino linker. The oligonucleotide was labeled with fluorescein at the 3 '-end. Accumulation of the oligonucleotide was monitored via the fluorescent label, and was found to occur in 4Tl murine mammary carcinoma cells in mice. Accumulation was also seen in keidney cells, but not in lever or heart. Accumulation was not seen in the carcinoma when the peptide portion of the conjugate was omitted.

Example 16: Accumulation using different homing peptide

Quantification of tumor-uptake was performed with 111 -Indium labeled PCAOs containing IdI antisense coupled to the peptide via a GMBS linker. Four different peptides were modified with a DOTA (1,4,7, 10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) chelator at the N-terminus during standard solid phase peptide synthesis. After conjugation to the antisense oligonucleotide the DOTA group was used to attach an ^{u 1}In-Km. The labeled PCAOs were injected into 4Tl -breast carcinoma bearing animals, and tumor accumulation was measured after 2h by resecting the tumor and quantifying gamma-radiation emitted from the sample.

The peptides used were HMGN2 (Seq. ID No. 3), CAPRPG (Oku et al., Oncogene, 2004, Seq. ID No. 8), RGD-4C (ACDCRGDCFCG, Seq,. ID No. 9) and CWIFPWIQL (Seq. ID No. 7). The results are summarized in Fig. 8. As shown, all four peptides resulted in accumulation of ^π 'In-labeled conjugates in 4Tl beast carcinomas, although to differing extents.

Example 17: Additional Homing Sequences

To identify peptide sequences displaying homing potential to specific tumors or to the vasculature of specific tumors, in vivo phage display panning was performed. Utilizing commercially available M13-based phage libraries (PhD-7 Prod# E8100S and PhD-C7C E8120S, New England Biolabs, Ipswich, MA) phage were isolated that were enriched after 3-4 rounds of panning in different tumor models. These phage particles display surface peptides that potentially confer homing and cellular uptake capabilities.

Angiosarcoma model

Panning was conducted in SVR (ATCC CRL-2280, a murine endothelial cell line) tumor allograft bearing mice. Tumors were resected 24h post phage injection

Enriched sequences:

Breast carcinoma model, tumor cell homing phage

Panning was conducted in 4Tl (ATCC CRL-2539) tumor allograft bearing mice. Tumors were resected 24h post phage injection.

Enriched sequences:

Breast carcinoma model, tumor vascular homing phage

Panning was conducted in 4Tl (ATCC CRL-2539) tumor allograft bearing mice. Tumors were resected 5-7h post phage injection.

Enriched sequences:

Seq ID Nos. 10-30 can be used in conjugates in accordance with the present invention.

To Applicant's knowledge, the present invention is the first successful targeted, in vivo delivery of an antisense molecule with demonstrated preclinical efficacy. While antisense and targeting peptides have been described before, they have never been fused in a way that allows both moieties to maintain their activities. Thus, the present invention provides the substantial advances over the art.

Claims

1. A peptide-oligonucleotide conjugate having the general formula: peptide-HBL-antisense in which peptide is a homing peptide which directs the conjugate to cells of a particular type, antisense is an antisense oligonucleotide having a sequence selected to provide sequence- specific inhibition of a target protein, and HBL is a heterobifunctional linker having reactivity towards amino and sulfhydryl groups.

2. The conjugate of claim 1 , in which HBL is SMCC, GMBS or EMCS.

3. The conjugate of claim 2, in which the antisense oligonculeotide targets a transcription regulating factor in a sequence specific manner.

4. The conjugate of claim 2 in which the antisense oligonucleotide targets IdI .

5. The conjugate of claim 4 in which the antisense oligonucleotide comprises Seq. ID No. 1 or 31 or an RNA counter-part thereof.

6. The conjugate of claim 2, in which the peptide directs the conjugate to tumor endothelium.

7. The conjugate of claim 6, in which the peptide portion of conjugate of the comprises a peptide of the sequence:

CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (Seq. ID No. 3).

8. The conjugate of claim 6, wherein the peptide portion of conjugate of the comprises a peptide of the sequence:

WIFPWIQL (Seq. ID No. 4) or WDLAWMFRLPVG (Seq. ID No. 5) .

9. The conjugate of claim 6, in which HBL is SMCC, GMBS or EMCS.

10. The conjugate of claim 6, in which the antisense oligonculeotide targets a transcription regulating factor in a sequence specific manner.

11. The conjugate of claim 6 in which the antisense oligonucleotide targets IdI .

12. The conjugate of claim 11 in which the antisense oligonucleotide comprises the sequence gcaccagctccttgaggcgtgag (Seq. ID No. 1) or an RNA counter-part thereof.

13. The conjugate of claim 1 , in which the antisense oligonculeotide targets a transcription regulating factor in a sequence specific manner.

14. The conjugate of claim 1 in which the antisense oligonucleotide targets IdI .

15. The conjugate of claim 14 in which the antisense oligonucleotide comprises the sequence gcaccagctccttgaggcgtgag (Seq. ID No. 1) or an RNA counter-part thereof.

16. The conjugate of claim 1, in which the peptide directs the conjugate to tumor endothelium.

17. The conjugate of claim 16, in which the peptide portion of conjugate of the comprises a peptide of the sequence:

CKDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (Seq. ID No. 3).

18. The conjugate of claim 16, wherein the peptide portion of conjugate of the comprises a peptide of the sequence:

WIFPWIQL (Seq. ID No. 4) or WDLAWMFRLPVG (Seq. ID No. 5) .

19. The conjugate of claim 16, in which HBL is SMCC, GMBS or EMCS.

20. The conjugate of claim 16, in which the antisense oligonculeotide targets a transcription regulating factor in a sequence specific manner.

21. The conjugate of claim 16 in which the antisense oligonucleotide targets IdI .

22. The conjugate of claim 21 in which the antisense oligonucleotide comprises the sequence gcaccagctccttgaggcgtgag (Seq. ID No. 1) or an RNA counter-part thereof.

23. The conjugate of claim 1, wherein the peptide portion of the conjugate comprises a sequence selected from Seq. ID Nos. 10-14.

24. The conjugate of claim 1, wherein the peptide portion of the conjugate comprises a sequence selected from Seq. ID Nos. 15-21.

25. The conjugate of claim 1, wherein the peptide portion of the conjugate comprises a sequence selected from Seq. ID Nos. 22-30.

26. A method of treating cancer by administration of a conjugate of any of claims 1 to 25.

27. The method of claim 26, further comprising administration of an inhibitor of hsp90.

28. The method of claim 27, wherein the inhibitor of hsp90 is 17-AAG.

29. The method of claim 26, wherein the cancer is a glioma.

30. The method of claim 29, further comprising administration of an inhibitor of hsp90.

31. The method of claim 30, wherein the inhibitor of hsp90 is 17- AAG.

32. The method of claim 26, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, glioma and other brain cancers, colorectal cancer, pancreatic cancer, liver cancer, and sarcoma.

33. The method of claim 32, further comprising administration of an inhibitor of hsp90.

34. The method of claim 33, wherein the inhibitor of hsp90 is 17-AAG.

35. Use of a conjugate in accordance with any of claims 1 to 25 in the formulation of the pharmaceutical composition for the treatment of cancer.

36. Use of claim 35, wherein the cancer is glioma.

37. Use of claim 35, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, lung cancer, glioma and other brain cancers, colorectal cancer, pancreatic cancer, liver cancer, and sarcoma.