PT100424A - PROCESS AND TEST SYSTEM FOR NEUROTROFIN ACTIVITY - Google Patents

Description

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DESCRIPTIVE MEMORY

Field of the Invention

This invention relates to pharmaceutical compositions and methods for the treatment of mammals bearing tumor cells which express neurotrophic factors and which utilize an autocrine loop mechanism for survival. More specifically, this invention relates to methods of disrupting the brain-derived neurotrophic factor signal transduction pathway (" BDNP ") and causing cell death in BDNF-expressing cells.

BACKGROUND OF THE INVENTION A. Neural Christian Derived Tumors The term neuroblastoma is used to denote the spectrum of neurogenic neoplasms derived from neuroblasts of the embryonic sympathetic, neural Christian cells and the mantle layer of the neural tube. Neuroblastoma tumors, the most commonly diagnosed neoplasms in children under 1 year of age, occur with a frequency of 1 in 10,000 births.

Neuroblastoma is considered the third most common malignancy in childhood, accounting for approximately 10% of all pediatric malignancies, and at least 15% of all cancer-related deaths in children.

Biochemically, neuroblastomas often contain elements of both adrenergic and cholinergic neurotransmitter pathways. They express neuron-specific enolase and neurofilament proteins and exhibit cell growth, in culture, adherent to the substrate, with formation of neuritis. Perhaps the most salient fact of human neuroblastoma is the amplification of the N-myc oncogene, which has been identified in 19 of 22 neuroblastoma cell lines and in approximately 31% of the tumor tissues of stage III and stage 4 neuroblastoma patients [Kohl et al., Science. 226: 1335 (1984)]. The SK-N-SH human neuroblastoma cell line and its SH-SY5Y derivative are of thoracic origin and not of the neural Christian and do not express amplified N-myc but express N-ras. 73 889 6526-089-118 -3-

Another class of tumors, small cell lung carcinoma (SCLC) shares a common developmental line with neuroblastomas, both apparently derived from neural progenitor cells originating in the neural Christian [Carney, Cancer Res .. 45: 2913 ( 1985); Gazdar et al., Cancer Res. 45: 2924 (1985)]. Small cell lung carcinoma accounts for about one-third of all lung cancers. Although expression of amplified levels of N-mvc is known in several neuroblastomas. SCLCs are generally characterized by activated levels of N-myc or L-mvc. Small cell carcinomas account for approximately 20-25% of all lung malignancies, producing an incidence of approximately 25,000-30,000 cases per year in the United States, and are by far the most aggressive lung malignancies. SCLC is often (70% -90%) of metastatic presentation. Small cell carcinomas are of neuroectodermal origin. These tumors have absorption and incorporation and decarboxylation properties of the amine precursor (APUD), and other neuronal characteristics, such as the production of neuroactive peptides. Paraneoplastic syndromes, such as subacute sensory neuropathy, occur more frequently among small cell victims than other lung cancers. B. Neurotrophins and Their Developers The development and function of the nervous system depends on proteins, called neurotrophic factors, originally defined by their ability to support the survival of neuronal cells. In addition to promoting neuronal survival, these factors can influence processes of proliferation and differentiation in the nervous system and may also have actions outside the nervous system. Much has been learned about the neuronal prototypic survival molecule, nerve growth factor (NGF), and, more recently, its two structural relatives, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). These three proteins, together 73 889 6526-089-118 -4-

with the newly identified neurotrophin-4 [Hollbook et al., Neuron, 6: 845-58 (1991)], comprise a family (designated " neurotrophins "), from which each member shares with the others about 55% to 60% amino acid sequence identity. Understanding the biological roles of these neurotrophic factors requires the characterization of the signal transduction pathways and the receptor they use to exert their effects. The trk family of tyrosine kinase protein receptors have been identified as biologically functional receptors for neurotrophins. BDNF is a neuronal survival molecule that is capable of binding to the trk receptor on the cell surface known as trkB, which has an intrinsic protein kinase activity and is primarily expressed in the nervous system [Kaplan et al., Nature. 350: 158 (1991); Klein et al. . Cell. 65: 189 (1991), Hempstead et al. Nature. 350: 678 (1991)]. The trkB gene encodes a protein that binds and mediates functional responses to both BDNF and NT-3 [PCT International Application WO 91/03568; Squinto et al. . Cell. 65: 885 (1991); Glass et al., Cell. 66: 405-413 (1991); Soppet et al., Cell. 65: 895 (1991)]. The recent introduction of trk receptors into fibroblasts has allowed the creation of cells that present biological responses to neurotrophic factors that mimic the actions of traditional growth factors. Introduction of the trkB receptor in NIH 3T3 cells that require certain growth factors (FGF or PDGF) for proliferation and survival, resulted in cells capable of surviving at exogenous, physiologically appropriate levels of BDNF or NT-3. Such models provide a powerful assay system that can be used to detect and / or measure neurotrophin activity, to identify agents exhibiting neurotrophin-like activity, and to identify antagonists that block ligand binding to neurotrophin receptors [Glass et al., supra1. The use of tyrosine kinase receptors by 73 889 6526-089-118

family of neurotrophic factors related to NGF, suggests that the signal transduction mechanisms used by neurons resemble fundamentally those used by other cell types in response to mitogenic factors. This finding is consistent with recent data in which neurotrophic factors may act as mitogens in certain contexts [Cattaneo and McKay, Nature. 347: 762 (1991); Glass et al., Supra). BDNF and NT-3 may serve as survival and / or mitogenic factors for neuronal precursors that have not yet reached a postmitotic phenotype.

Together with the trk family of tyrosine kinase proteins that have been identified as biologically functional receptors for neurotrophins, ERK kinases represent a newly identified and molecularly cloned family of extracellular protein-regulated signal kinases [Boulton et al. . Cell. 65: 663-75 (1991)]. ERK activity is rapidly activated in response to stimulation of cells by growth factor (i.e., insulin and NGF) and represents a class of intracellular Ser / Thr kinases that themselves are phosphorylated on tyrosine [Boulton et al., Supra]. It has been demonstrated in vitro that ERK tyrosine phosphorylation greatly increases its kinase activity. C. Antisense Technology Understanding the molecular events that guide the regulation of cell proliferation, differentiation, and survival should ultimately lead to the rational design of drugs specifically targeted to the treatment of various diseases including cancer, immunodeficiency, and neurodegeneration. Recently a powerful experimental tool has emerged that allows, by selective and effective means, inhibition of the expression of key gene products known to be involved in the control of proliferation, differentiation and survival of eukaryotic cells. The technique involves the use of antisense DNA or RNA molecules designed to provide translation blocking of these key regulatory proteins. This technology allowed the contribution -6

-6 C. X 73 889 6526-089-118 the function of the product of a single gene during important periods of cell transition, by creating a null mutant for a specific gene product.

Currently, there are two main approaches to achieving blocked translational antisense translation of protein synthesis. The first method makes use of stably transfected and promoter-directed genetic constructs designed to constitutively synthesize complementary antisense mRNA sequences. This technology generally results in blocking by hybridization, the translation of the protein into a given gene product. It has recently been suggested that RNase may play a role in this mechanism by cleaving the RNA / DNA duplexes formed between antisense mRNA and DNA. Other possible mechanisms include decreased nuclear processing or the inability of the RNA / DNA duplex to be efficiently translated. An alternative approach to blocking vector-driven antisense translation involves the synthesis of small synthetic 5 'or 3' anti-sense oligodeoxyribonucleotides (antisense DNA). Several papers have recently demonstrated the ability of antisense DNA to block translation of selected RNAs when added to eukaryotic cells in. vitro (review in Van der Krol et al., Biotechnology, 6: 958-973 (1988)). Several approaches have been taken to use oligonucleotides that are complementary to selected target sequences of cellular or viral nucleic acid to modulate the expression of the target nucleic acid sequence. There have been several works on the use of specific nucleic acid sequences to inhibit viral replication [see for example Goodchild et al., Proc. Natl. Acad. Know. USA. 85: 5507-5511 (1988); Wickstrom et al. . Proc. Natl. Acad. Know. USA. 85: 1028-1032 (1988); and Kawasaki, Nucl. Acids Res. 13: 4491 (1985)]. Several laboratories have attempted to develop modified oligonucleotides that are relatively membrane permeable and nucleases resistant. One approach involves the development of

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of nonionic oligonucleotide analogs. Examples of such analogues include methylphosphonates [Smith et al., Proc. Natl.

Acad. Know. USA. 83, 2787-2791 (1986); Agris et al., Biochemistry. 25: 6268-6275 (1986); Jayaraman et al., Proc. Natl. Acad. Know. USA. 78: 1537-1541 (1981)]; phosphorothioates [Agarwal et al., Proc. Natl. Acad. Know. USA. 83: 4314-4146 (1988); Matsukura et al. Proc. Natl. Acad. Know. USA. 84: 7706-7710 (1987); Marcus-Sekura et al., Nucl. Acids Res. 15: 5749-5763 (1987)]; and phosphoramidates [Agarwal et al. Proc. Natl. Acad. Know. USA. 83: 4143-4146 (1988)].

It has been speculated that phosphorothioates may, in addition to binding to complementary nucleic acid target sequences, also direct inhibition of primer binding to HIV reverse transcriptase [Matsakura et al., Proc. Natl. Acad. Know. USA. 84: 7706-7710 (1987)]. Antimold inhibition has also been described using polynucleotides, including partially thiolated polycytidylic acid [review in Stein and Cohen, Cancer Res. 48: 2659-2668 (1988)].

Another approach has involved conjugation of the oligonucleotide to a molecule which will increase the efficiency of absorption and incorporation of the oligonucleotide into the cell. Examples of these conjugates include cholesteryl-conjugated oligonucleotides [Letsinger et al., Proc. Natl. Acad. Know. USA. 86: 6533-6556 (1989)] and a poly-L-lysine conjugate [Lemaitre et al., Proc. Natl. Acad. Know. USA. 84: 648-652 (1987)]. Another example includes an oligonucleotide linked through a linker arm to a group that decreases the amphiphilic character of the final product, so as to increase the efficiency of membrane transport. [PCT Publication No. WO 88/09810, published December 15, 1988 ].

Another approach that has been followed involves the use of reactive oligonucleotides, i.e. antisense oligonucleotides bound to reactive agents that are capable of modifying the target nucleic acid. One of these groups of reactive agents are intercalating agents that can bind to the duplex by internal insertion

between adjacent base pairs, or binding to external nucleosides or phosphate elements, respectively. Examples of intercalators that have been linked to oligonucleotides and oligonucleotide analogues include acridine, anthldride, and photosensitizing psoralen [review in Zon, Pharm. Res., 5: 53-549 (1989)]. Other such groups of reactive groups coupled to oligomers include metal complexes such as EDTA-Fe (II), o-phenanthroline-Cu (I), or porphyrin-Fe (II) [review in Krol et al. Biotechniques. 6: 958-976 (1988)]. These compounds may generate hydroxyl radicals in the presence of molecular oxygen and a reducing agent. The resulting radicals may cleave the complementary strand following the attack on the target nucleic acid framework.

There are many recent publications dealing with inhibition by antisense oligonucleotides. For example, proliferation of malignant human melanomas has been inhibited in vitro by antisense oligonucleotides directed against basic fibroblast growth factors [Becker et al., EMBO J. 8, 3685 (1989)]. It was observed that formation of antisense RNA for part of the human N-mvc gene via an episomal replication expression vector, blocked the transdifferentiation of neuroectodermal tumor cell lines [Whitesell et al. . Mol. Cell. Biol. . 11 (3): 1360-1371 (1991)]. Antisense oligonucleotides for the tau protein gene associated with the neuronal microtubule, when added to culture media, inhibited the polarity of neuritis in primary cerebellar neurons [Caceres and Kosik, Nature. 343: 461 (1990)]. An antisense oligonucleotide for transforming beta-3 growth factor inhibited transformation of the mesenchymal epithelium of embryonic cardiac endothelial cells into explant cultures [Potts et al., Proc. Natl. Acad. Know. USA. 88: 1516-1520 (1991)]. D. Autocrine Ansas The involvement of the autocrine growth mechanism in neoplasia was first identified in 1980 [Sporn and Todaro, N. Encl. J. Med., 308: 878-80 (1980)]. The loops

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autocrine have been observed for various growth factor molecules and tumor cell lines. Certain tumor cells are known to synthesize and respond to growth factors that are required for normal cell growth and cell division. Through autocrine signaling, the cells respond to substances that they themselves produce. Autocrine loops can serve to accelerate or amplify a cellular response in tumor cells because that cell is less dependent on its environment for its existence.

In some cases, autocrine loops have been experimentally defined by the use of antisense approaches for autocrine loop rupture. In other words, the mere ability of an oligonucleotide, which is antisense to a particular factor, to adversely affect cell growth is indicative that that cell is synthesizing that factor and the factor is required for the growth or survival of the cell. In such situations, the existence of cellular receptors for the factor or even the release of growth factor into the environment may not be detectable.

Antisense oligonucleotides have been used to demonstrate that transforming beta-growth factor serves as the autocrine factor of cell differentiation responsible for transformation of epithelial cells into mesenchymal cells [Potts et al., Proc. Natl. Acad. Know. USA. 88: 1516 (1991)]. Antisense approaches have been used to demonstrate that basic fibroblast growth factor appears to be necessary for the proliferation by autocrine stimulation of both human melanomas [Becker et al. . EMBQ J., 8: 3685 (1989)] and transformed human astrocytes [Morrison et al. . J. Biol. Chem., 266: 728 (1991)]. Growth hormone anti-sense oligonucleotides inhibit lymphocyte proliferation [Weigent et al., Endocrinol. 128: 2053 (1991)]. E. Protein-Ouinase Inhibitors Specific inhibitors of protein phosphorylation have been used to study the effect of various kinases and their

Actions on the phosphorylation of key cellular proteins for the biological activity of nerve growth factor in their target cells. The guinase inhibitor K252a, isolated from a culture broth of Nocardiosis sp. and derivatives thereof, are described in U.S. Patents 4,555,402; 4,877,776; and 4,923,986, which documents are incorporated herein by reference, K252a and staurosporine were initially characterized as potent inhibitors of in vitro protein kinase c (PKC) and nucleotide-dependent cyclic kinase [Kase et al., Biochem. Biophys. Com. 142: 436-440 (1987)], but are now known to have broader actions including inhibition of tyrosine-specific protein kinases [Fujita-Yamaguchi and Kathuria, Biochem. Biophys. Comm. 157: 955-962 (1988); O'Brian and Ward, J. Natl. Canc. Instit. 82: 1734-1734 (1990)]. Nanomolar concentrations of K252a and its derivatives have been shown to inhibit in some rather selective manner the protein * guinase C, cyclic AMP and cyclic GMP-dependent protein kinases, myosin light chain kinase, and phosphodiesterase-dependent of calmodulin [Kase et al., Biochem. Biophys. Res. Commun. 142: 43640 (1987); Nakanishi, J. Biol. Chem., 263: 6215-19 (1988); Kase et al. . J. Antibiotic. 39: 1059-60 (1986)]. the mechanism of selective kinase inhibition appears to be related to competition for the binding site of adenosine-5, triphosphate (ATP) in the enzyme.

Although K252a and eataurospores do not appear to decrease FGF or EGF responses in PC12 cells, they are able to block the earlier detectable NGF-induced signaling processes, including NGF-induced tyrosine phosphorylation. K252a has also been shown to inhibit NGF induced excrescences of neurites from primary cultures of dorsal root embryonic explants as well as completely blocks NGF survival activity in primary cultures of chick embryo sympathetic neurons [Matsuda and Fukuda, Neurosis. Lett., 87: 11-17 (1988); Borasio, Neurosci. Lett., 108: 207-12 (1990)].

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Another class of tyrosine protein kinase inhibitors, the class of thiazolidinedione inhibitors, has demonstrated specific epidermal growth factor (EGF) -induced receptor autophosphorylation and has been shown to be an inhibitor of EGF-dependent cells [Geissler et al. . " Thiazolidine-Diones, " J. Biol. Chem., 265: 22255-22261 (1990)]. These inhibitors are analogous to K252a in its specific mechanism of disruption of growth factor mediated cell changes.

There is in the art a need for pharmaceutical processes and compositions capable of reducing in vivo growth of BDNF-expressing tumor cells, such as neuroblastomas, and of inhibiting tumor progression. In particular, there is a need for means of disrupting the autocrine loop of BDNF survival of BDNF-expressing tumor cells.

Summary of the Invention The present invention is directed to a method of treating mammals bearing a tumor cell of a type characterized by the expression of a BDNF. In particular, the present invention relates to the identification of an autocrine loop of survival in tumor cells expressing BDNF and means for disruption of the autocrine loop in order to cause cell death. The present invention is further directed to recombinant cells which serve as a template system for cells, including tumor cells, which are dependent on an autocrine loop to survive. Such recombinant cells provide a means for the screening of compounds for therapeutic efficacy in the treatment of tumors that utilize autocrine survival loops.

In one aspect of the invention, recombinant cells expressing both BDNF and the trkB receptor, and thus depend on an autocrine loop of BDNF for survival, are used to identify agents that disrupt the autocrine loop of BDNF and 73 889 6526-089-118

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which can be used to treat tumor cells that similarly depend on such autocrine loops for survival and / or proliferation.

In one aspect, the invention is directed to nucleic acids of at least six nucleotides that are antisense for a gene or cDNA encoding BDNF or a portion thereof. " Antisense ", as used herein, refers to a nucleic acid capable of hybridizing to a portion of BDNF RNA (preferably mRNA) by virtue of some complementarity of the sequence.

The antisense nucleic acids of the invention, which are used to disrupt an autocrine loop of BDNF survival, may be oligonucleotides which are double stranded or single stranded, DNA or RNA or a modification or derivative thereof, which can be directly administered to a cell, or can be produced intracellularly by transcription of introduced exogenous sequences.

In another aspect, this invention is directed to the use of staurosporine, K252a and its derivatives, or other protein kinase inhibitors, to disrupt the autocrine loop of BDNF survival.

The antisense nucleic acids for BDNF and K252a or derivatives thereof provided by this invention can be used for the treatment of tumors whose tumor type cells demonstrate expressing BDNF.

In one embodiment, the invention is directed to methods for inhibiting the expression of a BDNF nucleic acid sequence in a eukaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense nucleic acid for BDNF of the invention.

In another embodiment of this invention, staurosporin, K252a or derivatives thereof or other protein kinase inhibitors, can be used to disrupt the autocrine loop of BDNF at

Cell surface receptor level by inhibition of BDNF receptor phosphorylation.

In another embodiment, the identification of cells expressing functional BDNF or other neurotrophin receptors can be accomplished by observing the ability of a neurotrophin to " such cells from the cytotoxic effects of an antisense nucleic acid for BDNF.

Another aspect of the invention provides for the diagnosis of human neuroblastoma or small cell lung carcinoma through the detection of BDNF expression in cells obtained from patients.

In addition, the invention provides pharmaceutical compositions comprising an effective amount of the BDNF antisense nucleic acids of the invention in a pharmaceutically acceptable carrier. Also provided are methods for treating various diseases and disorders comprising administering the pharmaceutical compositions of the invention.

In a further aspect, there is also provided a pharmaceutical composition comprising, as its active ingredient, K252a, staurosporine or a related compound, in a pharmaceutically acceptable carrier. This composition may be used in the treatment of various diseases and disorders related to tumor cells expressing BDNF.

A further aspect of this invention is a pharmaceutical composition comprising as an active ingredient, K252a, staurosporine, or a related compound, and as the second active ingredient the BDNF antisense nucleic acids of the invention. Alternatively, staurosporine, K252a or other protein kinase inhibitor may be combined with another conventional pharmaceutical agent useful in treating or preventing disorders associated with BDNF-expressing tumor cells.

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Yet another aspect of this invention is a method for treating patients having small cell lung neuroblastoma or carcinoma by administering an effective amount of the materials and compositions described above.

Yet another aspect of this invention is a process of stimulating neurite outgrowth by administering particularly low doses of a protein kinase inhibitor such as K252a.

Other aspects or advantages of the present invention are further set forth in the following detailed description of the preferred embodiments of the present invention.

Description of Figures

Figure 1. Inhibition of BDNF synthesis by antisense oligonucleotides in an in vitro translation system of a wheat seed lysate. BDNF mRNA was synthesized in vitro using the expression plasmid constructed pC8hB, [see PCT Publication No. WO 91/03568 published March 21, 1991] which contains the bacterial T7 promoter for in vitro efficient transcription by T7 RNA polymerase , according to the manufacturer's instructions (Promega, Madison, WI). BDNF mRNA was purified and then placed in an in vitro translation system of a wheat seed lysate (Promega) in the absence or presence of BDNF oligonucleotides. Synthesis of the BDNF protein was followed by the use in the reaction of 35S-labeled methionine. Control oligonucleotide refers to the use of a random mote 18 unrelated to the human BDNF sequence.

Figure 2. Effects of 3β-BDNF on cell viability in culture. The percentage of cell viability (y-axis) is presented for different concentrations of 3'-AS-BDNF (x-axis, in micromolar) added to cultured neuroblastoma cells. A: LA-N-5 cells; B: LA-N-1 cells; C: SK-ES cells; D cells: SH-SY5Y cells.

Figure 3. Effect of the joint addition of various neurotrophins with 3β-AS-BDNF on neuroblastoma cell lines. The indicated neuroblastoma cell lines were simultaneously incubated with 50 μ 3 3 'AS-BDNF and without neurotrophin (blank squares with center point), with BDNF (filled diamonds), NT-3 (blank squares), or NGF (white lozenges). Axis of yy: percentage of cell viability; xx axis: hours in culture. Figure 3A; SH-SY5Y cells; Figure 3B: LA-N-1 cells; Figure 3C: LA-N-5 cells; Figure 3D: CHP-134 cells; Figure 3E: CHP-404 cells.

Figure 4. Northern blot analysis of total cellular RNA (10 μg per lane) derived from small cell lung carcinoma or adult rat brain (lane 1) cell lines. Northern blotting was hybridized with a human BDNF probe. Small cell lung carcinoma cell lines were as follows: H82 (lane 2), H209 (lane 3), H345 (lane 4), H378 (lane 5), H510 (lane 6), and N417 (lane 7 ).

Figure 5. Comparisons of BDNF expression in both human and rodent tumor cell lines by Northern blot (RNA). Total RNA (10 μg) from each cell line was fractionated, transferred to membranes and hybridized with 32 P-labeled BDNF as previously described [Maisonpierre, et al., Science 247: 1446 (1990)]. The neuroblastoma cell lines in Tables B and C are represented by LAN5, SY5Y and N18TG2.

Figure 6. Morphological effects of sense and antisense BDNF oligomers on LA-N-5 neuroblastoma. Light photomicrographs of untreated LA-N-5 neuroblastoma cells (Table A), treated with 10 μM 3'-AS-BDNF oligomer (Table B), with 10 μM 3'-S-BDNF oligomer (Table C) or with 10 μg 3 BSA-BDNF and 100 ng / ml recombinant human BDNF (Table D): were obtained with the other antisense BDNF oligomer, PS-AS-BDNF, similar results to those shown in Table B. LA-N-5 neuroblastoma cells were plated on Costar plates of 6

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wells at a density of 3 x 105 cells per well in RPMI 1640 (Irvine Scientific) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 2% streptomycin (P / S) and 2 mM glutamine. Eighteen hours after seeding, cells were transferred to a defined, serum-free medium [Zhan et al., Mol. Cell. Biol. 6: 3541 (1986) and treated for 72 hours with the reagents described above. Constructed BDNF was produced in CHO cells and purified from conditioned medium from CHO cells to homogeneity as previously described [Squinto et al., Cell 65: 885 (1991)].

Figure 7. Dual staining flow cytometric assay to quantify the DNA and protein contents of LA-N-5 neuroblastoma cells. LA-N-5 neuroblastoma cells were seeded in 10 cm dishes at a density of 1 x 106 cells per dish and were cultured as described in Figure 6. Cells were either not treated (Table A) or were treated with only 3 With BSAF (Table B), with 3'-AS-BDNF with 100 ng / ml BDNF (Table C), or with high concentrations (100 μΜ) of 3, -S-BDNF of control (Table D). Following these treatments the cells were harvested and resuspended in PBS-versen (PBS with glucose and EDTA) to give suspensions with single cells. The cells were stained with 1 μg / ml DAPI (for the left-side DNA content of the Tables) and 10 μg / ml of sulforhodamine 101 (for the protein content - right side of the Tables) and analyzed by flow cytometry as described [Del Bino, et al. Exp. Cell Res. 193: 27 (1991); Jakobisiak, et al. Proc. Natl. Acad. Know. USA 88: 3628 (1991)]. At least 5 x 103 cells were counted for each analysis. The line drawn on the protein profiles underscores the decrease in fluorescence observed in Table 4 (right side) relative to Tables A-C (right side). The arrow at B indicates a population of apoptotic cells. The phases of the cell cycle are indicated. The percentage of cells in the S-phase was as follows: Table A - 25.5%; Table B - 16.2%; Table C -30.1%; Table D - 24.9%.

Figure 8. Dose response death curves for I oligomers 73 889 6526-089-118 -17-

(S) and antisense BDNF (AS) in human neuroblastomas and 3T3 autocrine recombinant fibroblasts. Human neuroblastoma cells (LA-N-5, Table A, SH-SY5Y, Table B) were cultured as described for Figure 6 whereas the autocrine 3T3 BDNF cells (Table C) were cultured in factor-deficient media as described [Glass et al. . Cell 66: 405 (1991); Zhan et al. . Mol. Cell. Biol. r supra]. All cells were plated at a density of 2 x 10 4 cells per well in a Costar 24-well plate. Various concentrations of 3β-AS-BDNF or control oligomers (0.1, 5, 10, 50, 100 and 250 Âμ) were added to the cultures for 4 hours in the presence or absence of recombinant human BDNF (100 ng / ml) using serum-free EMEM (Tables A and B) or growth factor deficient media (Table C) to allow uptake and incorporation of oligonucleotide into cells. Insulin, transferrin and selenium (ITS) were then immediately added to the cultures and cell viability measured 72 hours later by determination of the glucose concentration that remained in the culture media. Full squares - 3'AS; Blank squares - 3'AS and BDNF; Full circles - 3'S; Circles in white - 3'S and BDNF.

Figure 9. Identification of trk receptors, constitutively autophosphorylated in neuroblastoma cell lines. Table A, anti-phosphotyrosine immunoblotting of autophosphorylated trkB receptors that were immunoprecipitated specifically from total protein lysates prepared from approximately 3x10 3 BXF-treated and BDNF-treated trkB [3T3 (trkB)] NIH3T3 cells, or from 2-5 x 10 6 untreated neuroblastoma cells. In Table B, N18TG2 neuroblastoma cells were not treated or pre-treated with 200 nM K252a prior to the preparation of cell lysates and trk-specific immunoprecipitation. Table C, anti-phosphotyrosine immunoblot of total protein lysates prepared from NIH3T3 (3T3) cells, 3T3 cells (trkB) treated with BDNF or untreated 3T3 (autocrine) cells. The position of the trkB is marked with an arrow,

Whereas the molecular weight standards (KD) are indicated on the left side of the figure. Full squares - 3'AS; Blank squares - 3'AS and BDNF; Full circles - 3'S; Circles in white - 3'S and BDNF.

Figure 10. Identification of trk receptors constitutively autophosphorylated in neuroblastoma cell lines. Table A. Anti-phosphotyrosine immunoblotting of autophosphorylated trkB receptors which were immunoprecipitated specifically from total protein lysates prepared from approximately 3x10 6 truB-expressing and BDNF-treated NIH3T3 cells or from 2-5x10 untreated neuroblastoma cells. In Table B, N18TG2 neuroblastoma cells were not treated or pre-treated with 200 nM K252a prior to the preparation of cell lysates and trk-specific immunoprecipitation. Table C, anti-phosphotyrosine immunoblot of total protein lysates prepared from NIH3T3 (3T3) cells, NIH3T3 (trkB) treated with BDNF or untreated (autocrine) 3T3 cells. The position of the trkB is marked with an arrow, whereas the molecular weight standards (KD) are indicated on the left side of the figure.

Figure 11. Differential effect of K252a on neuroblastoma (Table A) and on 3T3 cell lines (Table B) whose survival depends on, or is related to, an autocrine loop of BDNF survival. Neuroblastoma cells (Table A) and 3T3 cells (Table B) were seeded in 24 well plates as described in Figure 7. After seeding, all cells were transferred to growth factor deficient media. The 3T3 progenitor cells were maintained in 50 pM FGF. Cells were treated for 48 hours at various concentrations of K252a (ranging from 0 to 250 nM). Cell viability for all cell lines was determined by assaying glucose utilization on duplicate samples. Table A; Blank squares - SH-SY5Y (without BDNF); Squares filled -LA-N-5 (with BDNF); Circles filled - N18TG2 (with BDNF). Table B; Squares in white - 3T3 (with FGF); Squares to full - 3T3

73 889 6526-089-118 -19 autocrine (with BDNF).

Figure 12. Differential effect of K252a on small cell lung carcinoma (NCI-H69), pulmonary adenocarcinoma (Calu-3), 3T3 (autocrine) and neuroblastoma cells (N18TG2). The cells were seeded in 24-well plates as described in Figure 7. Cells were treated for 48 hours with 0.50, 0.50 and 100 nM K252a. Cell viability for all cell lines was determined by the glucose assay assay on duplicate samples. Histograms (from left to right) are shown as follows: Histograms filled; NCI-H69; Histogram with full diagonal lines; N18TG2; Histogram with dotted; 3T3 (autocrine); Histogram with thin diagonal lines; Calu-3.

Detailed Description of the Invention The present invention provides processes and pharmaceutical compositions for the therapeutic treatment of mammals bearing tumor cells which express neurotrophins to inhibit or interfere with the growth of tumor cells and their progeny. The compositions and methods of the present invention involve administering to the affected mammal an effective amount of a substance that interferes with the autocrine survival loop of the tumor cells.

More specifically, two examples of mechanisms are provided which can be used to interfere with an autocrine loop of BDNF survival and thus cause cell death in BDNF-expressing tumor cells.

One mechanism for practicing the present invention involves the use of nucleic acids of at least six nucleotides that are antisense for a gene or for the cDNA encoding BDNF or a portion thereof. " Antisense " as used herein refers to a nucleic acid capable of hybridizing to a portion of BDNF RNA (preferably mRNA) by virtue of some complementarity of the sequence.

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Another mechanism involves the use of the compound staurosporin, K252a or derivatives thereof, which are described in Murakata et al. U.S. Patent Nos. 4,923,986 and European Patent Applications Nos. 303,697, published February 22, 1989 and Na 323,171, issued July 5, 1989, or other protein kinase inhibitors.

Additionally, the invention provides a recombinant autocrine loop cellular model that embodies many characteristics of tumor cells (such as neuroblastomas and SCLC) that utilize autocrine survival loops. A cell using an autocrine survival loop, as used herein, refers to a cell expressing a molecule that is necessary for its own survival.

Autocrine Antisense Interruption A. Inhibition of BDNF Expression

The antisense nucleic acids of the invention interrupt the autocrine loop of BDNF at an intercellular level, by preventing the synthesis of BDNF by the cell which depends on the expression of BDNF to survive. The mechanism of K252a and related compounds to disrupt the autocrine loop of BDNF occur at the BDNF receptor level by preventing activation and phosphorylation of the trk B receptor. Another class of tyrosine protein kinase inhibitors, the class of inhibitors thiazolidinedione [Geissler et al., J. Biol. Chem., 265i22255-22261 (1990)] may act similarly to K252a. Other protein kinase inhibitors such as calfostin C, staurosporine, K252b, KT5720, KT5823 and KT5926 (Kamiya Biomedical Company, Thousand Oaks, California) may also be used. Other mechanisms of BDNF autocrine loop interruption are also encompassed by this invention.

The antisense nucleic acids of this invention may be oligonucleotides which are double stranded or single stranded, RNA or DNA or a modification or derivatives thereof, which can be directly administered to a cell, or which can be produced intracellularly by sequence transcription

introduced.

The BDNF antisense nucleic acids provided by the present invention may be used in the treatment of tumors, wherein the cells of such a tumor type can demonstrate in vitro or in vivo expression of the BDNF gene. Such demonstration can be done by detecting BDNF or BDNF protein RNA. According to the invention, the antisense oligomers of BDNF not only prevent the growth of such tumors, but may also result in the death of tumor cells by an unusual mechanism involving programmed or "apoptotic" death, which is characterized by the loss of DNA before loss of cellular protein. [Arends et al., Am. J. Pathol. 136: 593 (1990)]. The invention further provides pharmaceutical compositions comprising an effective amount of the BDNF antisense nucleic acids of the invention, in a pharmaceutically acceptable carrier. Also provided are methods for treating various diseases and disorders comprising administering pharmaceutical compositions of the invention.

In another embodiment, the invention is directed to methods for inhibiting the expression of a BDNF nucleic acid sequence in a eukaryotic cell, comprising providing the cell with an effective amount of a composition comprising a BDNF antisense nucleic acid of the invention.

In another embodiment, the identification of cells expressing functional BDNF or other neurotrophin receptors can be accomplished by observing the ability of a neurotrophin to " save " these cells from the cytotoxic effects of an antisense BDNF nucleic acid.

Another aspect of the invention provides for the diagnosis of human neuroblastoma or small cell lung carcinoma by detecting expression of BDNF in cells obtained from patients. Such detection can be performed by detecting RNA

of BDNF or expression of the protein.

The antisense nucleic acids of the invention are at least six nucleotides and are preferably oligonucleotides (ranging from 6 to about 50 nucleotides). The oligonucleotides may be from DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide may be modified at the base portion, the sugar moiety or the phosphate structure. The oligonucleotide may include other linked groups, such as peptides or agents that facilitate transport across the cell membrane [see e.g. Letsinger et al., Proc. Natl. Acad. Know. USA. 86: 6553-6556 (1989); Lemaitre et al. . Proc. Natl. Acad. Know. USA. 84: 648-652 (1987); PCT Publication No. W088 / 09810, published December 15, 1988] or the blood-brain barrier [see eg PCT Publication No. WO 08/10134, published April 25, 1988], hybridization triggered cleavage agents . Krol et al. BioTechniaues. 6: 958-976 (1988)] or intercalating agents [see e.g. Zon, Pharm. Res. 5:539-549 (1988)].

In a preferred aspect of the invention, there is provided an antisense oligonucleotide of BDNF, preferably single stranded DNA. In a more preferred aspect, such an oligonucleotide comprises an antisense sequence for the last 6 codons of human BDNF. The oligonucleotide can be modified at any position in its structure with substituents commonly known in the art. The BDNF antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluoro-uracil, 5-bromo-uracil, 5-chloro-uracil, 5-iodo 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylamino-methyl-2-thio-uridine, 5-carboxymethylamino-methyl-uracil, dihydro-uracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,

5-methylaminomethyluracil, 5-methoxymethyluracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), 5-methylaminomethyluracil, 5-methoxyaminoethyl- , vibroxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester , 5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine .

In another embodiment, the oligonucleotide comprises at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinosis, xylulose and hexose.

In yet another embodiment, the oligonucleotide comprises at least one modified phosphate structure selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a 15-phosphoramidate, a phosphorodiamidate, a methylphosphonate, a phosphotriesteralkylic acid and a formacetal or analogue thereof .

In yet another embodiment, the oligonucleotide is an α-anomeric oligonucleotide. An Î ± -anomeric oligonucleotide forms double-chain specific hybrids with complementary AKN in which, unlike the usual β units, the strands are parallel to each other [Gautier et al., Nucl. Acids Res. 15: 6625-6641 (1987)]. The oligonucleotide may be conjugated to another molecule, e.g., a peptide hybridization-enhancer crosslinking agent, a cleavage agent driven by hybridization with transport agent, etc.

The oligonucleotides of the invention may be synthesized by standard procedures known in the art, e.g. by use of an automated DNA synthesizer (such as those commercially available from Biosearch, Applied Biosystems,

73 889 6526-089-118 etc.). As examples, phosphorothioate oligos may be synthesized by the method of Stein et al., Nucl. Acids Res. 16; 3209 (1988), methylphosphonate oligos can be prepared using controlled pore glass polymer supports [Sarin et al. . Proc. Natl. Acad. Know. USA. 85: 7448-7451 (1988)], etc.

In a specific embodiment, the BDNF antisense oligonucleotide comprises catalytic RNA, or a ribozyme [see, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., Science 247; 1222-1225 (1990)]. In another embodiment, the oligonucleotide is a β-O-methylribonucleotide [Inoue et al. . Nucl. Acids Res. 15: 6131-6148 (1987)], or a chimeric RNA-DNA analogue [inoue et al .. FEBS Lett. 215: 327-330 (1987)].

In an alternative embodiment, the BDNF antisense nucleic acid of the invention is produced intracellularly by transcription of an exogenous sequence. For example, a vector may be introduced in vivo so that it is incorporated by a cell, into which the vector, or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the BDNF antisense nucleic acid. Such a vector may remain episomal or become integrated into the chromosome, as long as it can be transcribed to produce the desired antisense RNA. Such vectors may be constructed by recombinant DNA technology methods, common in the art. Vectors may be plasmid, viral, or other known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the BDNF antisense RNA can be performed by any promoter known in the art to act on mammalian, preferably human, cells. Such promoters may be inducible or constitutive. Such promoters include, but are not limited to: SV40 early promoter region [Bernoist and Chambon, Nature. 290: 304-310 (1991)], the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus [Yamamoto et al., Cell. 22: 787-797 (1980)], the promoter

73 889 6526-089-118 -25 of herpes thymidine kinase [Wagner et al., Proc. Natl. Acad. Know. USA. 78: 1441-1445 (1981)] r < the regulatory sequences of the metallothionein gene [Brinster et al. Nature. 296: 39-42 (1982)]; etc.

The antisense nucleic acids of the invention comprise complementarity to at least a portion of an RNA transcript of a BDNF gene, preferably a human BDNF gene. However, absolute complementarity is not required, although it is preferred. A &quot; complementary sequence for at least a portion of an RNA &quot; as referred to herein means a sequence having sufficient complementarity to be able to hybridize to the RNA, forming a stable duplex (or triplex, in the case of antisense nucleic acids of double-stranded BDNF). The ability to hybridize will depend both on the degree of complementarity and on the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the greater the number of base mismatches that it may contain with a BDNF RNA and even though forming a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatches using standard procedures to determine the melting point of the hybridized complex. B. Interruption of Recentor Phosphorylation The compound known as K252a is commercially available from the Kamiya Biomedical Company in Thousand Oaks, California and is otherwise described in the references cited above. The physiologically active substance K252a is a derivative of a K252 substance which has been produced by culturing a microorganism of the genus Nocardiosis [Matsuda et al. U.S. Patent 4,555,402]. Ο K252 is defined in Murakata et al. U.S. Patent 4,877,776 as a compound represented by the formula:

J 73 889 6526-089-118 -26-

(I.e.

in which R1 and R2 are H or OH; X is COOH, COOR or CH 2 OH; Y is H, R or COR? and Z is OH, OR or SR, where R is a lower alkyl. K252a has been shown to inhibit the growth of human uterine cancer HeLa cells, human breast cancer MCF7 cells, human colon adenocarcinoma COLO320DM cells and human lung carcinoma PC10 cells, by the protein kinase inhibitory activity.

K252 derivatives are disclosed in Murakata et al. U.S. Patent No. 4,923,986 as compounds of the formula: wherein W1, W2, R1, R2, R3, R4, X and Y represent various substituents.

Are limited by theory or mechanisms, our results indicate that staurosporine and its derivatives and K252a and its derivatives operate by interfering with the phosphorylation of the neurotrophin receptor. More specifically, the trk B receptor tyrosine kinase is inactivated by disruption of the autocrine BDNF loop at the cell surface receptor level. Suppression of the phosphorylation of cellular proteins is believed to be due to the direct effect of the specific interference of K252a or staurosporine or its derivatives with BDNF-mediated cellular responses. Other protein kinase inhibitors, such as thiazolidinediones, which inhibit EGF-induced receptor phosphorylation, may act in a similar fashion.

to interfere with BDNF-mediated cellular responses. Therapeutic Utility

The materials of this invention may be used to treat tumors of a type shown to express BDNF. Such tumors include, but are not limited to, neuroblastoma, small cell lung carcinoma and some neuroepithelial tumors. In one embodiment, an antisense single stranded DNA oligonucleotide for BDNF is used in the treatment of neuroblastoma. In another embodiment, staurosporine or K252a is used in the treatment of neuroblastoma.

Other types of tumors expressing BDNF RNA can be identified by various methods known in the art. Such processes include, but are not limited to, nucleic acid hybridization specific for BDNF, e.g. by Northern blotting, dot blot hybridization, by observing the ability of the RNA to be translated in vitro into BDNF, etc. In a preferred aspect, a patient's primary tumor tissue for BDNF expression may be analyzed prior to treatment.

They may be aministered to a patient having a tumor which is of a type expressing BDNF RNA, pharmaceutical compositions of the invention, comprising an effective amount of a substance that interferes with the autocrine BDNF survival loop, in a pharmaceutically acceptable carrier.

The therapeutic and pharmaceutical compositions of the present invention for inhibiting the growth of BDNF-expressing tumor cells thus comprise a therapeutically effective amount of a substance capable of interfering with the autocrine BDNF loop in a mixture with a pharmaceutically acceptable carrier. Pharmaceutical compositions containing tumoricidal activity may be used in conventional formulations such as, for example, solutions, syrups, emulsions, injectables, tablets, capsules, or suppositories.

Suitable carriers are well known to those skilled in the art of pharmacology. Examples of carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, sgualene and water. Additionally, the carrier or diluent may include a retardant material such as glycerol monostearate or glycerol distearate alone or with a wax. Optionally, suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabilizers are well known to those skilled in the art and include, for example, citric acid and other pH adjusting agents, chelating or sequestering agents and antioxidants.

Formulations of the pharmaceutical composition containing K252a, staurosporine, or a derivative thereof, or any other protein kinase inhibitor may conveniently be presented in the form of a dosage unit and may be prepared by any of the standard procedures. Alternatively, the composition may be in a form adapted for slow in vivo release, as is known in the art. All processes include the step of bringing together the active ingredient with the carrier which may comprise one or more accessory ingredients. The amount of the substance that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and may be determined by standard clinical techniques. In one embodiment of this invention, it would be desirable to determine the antisense cytotoxicity of the tumor type to be treated in vitro. for example, in the test systems described in the examples below. and then into useful animal model systems, prior to testing and use in humans.

Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. In addition, it may be

It is desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an ommaya reservoir.

In addition, it may be desirable to administer the pharmaceutical compositions of the invention locally in the area in need of treatment; this can be achieved, for example, and is not limited to, by local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being a porous, nonporous or gelatinous material , including membranes, such as sialastic membranes, or fibers. The invention also provides pharmaceutical compositions comprising substances that interfere with an autocrine loop of BDNF administered via liposomes, microparticles or microcapsules. In various embodiments of the invention it may be useful to use such compositions to achieve sustained release of the substances. In a specific embodiment, it may be desirable to use antibody-directed liposomes for specific identifiable tumor antigens (e.g., cell surface antigens selective for neuroblastoma or SCLC) [Leonetti et al. Proc. Natl. Acad. Know. USA. 87: 2448-2451 (1990); Renneisen et al. . J. Biol. Chem., 265: 16337-16342 (1990)]. K252a or staurosporine or derivatives thereof, as well as other protein kinase inhibitors, may also be employed according to the methods and compositions of this invention, alone or in combination with other therapeutic or diagnostic agents useful in the direct or adjunctive treatment of certain cancers. It is contemplated that K252a or a derivative thereof may be used in combination with the BDNF antisense nucleic acids of this invention. Other agents, e.g., antimetabolites, alkylating agents, vinca alkaloids,

I 73 889 6526-089-118 30

Anti-neoplastic antibiotics, platinum derivatives, substituted ureas, adrenocorticosteroids, cytokines, interleukins, interferons or antibodies, may also be employed in conjunction with such kinase inhibitors to treat a variety of cancers characterized by cells which express BDNF and related diseases. The dosage regimen involved in administering an effective amount of, for example, K252a in a method for treating the conditions described below will be determined by the attending physician, considering various factors that modify the action of the drugs, e.g., condition, body weight, sex and diet of the patient, tumor severity, time of administration and other clinical factors. The dosage of the compositions of the invention used to treat the disease-specific condition described herein may vary depending upon the particular disease and stage of the disease.

It is further contemplated that pharmaceutical compositions containing K252a or a derivative thereof, staurosporine, or other kinase inhibitors also contain another conventional therapeutic agent, such as. cyclophosphamide, cytokines, interleukins, interferons or antibodies, as mentioned above. It is especially contemplated that the antisense oligonucleotides of this invention can be combined with pharmaceutical compositions containing protein kinase inhibitors. When these agents are combined in a pharmaceutical composition, it is anticipated that each active ingredient will be present in the combined composition in the same concentration or at a slightly lower concentration than that agent would have if given alone. The therapeutic mechanism of the compositions and methods of the present invention differs in principle from that of the vast majority of the drugs presently being used for treatment of tumors associated with BDNF expression. Substances that interfere with the autocrine loop of BNDF survival, alone or in combination with other known tumourocidal agents, have activity

It is highly specific so that patients do not suffer from the many disadvantages of conventional therapy for cancer.

BDNF-expressing tumors amenable to treatment by the instant invention and compositions include, but are not limited to, neuroblastoma, small cell lung carcinoma, and some neuroepithelial tumors. Other tumor types expressing BDNF can be identified by various methods known in the art. According to the method of the present invention, a patient's primary tumor tissue can be analyzed for BDNF expression prior to treatment, if desired. Regardless of the mammalian disorder treatment presented herein, the methods and compositions of this invention may be used for veterinary purposes in the treatment of BDNF-expressing tumors, which afflict horses, pigs, cattle and poultry, for example. Such disorders may be treated using amounts of the compound that may be used in the treatment of the above mammalian disorders.

Diagnostic Utility Most human neuroblastoma cell lines express some level of human BDNF mRNA. BDNF mRNA expression appears to be unique in neuroblastoma, with only a few exceptions (such as small cell lung carcinoma and some neuroepithelial tumor cells). These results suggest that BDNF mRNA expression may serve clinically as a useful marker for human neuroblastoma, as well as for SCLC and for some neuroepithelial tumors. Since the best clinical marker for neuroblastoma to date is N-myc amplification and N-mvc is only amplified in approximately 30% of all late stage neuroblastomas (Stages III and IV), mRNA expression of BDNF may be a more useful and broader spectrum clinical marker for both early-stage and late-stage neuroblastomas.

73 889 6526-089-118 -32-

Identification of Cells Expressing Functional BDNF or Other Neurotrophin Receptors

Certain neuroblastoma cell lines can be efficiently saved from cytotoxic effects of BDNF antisense oligonucleotides by the addition of either BDNF / NGF or NT-3 (see Section 6 below). Thus # some neuroblastomas can be expected to express functional receptors for BDNF, NGF or NT-3 based on the ability of these individual ligands to salvage one of these types of antisense cytotoxicity 5 cells. For example # our results (Example 1. # Section 1.1) suggest that neuroblastoma cells LA N-5 express functional receptors for NGF # BDNF and NT-3, whereas LA-Ni cells express only functional receptors for BDNF # and that CHP-134 and CHP-404 neuroblastoma cells express both NGF and BDNF receptors # but fail at NT-3 receptors.

Genetic Engineering of a Model Cell System That Imitates Autocrine Ankle-Dependent Tumor Cells.

Despite the identification of an autoregressive BNDF-dependent survival loop in neuroblastomas, the properties of cells reflecting such a survival loop, such as detectable levels of trkB mRNA or detectable levels of a constitutively phosphorylated trk receptor, were not detected immediately. Previous studies of autocrine loops involving conventional mitogenic factors have demonstrated that these loops can function in the absence of detectable secretory factor with occasional intracellular receptor activation [Zhan and Goldfarb, Mol. Cell. Biol. 6: 3541 (1986)]. In addition, chronic autocrine stimulation may result in substantial regulation in the lower range of receptor autophosphorylation, as well as rapid recycling of the involved receptor, both of which may hinder or prevent the detection of constitutively phosphorylated receptors. Similarly, continuous exposure of neuronal cells to NGF, as required for survival, eventually results in substantial regulation in the lower range of the activated trkA receptor [Kaplan et al. . Science 252: 554 (1991)]. 73 889 6526-089-118 33-

To overcome these problems and to provide a cellular model using an autocrine BNDF loop, in which the interruption of that loop can be easily detected in vitro. thus providing a useful system for screening compounds with the ability to disrupt such an autocrine loop, a recombinant cell system has been created. This system utilized a BDNF / trkB-mediated autocrine loop. This system is based on a variant of the NIH 3T3 fibroblast cell line whose growth and survival in defined media requires either fibroblast growth factor (FGF) or platelet derived growth factor (PDGF) [Lee and Donoghue, J. Cell Biol. 113, 361 (1991)]; the death of these fibroblasts due to factor deprivation is also apoptotic [Ernfors et al., Neuron 5:51 (1990)]. When these cells are stably transfected with the trkB receptor, BDNF can replace FGF or PDGF [Glass et al., Cell 66: 405 (1991)]. Co-transfection of these cells with both trkB and BDNF leads to an NIH3T3 cell [referred to as 3T3 (autocrine) or MBx] that can survive in defined media without the addition of exogenous growth factor (ie, they become autocrine for BDNF). Surprisingly, these autocrine NIH3T3 cells are, in many ways, similar to neuroblastomas dependent on an autocrine loop of BNDF survival. For example, they exhibit a similar sequence specific susceptibility for antisense oligomers of BDNF, which may be overcome by exogenous BDNF. In addition, these cells do not secrete detectable levels of BDNF to the media nor have detectable levels of a constitutively phosphorylated trkB receptor.

In addition, K252a and staurosporine act on BDNF / trkB transfected NIH3T3 cells, grown in defined media, in a manner that is very similar to its effect on neuroblastomas, thus confirming that such cells provide a useful assay system for the identification of agents that can be used to kill autocrine loop-dependent tumor cells by rupturing the autocrine loop. Other autocrine loop model systems, which are constructed to encode and express a particular factor, as well as the receptor 73 889 6526-089-118 34-

for this factor, can also be created and used, as contemplated herein, to identify agents that disrupt such autocrine loops. Such factors include, but are not limited to, nerve growth factor, neurotrophin-3, neurotrophin-4, and ciliary neurotrophic factor.

In order that the invention disclosed herein may be better understood in its entirety, the following examples are set forth. It is to be understood that these examples are for illustrative purposes only and should not be construed as limiting this invention in any way.

EXAMPLES

Example 1

Experimental Checks: Neuroblastoma

As described herein, we have shown that antisense oligonucleotides directed against BDNF are cytotoxic in vitro to neuroblastoma cell lines, thus demonstrating that human neuroblastoma cells require BDNF as a molecule of autocrine survival and that these neuroblastoma cells can be saved of the cytotoxic effects of antisense BDNF through administration of exogenous BDNF. Since most of these neuroblastoma cells do not express detectable levels of trk B mRNA, our data imply that there may be additional receptors for BDNF. 1.1 Neuroblastoma Cell Lines from Humans and Rodents, Expressing BDNF mRNA.

We have investigated a panel of human and rodent tumor cell lines by Northern blotting approaches for BDNF mRNA expression [Maisonpierre et al., Science. 247: 1446-1451 (1990)]. Table I and Figure 5 summarize these results and indicate that most neuroblastoma cell lines (17 out of 18 tested) express BDNF mRNA and that BDNF mRNA expression appears to be somewhat unique to neuroblastoma . For example, only 3 of 12 neuroepitheliomas or Ewing's sarcomas express BDNF mRNA. No non-neural tumor tested revealed to express BDNF mRNA.

Cell lines from non-neuronal tumors tested included retinoblastomas, melanomas, carcinomas (cervical and breast), and leukemias. The only neuroblastoma cell line that did not express BDNF mRNA was SH-SY5Y which is unique in the sense that it is thoracic as opposed to the derived origin of neural Christian. Dr. Mark Israel (UCSD) provided RNA blots from some of these neuroblastoma cell lines.

Table 1

Expression of BDNF mRNA in Human Cell Lines * Cell Line BDNF Expression

Neuroblastoma 382 + GICAN + NB-69 + CHP-404 + CHP-234 + CHP-134 + CHP-126 + NMB + KCNR + NGP + BE2 + KAN + GI + AS + LA-N-1 +/- LA-N -5 + IMR-32 + SH-SY5Y-I 73 889 6526-089-118 -36-

Table 1 (cont.)

Cell Line BDNF Expression

Neuroepithelioma / Ewing SK-N-MC CHP-100 A4573 5838 EW-1 71 6647

N1050 32 DW + +

SK-N-LO LI

Non-Neuronal Cell Lines Y79 FOI BU Mol HL-60 COLO-320 HELA U937 K562 MCF7 * The data are a summary of Northern blot results of human cell lines probed with human BDNF. (+) indicates positive expression of hBDNF mRNA and (-) indicates absence of BDNF mRNA. (+/-) indicates a very low expression level. (Current Northern Transfer Data are shown in the BDNF patent). 73 889 6526-089-118 -37-

Antisense oligonucleotides Inhibition of in vitro translation of BDNF mRNA Three complementary antisense oligonucleotides were synthesized for various regions of the human BDNF gene [see PCT International Publication No. WO 91/03568, published March 21, 1991], as follows. Table II below. Each oligonucleotide (&quot; oligo &quot;) was constructed as a single 18 and the complementary sense oligonucleotides served as controls in all experiments. The 5 'anti-sense oligo (5'-AS-BDNF; SEQ ID NO: 1) consisted of the human BDNF DNA sequence, starting 3 nucleotides upstream of the presumed ATG start codon and extending to 4 downstream codons of this initiation methionine. The second antisense oligo corresponded to a BDNF DNA sequence around the dibasic residue processing site (PS-AS-BDNF; SEQ ID NO: 3) and the third antisense oligo (the S-AS-BDNF ; SEQ ID NO: 5) corresponded to the last 6 codons of human BDNF

Table II

Oligo-nucleotides of BDNF Antisense SEQ. ID NO: 1 5'-AS-BDNF 5'-GAA AAG GAT GGT CAT CAC-3 '1 -129 to -124 5'-S-BDNF 5'-GTG ATG ACC ATC CTT TTC- 124 SEQ. BDNF 5'-CGC CAG TCT GAC CCT GCC-3 '4 (Arg) (Lys) 5'-GGC 5'GGC AGG GTC AGA GTG GCG-3'3 1 to +5 PS-S- -1 to +5 SEQ. ID NO: 5 3'-AS-BDNF 5'-CTA TCC CCT TTT AAT GGT-3 '5 +113 to +119

3'-S-BDNF 5 / -TTG ACC ATT AAA AGG GGA-3 '+113 to +119 AS - refers to the antisense sequence; s sense sequence; refers to

73 889 6526-089-118 -38-

PS - refers to the dibasic amino acid processing site. The presumed ATG start codon is highlighted in the 5'-S-BDNF sequence. The Arg-Lys dibasic residue codons are indicated in the PS-S-BDNF sequence. All oligonucleotides were synthesized on a nucleic acid synthesizer from Applied Biosystems.

Each antisense BDNF oligonucleotide was first tested for its ability to inhibit BDNF synthesis using an in vitro translation system of wheat seed lysate (Figure 1). Synthesis of BDNF (+/- antisense or sense oligonucleotide) was followed in this assay system by metabolic labeling with 35S-methionine, polyacrylamide gel electrophoresis, and fluorography (Figure 1). It was observed that a random mere 18 (control oligo) had no inhibitory effect on the synthesis of BDNF in. vitro. at both 1 and 6 μ concentra concentrations. The oligo 5'-AS-BDNF (SEQ ID NO: 1) had a slight inhibitory effect on the synthesis in. vitro inhibition of BDNF at a concentration of 1 μΜ, and profound inhibitory effects at 6 μΜ. 3'-AS-BDNF (SEQ ID NO: 5) and PS-AS-BDNF (SEQ ID NO: 3) both effectively inhibited BDNF synthesis in concentrations of 1 μΜ and 6 μΜ. Complementary sense oligos had little or no effect on the in vitro synthesis of BDNF at 1 μg but had some inhibitory activity at very high concentrations (6 μg) (representative data presented for oligo 57-S-BDNF (SEQ ID NO : 2), Figure 1).

1.3 3 'Antisense oligonucleotides for BDNF. but not 5 'Antisense or Meaningful for BDNF. are Cytotoxic for Neuroblastoma Cells aue Express BDNF

We tested the effects of antisense BDNF oligonucleotide (5'-AS-BDNF, PS-AS-BDNF, and 3β-AS-BDNF) on cell viability when added directly to human neuroblastoma cell cultures, in vitro.

Human neuroblastoma cells were cultured in Eagle's modified essential medium (EMEM) with 10% fetal bovine serum (FBS), 2 mM glutamine and penicillin and streptomycin, 1% 73 889

6526-089-118 of each (complete media). For the antisense assays, the cells were plated in Costar 24-well plates at a seed density of 2 x 10 4 cells / well. Absorption and incorporation of antisense oligonucleotides was performed by adding the antisense oligos directly to the cells in serum-free EMEM. The concentration of sense or antisense oligonucleotides added to the cell culture media ranged from 0.1 to 50 μΜ (Figure 2). After an incubation period of 4 hours with the respective oligonucleotide, ITS supplement (insulin, transferrin and selenium) was added to cell culture wells and cell viability was analyzed 96 hours after addition of the oligo. We analyzed and averaged wells in duplicate. Cell viability was analyzed by trypan blue staining. (Figure 2). the morphological effects of sense or antisense BDNF oligomers on LAN-5 neuroblastoma cells are shown in the light photomicrographs in Figure 6. As shown in Figure 2, the LA-N-5 neuroblastoma cell line was the most sensitive to the cytotoxic effects of the 3'-AS-BDNF oligonucleotide (SEQ ID NO: 5). More than 80% of these cells were killed with 1 μΜ of 3'-AS-BDNF within 96 hours. LA-N-1 neuroblastoma cells were somewhat less sensitive than LAN-5 cells, since only 40% of these cells were killed with 1 μΜ of 3'-AS-BDNF in 96 hours. Interestingly, LA-N-1 cells express less BDNF mRNA than LA-N-5 cells. SK-ES and SH-SY5Y cells (which do not express BDNF mRNA) were resistant to the cytotoxic effects of 3'-AS-BDNF, even at concentrations of 50 μΜ. Some loss of cell viability at 50 μ 3 3 'AS-BDNF concentrations was observed for these resistant cell lines, but the same loss of cellular activity was observed when these cells were treated with 50 μΜ of the control oligonucleotide 3'- sense-BDNF (3'-S-BDNF). In fact, no loss of cells greater than 30% was observed in any of the four cell lines when treated for 96 hours in serum-free media with 50 μM 3'-S-BDNF. Finally, no loss of cell viability was observed in any of the neuroblastoma cell lines when treated with the 5'-AS-BDNF oligo (SEQ ID NO: 1) but PS-AS-BDNF (SEQ ID NO: 3) He gave

virtually identical results with 37-AS-BDNF (SEQ ID NO: 5).

Antisense oligoners of BDNF, but not control oligomers, had profound effects on cell morphology while adding to cultures of neuroblastomas at low concentrations (Figure 6A-C). As would be expected if these effects were derived from the rupture of an autocrine loop of BDNF, changes in cell morphology mediated by antisense could be avoided by the addition of exogenous BDNF (Figure 6D). 1.4 Neuroblastoma Cells Expressing BDNF May Be Selectively Saved from the Cytotoxic Effects of 37-AS-BDNF Oliaonucleotide by Co-Addition of Neurotrophins to the Cell Culture System.

Figure 3 (EA) shows the results of co-addition experiments in which either BDNF, N6F, or neurotrophin 3 (NT-3) (100 ng / ml of each purified recombinant factor obtained from CHO cells transfected with the respective gene from neurotrophin (see PCT International Publication No. W091 / 03568) was added simultaneously to the oligonucleotide 3'-AS-BDNF (SEQ ID NO: 5) (50 μ) to various neuroblastoma cells (ie, SH-SY5Y (A), LA-N-1 (B), LA-N-5 (C), CHP-134 (D), CHP-404 (E)) Cell viability was determined in duplicate wells of a 24 well plate, staining with trypan blue at 24 hour intervals after the addition of 3β-AAS-BDNF +/- neurotrophic factor. The number of cells was also recorded with a hemocytometer. As shown above, the uptake and incorporation of oligonucleotides was performed by addition of the oligo directly into the cell culture system under serum-free conditions for 4 hours. ura 3A demonstrate that 37-AS-BDNF has no cytotoxic effects on SH-SY5Y cells that are negative for BDNF mRNA. Both CHP-134 and CHP-404 cells (Figures 3D and E, respectively) are sensitive to the cytotoxic effects of 37-AS-BDNF and each of these cell lines can be saved (70-90%) by the co-addition of both BDNF as NGF, but not NT-3. LA-N-1 cells are only saved from the cytotoxicity of 37-AS-BDNF by BDNF (Figure 3B) whereas

LA-N-5 cells are saved by all three neurotrophins (Figure 3C). Although not shown in Figure 3, 3'-AS-BDNF oligonucleotide was found to be cytostatic as well as cytotoxic in LA-N-1, LA-N-5, CHP-134, and CHP-404 cells, but not in SH-SY5Y cells. 1.5 Treatment of Neuroblastomas with BDNF Antisense Oliaomers Causes Apoptotic Death

In contrast to the effects resulting from the disruption of autocrine growth factor loops previously described [Becker, et al., EMBO J., 3685 (1989); Morrison, J. Biol. Chem. 266: 728 (1991); El-Badry, et al. J. Clin. Invest. 87: 648 (1991); Selinfreund, et al., J. Cell Biol. 111: 2021 (1990)], even those known to be operative in neuroblastoma, the antisense BDNF oligomers not only prevented the growth of neuroblastoma but also resulted in the death of neuroblastoma tumor cells (Figure 6B). If BDNF does indeed function in these cells as a neuronal survival molecule, it would be expected that death due to rupture of an autocrine loop of BDNF could occur, by mechanisms similar to those previously described for neuronal cell death following neurotrophic factor deprivation [Martin, et al. J Cell Biol 106: 829 (1988); Scott, et al. J. Neurobiol. 21: 630 (1990); Batistatou, et al. J. Cell Biol. 115: 461 (1991); Rukenstein, et al. J. Neurosci. 11: 2552 (1991)]. Although the morphological patterns presented by neurons that undergo naturally occurring cell death may vary [Server, et al. in Apootosis. The Molecular Basis of Cell Death. pp. 263-279 (1991)], neuronal death can generally be marked by some of the same biochemical changes that characterize programmed cell death in other systems such as the thymus and prostate (Batistatou, supra; Rukenstein, supra; Wylie et al., Int In particular, the activation of endogenous calcium-dependent endonucleases that result in the loss of DNA prior to the loss of cellular protein, may be a general trait of programmed or "apoptotic" death &quot; , [Arends, et al., Am. J. Pathol., 136: 593 (1990)] We took advantage of a double staining flow cytometric assay (ie, for DNA and

73 889 6526-089-118 -42-

protein), to distinguish between apoptosis and necrosis [Del Bino, et al. Exp. Cell Res. 193: 27 (1991); Jakobisiak, et al., Proc. Natl. Acad. Know. USA. 88: 3628 (1991)]. The DNA profile of human neuroblastoma cells LA-N-5 is typical of many normal cycle cell lines, with a large percentage of the cell population in the G1 phase of the cell cycle, and the remainder of the population in the S phase or the phase G2 + M (Figure 7A). Treatment with antisense BDNF oligomers results in the appearance of an apoptotic population of LA-N-5 cells, characterized by a significantly reduced DNA content in the absence of protein loss (Figure 7B); these changes are accompanied by a decrease in the percentage of cells in the S phase, as is generally observed in apoptotic populations (Del Bino, supra). BDNF salvage of antisense treated autocrine cells prevents the appearance of the apoptotic population, as previously observed in Figure 6D (Figure 7C). Although neuroblastoma cells are not sensitive to low concentrations of sense BDNF or random sequence oligomers, these oligomers resulted, when present in low concentrations (see below), in neuroblastoma cell death, as well as death of non-dependent cells of BNDF autocrine loops. In contrast to the apoptotic profile exhibited by neuroblastoma cells treated with low concentrations of antisense oligomers of BDNF, high concentrations of sense oligomers resulted in DNA and protein profiles consistent with necrosis - loss of cellular protein is apparent with no effect on DNA content or the percentage of cells in the S phase (Figure 7D).

To verify that the antisense oligoners of BDNF operate in a sequence dependent manner to specifically kill neuroblastoma cells requiring an autocrine loop of BDNF, we compared the viability of neuroblastoma cell lines when exposed to varying concentrations of oligomers either with or without sense antisense. Dosage response studies revealed that antisense BDNF oligomers were surprisingly more potent than control oligomers in their ability to kill a neuroblastoma expressing BDNF, LA-N-5 (Figure 8A). In contrast, antisense and control oligomers were equally ineffective in killing the single neuroblastoma negative for BDNF, SY5Y (Figure 8B). Although addition of exogenous BDNF had not altered the effects of either the sense oligomer or the antisense oligomer on SY5Y cells (Figure 8B), exogenous BDNF markedly displaced the death curve of antisense oligomers in LA-N- 5 equalizing that of the control sense oligomers (Figure 8A). While the Ewing sarcoma negative cell line for BDNF, SK-ES, was similar to SY5Y in its insensitivity to antisense BDNF oligomers, the examination of 4 additional BDNF-positive cell lines (CHP-134, N18TG2 , CHP-404 and LA-N-1) revealed remarkable susceptibility to antisense BDNF oligomers as well as a BDNF-scavenging ability, which was essentially indistinguishable from that of LA-N-5 cells (results not shown) .

Thus, the antisense BDNF oligomer acts in a sequence specific manner and only in neuroblastomas which express BDNF. In addition, the toxicity of the antisense oligomer is reduced at the level of control oligomers by the addition of exogenous BDNF. Together with our observation that the death of neuroblastoma caused by antisense BDNF oligomers occurs by apoptosis, whereas death due to higher concentrations of control oligomers is necrotic, these results unequivocally demonstrate that the antisense oligomers of BDNF selectively activate apoptotic cell death in neuroblastoma, by rupturing the autocrine loop of survival dependent on the continuous synthesis of BDNF. 1.6 Conclusion Most human neuroblastoma cell lines express a certain level of human BDNF mRNA and the expression of BDNF mRNA appears to be unique for neuroblastoma with only a few exceptions (such as small cell lung carcinoma and some neuroepithelial tumors ). These results suggest that BDNF mRNA expression may serve as a clinically useful marker for human neuroblastoma. Since the best 73 889 6526-089-118 -44-

clinical marker to date for neuroblastoma is N-myc amplification and N-myc is only amplified in approximately 30% of all late stage neuroblastomas (Steps III and IV), expression of BDNF mRNA may be a more useful and broader spectrum clinical marker for both early-stage and late-stage neuroblastomas.

Oligonucleotides 37-AS-BDNF (SEQ ID NO: 5) and PS-AS-BDNF (SEQ ID NO: 3) are cytostatic and cytotoxic only in human neuroblastoma cells expressing BDNF mRNA. These results imply that neuroblastomas positive for BDNF mRNA require an autocrine loop of BNDF for their own proliferation and survival. Our results suggest that at least some neuroblastomas can be killed specifically and effectively by treatment with antisense BDNF oligonucleotides.

Certain neuroblastoma cell lines can be efficiently saved from the cytotoxic effects of antisense BDNF oligonucleotides by the addition of either BDNF, NGF or NT-3.

Example 2

Experimental Checks: Small Cell Lung Carcinoma

2.1 Small Cell Lung Carcinoma Cell Lines express BDNF mRNA

In order to better understand the potential role of BDNF as an autocrine survival factor for small cell lung carcinoma tumors, we used a Northern blot approach to examine the expression of BDNF mRNA in various lung carcinoma cell lines of small cell.

Total RNA samples prepared from six different cell lines of small cell lung carcinoma were obtained from the laboratory of Dr. Jim Battey at NIH. The cell lines shown in Figure 4 are as follows: H82 -45-

, H345 (lane 4), H378 (lane 5), H510 (lane 6), and N417 (lane 7). 10 μg of RNA from each cell line was used for Northern blotting, and the level of BDNF mRNA was directly compared to adult rat brain (lane 1). We have found that all six small cell lung carcinomas expressed some BDNF mRNA. Two transcripts were detected, as previously demonstrated for neuroblastoma cell lines and tissues [Maisonpierre et al., Science. 247: 1446-1451 (1990)]. The small cell lung carcinoma cell line H378 (lane 5) expressed particularly high levels of BDNF mRNA: approximately 2 to 3 times that expressed in adult rat brain (lane 1).

2.2 Antisense oligonucleotides 3 'for BDNF but not With 5' Sense or Antisense for BDNF. are Cytotoxic to Pecmena Cell Lung Carcinoma Cells Expressing BDNF

Antisense BDNF nucleotides prepared as shown in Example 1.2 were added directly to SCLC cell cultures (H345 and H378) in vitro. The assays were conducted as set forth in Example 1.3. The concentration of sense or antisense oligonucleotide added to the cell culture media ranged from 0.1 to 50 μΜ (Figure 8). After an incubation period of 4 hours with the respective oligonucleotide, ITS supplement (insulin, transferrin and selenium) was added to the cell culture wells, and cell viability was analyzed 96 hours after the addition of the oligonucleotides. Wells were analyzed in duplicate by trypan blue staining. As shown in Figure 9, both H345 (8A) and H378 (8B) cells were extremely sensitive to the cytotoxic effects of the 3 'antisense oligonucleotide for BDNF (SEQ ID NO: 5) but not those of the 5' sense oligonucleotide . 2.3 SCLC Cells Expressing BDNF Can Be Selectively Saved from the Cytotoxic Effect of 3r-AS-BDNF Oligonucleotide by BDNF Co-Addition to the Culture System of

73 889 6526-089-118 -46-

Cells Figure 9 (A & B) shows the result of the co-addition experiments in which BDNF (100 ng / ml, obtained from CHO cells transfected with the respective neurotrophin gene) was added simultaneously with the 3 'oligonucleotide -AS-BDNF (SEQ ID NO: 5) to SCLC cell cultures H345 and H378. As shown in Figure 9, both cell lines were saved by the co-addition of BDNF. 2.4 Conclusion

In addition to human neuroblastomas, SCLC cell lines express some level of human BDNF mRNA. These results suggest that the expression of BDNF mRNA may serve clinically as a useful marker for SCLC.

Oligonucleotides 3'-AS-BDNF (SEQ ID NO: 5) and PS-AS-BDNF (SEQ ID NO: 3) are cytostatic and cytotoxic in both SCLC cell lines tested. These results imply that SCLC lines positive for BDNF mRNA require an autocrine loop of BNDF for their own proliferation and survival and that such cells can be killed effectively by treatment with antisense BDNF oligonucleotides.

Example 3 3.1 0 K252a Blocks the Transduction Path of NGF signal, but not that of FGF. in PC12 cells

In order to establish that K252a could act to specifically block neurotrophin-mediated cellular responses, we examined the tyrosine phosphorylation profile of total protein lysates prepared from PC-12 cells that had been stimulated with NGF or FGF in presence or absence of three structurally related protein kinase inhibitors: K252a (isolated from the microbacterium Norcardiopsis sp.). staurosporine (isolated from Streptomoves sp.) or H-7 (1- (5-isoquinolinesulfonyl) -2-methylpiperazine dihydrochloride).

We compared tyrosine phosphorylation in total lysates

73 889 6526-089-118 preparations of PC-12 cells that were pretreated with kinase inhibitors, K252a, staurosporlen or H-7 for 15 minutes, and then administered NGF or FGF for 5 minutes. PC-12 cells were grown to approximately 70% confluency on 100 mm tissue culture plates with serum-containing medium (DME supplemented with 6% equine serum, 6% calf serum, 1% glutamine, 1% % penicillin, 1% streptomycin). Growth factors and inhibitors were diluted in the same medium and administered to the cells in 200 microliter aliquots.

After incubation, the cells were washed twice at 4øC with phosphate buffered saline containing 1 mM sodium orthovanadate. Complete aspiration of the wash buffer was followed by cell lysis using 500 microliters of supplemented RIPA buffer (calcium and magnesium-free phosphate buffered saline, but containing 1% NP40, 0.5% DOC, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM PMSF, 0.14 TlU / mg aprotinin). The plates were mixed using a Vari mixer at 4 ° C for 15 minutes to lyse the cells. The cell lysate was transferred to a 2.2 ml Eppendorf tube and centrifuged for 15 minutes at 4 ° C. We removed and laid the pellet on the ice, using a sterile toothpick. The supernatant represented the total lysate and was prepared with protein application dye in the ratio of 1x to 5x, respectively. The lysate was boiled at 95Â ° C for 3 to 5 minutes, separated on a 10% SDS-polyacrylamide gel, transferred to Immobilon (Millipore) and then challenged with anti-phosphotyrosine antibodies (UBI) at 1: 1000. For autoradiography detection, one microliter / ml of 125 I-conjugated goat anti-mouse IgG was used. (SA = 13 μΟ / ταΙ).

Autoradiography analysis of the resulting electrophoresis gel showed that, in comparison to untreated PC-12 cells, 0.01% or 0.02% DMSO was non-toxic to cells and did not alter the phosphorylation pattern. In addition, the electrophoresis was run with samples from each of the following cells which were treated with inhibitors only [100 nM K8-8-8, 739, 6526-089-118 and 200 nM, 100nM staurosporine and 25-7 H-7]; cells that were treated with only [100 ng / ml NGF; 10, 50, 100 and 200 ng / ml FGF]; cells that were pretreated with inhibitors followed by administration of 100 ng / ml of NGF [100 nM and 200 nM K252a, 100 nM staurosporin, 25 μM H-7] and cells that were pretreated with inhibitors followed by administration of 50 ng / ml FGF [100 nM K252a and 200 nM, 100 nM staurosporine, 25 nM H-7]. The inhibitors were resuspended by the manufacturer in DMSO and added to the cells at a final concentration not exceeding 0.02%.

Compared with the untreated control, administration of NGF (100 ng / ml) to PC-12 cells for 5 minutes resulted in rapid tyrosine phosphorylation of ERK1 (43 Kd) and ERK2 (41 Kd) tyrosine together with a high protein molecular weight (140 Kd) which was presumed to be the TrkA receptor. Stimulation of PC-12 cells with 10-100 ng / ml FGF resulted only in detectable tyrosine phosphorylation of ERK2 whereas at 200 ng / ml FGF this effect was inhibited. Treatment alone with the inhibitors or with DMSO (control vehicle) did not affect the phosphorylation profile. H-7 did not block signal transduction pathways either NGF or FGF. Staurospore and K252a significantly blocked the NGF pathway as reflected in the loss of TrkA tyrosine phosphorylation, ERK1 and ERK2, but not the FGF pathway since ERK2 phosphorylation remained at the control level.

In conclusion, we have observed that NGF and FGF stimulate early cell responses in PC-12 cells via independent signal transduction pathways, differentiated by the specificity of NGF-mediated responses to staurosporine and K252a.

Example 4 4.1 0 K252a Blocks BDNF Stimulation of Phosphorylation of

TrkB and ERK tyrosine 73 889 6526-089-118

-4-

To determine whether the action of K252a could also block the signal transduction pathway of BDNF, we constructed 3T3 cells to express a functional trkB receptor. We have previously demonstrated that these trkB-expressing 3T3 cells are BDNF-dependent for their survival and proliferation in defined media [D. J. Glass et al., Cell. 66: 405-413 (1991)]. The same panel of inhibitors used in assays with PC-12 cells were administered to trkB-expressing 3T3 cells.

We compared tyrosine phosphorylation in total neuroblastoma cell lysates (N18TG2) and trkB expressing 3T3 cells that were pretreated with the inhibitors used in Example 3 for 15 minutes, but then administered 100 ng / ml BDNF for 5 minutes. We processed and immunoblotted the lysates with anti-phosphotyrosine antibodies as described in Example 3. BDNF-treated and untreated cell lysates were compared to cells that were pretreated with inhibitors only [25 μM H-7, 100 nM K252a and 100 nM staurosporine] , and cells that were pretreated with inhibitors and then administered 100 ng / ml BDNF [25-7 H-7, 100 nM K252a and 100 nM staurosporine].

Immunoblots of autophosphorylated trkB receptors, immunoprecipitated from the total protein lysates of treated and untreated BDNF treated (autocrine) 3T3 cells, and treated and untreated neuroblastoma cells (N18TG2) are shown in Figure 10. These results indicated that administration of BDNF to these cells resulted in rapid phosphorylation of trkB and ERK2 as compared to the untreated control. According to the PC-12 cell data presented in Example 3, the tyrosine phosphorylation pattern revealed that pretreatment of cells with K252a and staurosporine, but not with H-7, abolished tyrosine phosphorylation of trkB stimulated by BDNF. Inhibitors alone did not appear to alter the phosphorylation profile of tyrosine. Staurospore completely blocked phosphorylation of EKK2, but with 100 nM K252a, tyrosine phosphorylation of ERK2 was still immediately detectable. Since 100 nM K252a blocked

73 889 6526-089-118 -50-50% of the phosphorylation of ERK1 and ERK2 in 20 PC-12 cells, minimal stimulation of the trkB receptor by BDNF (not detectable with this assay) may be sufficient to impart an intracellular response.

Example 5 5.1 0 K252a Stops Trk Signal Transduction and Causes Death on Trk Receptor Activation Dependent Cell Lines for Survival.

Our findings with trkB-expressing 3T3 cells indicate that K252a may cause rupture of an autocrine loop of BDNF survival by inhibiting phosphorylation and trkB receptor activation in response to BDNF. We examined this hypothesis using 4 human neuroblastoma cell lines as well as the 3T3f fibroblast model cell system from a small cell lung carcinoma cell line (NCI-H69-1-1) and a lung adenocarcinoma cell line ( Calu-3). The 3T3 cell line chosen for these studies is FGF-dependent for survival in defined serum-free media. We have previously demonstrated that trkA-expressing 3T3 cells survive in defined media supplemented with NGF whereas 3T3 cells expressing trkB survive in defined media supplemented with BDNF [Glass et al. . Cell 66: 405-413 (1991)]. 3T3 cells expressing both trkB and BDNF, survive in defined media only and serve as a useful model cell system for autocrine survival, resembling human neuroblastoma tumor cell lines. Of the neuroblastoma cell lines, both LA-N-5 cells and SK-N-LO cells express BDNF mRNA, whereas SH-SY5Y and SK-ES cells do not express BDNF mRNA. However, trkB expression at the mRNA level was only detectable in the LA-N-5 cell line.

In order to examine the effects of the K252A protein kinase inhibitor on the survival of human neuroblastoma cells, small cell lung carcinoma cells and trkB expressing 3T3 cells, we have developed a &quot; &amp; 73 889 6526-089-118 cell viability based on glucose utilization. The principle underlying this assay derives from the fact that viable cells will metabolize the supplied glucose in their growth medium while the dead cells do not. Thus, the concentration of glucose in the growth media is inversely related to the number of viable cells in the culture. The cells were seeded in 24 well plates at a density of approximately 2 x 10 4 cells per well. Human neuroblastoma cells, SCLC cells and lung adenocarcinoma cells were cultured in RPMI 1640 with fetal bovine serum, while the 3T3 cells were cultured in defined serum-free media with the appropriate neurotrophin (ie, FGF, BDNF or NGF at 500 pM ) [Glass, et al., Cell supra]. The TrkB / BDNF (MBx) autocrine 3T3 cells were cultured in only defined media. All cell culture media contained 450 mg / dl of glucose at the time of cell seeding. At 36 hours after seeding, K252a (solubilized in DMSO-see Example 1) was added at concentrations of 0, 10, 50, 100, 250, 500, 1000, and 2000 nM. All assays were performed in triplicate. Cellular morphology was monitored visually and cell viability was assessed by the glucose utilization assay after 6 days in culture. The glucose concentration (mg / dl) was determined by transferring a 50 μl aliquot of the growth media to blood glucose bands and reading these strips 2 minutes later on a blood glucose monitor.

The mean of the glucose readings and then plotted against the concentration of K252a was calculated to estimate the LD50 for each drug in each cell line. Table 3 shows the LDgg values for each cell line. Our results (Figure 11) demonstrate that K252a is cytocidal to human neuroblastoma cells and that this tyrosine kinase receptor inhibitor is effective for neuroblastoma cell lines that require an autocrine BDNF loop for survival in culture (ie SK-N-LO and LA-N-5). For example, LA-N-5 cells are approximately 20 to 150 times more sensitive to the cytocidal effects of K252a than SK-ES cells 73 889 6526-089-118

Or SH-SY5Y, respectively. SK-N-LO cells are also more sensitive to K252a (2.5 to 19-fold) than SK-ES or SH-SY5Y cells. Since SK-N-LO cells apparently do not express trkB, our data also suggest that these cells may express a single trk-like BDNF receptor and sensitive to K252a or, alternatively, that the level of trkB expression in these cells is below detectable limits. In addition, our data demonstrate that protein kinase inhibitors like K252a are cytocidal to small cell lung carcinoma cells but are not cytocidal to lung adenocarcinoma cells. Comparison of the effect of K252a on autocrine 3T3, neuroblastoma cells (N18TG2), small cell lung carcinoma (NCI-H69) and lung adenocarcinoma cells (Calu-3) is shown in Figure 12. TABLE 3

Cell Neuroblastoma SK-ES SH-SY5Y LA-N-5 SK-N-LO Fibroblast Cells

Extrinsence of neurite λ 50 200 nm 1500 nm 10 nm 80 nm

(5 days) NE NE <25 nm NE MBx 25 nm MG87 (+ FGF) 175 nm MG87 trkA (+ NGF) 30 nm MG87 trkB (+ BDNF) 30 nm Small Cell Lung Carcinoma Cells NCI-H69 100 nm Lung Adenocarcinoma Cells

CaLu-3 750 nm MG87 cells are 3T3 cells that require fgf for survival in defined medium. MG87 trk-A cells require NGF. MG87 trk-B cells require BDNF. NE = No Effect

73 889 6526-089-118

We have also shown that K252a and staurosporine are more effective (approximately 6-fold) for 3T3 fibroblasts expressing trk receptor kinases (MBx, MG87 trkA and MG87 trkB) that require neurotrophins for survival in defined media, relative to 3T3 progenitor cells (MG87) which survive on defined media supplemented with FGF. These data with the transfected 3T3 cells provide a convincing argument on how the cytocidal effects of K252a and staurosporine are most pronounced for the trk receptor family relative to the tyrosine kinase of FGF receptors. Cumulatively, our data support the hypothesis that K252a selectively disrupts the neurotrophin / trk receptor transduction pathway and that K252a and staurosporine are effective inhibitors of the autoregression of trkB / BDNF survival.

Finally, we observed that at concentrations of K252a of approximately 25 nM or less, a pronounced antiproliferative effect, as well as a significant neuritis outgrowth, could be detected in LA-N-5 neuroblastoma cells within 4-5 days of culture. These morphological changes are reminiscent of NGF induction of cell differentiation in LA-N-5. These observations regarding low concentrations of K252a suggest that this drug may function as a partial agonist of the trk receptor signal transduction pathway.

As described herein, we have shown that K252a may be an important selective antagonist of the neurotrophin signal / trk receptor transduction pathway and thus may be potentially therapeutically useful for the killing of neuronally-derived tumor dependent cells an autocrine loop of BDNF survival. We hope that other cancer cell lines expressing BDNF can be affected by K252a in a similar way. For example, some neuroepithelial tumors would be expected to be adversely affected by K252a and other protein * kinase inhibitors.

While certain embodiments of the invention have been

In particular disclosed, it will be obvious to those skilled in the art that many modifications and variations may be made. Thus, the present invention is not to be construed as limited by any of the particular embodiments presented, rather its scope should be defined only by the following claims.

DEPOSITS The following cell line was deposited with the Amerian Type Culture Collection, 12301, Parklawn Drive, Rockville, Maryland 20852.

DEPOSIT

ACCESS NUMBER MBx -55- 73 889 6526-089-118 (1) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA

(xi) SEQ ID NO: 1: GAAAAGGATG GTCATCAC (1) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA

(xi) SEQ ID NO: 2: GTGATGACCA TCCTTTTC (1) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA (xi) : SEQ ID NO: 3:

GGCAGGGTCA GAGTGGCG 73 889 6526-089-118 C! // Μ, -56-

t (1) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA (xi) : SEQ ID NO: 4:

CGCCAGTCTG ACCCTGCC (1) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA

(xi) Description of the Sequence: SEQ ID NO: 5: CTATCCCCTT TTAATGGT (1) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS (A) Length: 18 base pairs (B) Type: Nucleic acid (C) Chain type: simple (D) Topology: unknown (ii) Type of molecule: cDNA (xi) : SEQ ID NO: 6:

TTGACCATTA AAAGGGGA

Claims (44)

I 73 889 6526-089-118 -57- An isolated oligonucleotide, characterized in that it consists of at least six nucleotides and comprises a sequence complementary to at least a portion of an RNA transcript of a brain derived neurotrophic factor gene, oligonucleotide that is hybridizable to the RNA transcript. The oligonucleotide according to claim 1, characterized in that the brain derived neurotrophic factor gene is a human gene. The oligonucleotide according to claim 2, characterized in that it consists of at least 18 nucleotides. The oligonucleotide according to claim 3, characterized in that it is selected from the group consisting of: 5'-GGC AGG GTC AGA GTG GCG-3 '5'-CTA TCC CCT TTT AAT GGT-3'. The oligonucleotide according to claim 2, characterized in that it contains a sequence complementary to a portion of the brain derived neurotrophic factor RNA containing the codons encoding the Arg-Lys processing site. The oligonucleotide according to claim 2, characterized in that it contains a sequence complementary to a portion of the brain derived neurotrophic factor RNA which lies within the last 6 codons of the RNA. The oligonucleotide according to claim 2, characterized in that it contains at least one modified nucleotide. The oligonucleotide according to claim 7, characterized in that it contains at least one modified base part. The oligonucleotide according to claim 7, characterized in that it contains at least one modified sugar moiety. 73 889 6526-089-118 -58 The oligonucleotide according to claim 7, characterized in that it contains at least one modified phosphate backbone. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 1; and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the oligonucleotide of claim 2; and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the oligonucleotide of claim 3; and a pharmaceutically acceptable carrier. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 4; and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the oligonucleotide of claim 5; and a pharmaceutically acceptable carrier. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 6; and a pharmaceutically acceptable carrier. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 7; and a pharmaceutically acceptable carrier. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 8; and a pharmaceutically acceptable carrier. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 9; and a pharmaceutically- 73 889 6526-089-118 -59- acceptable. Pharmaceutical composition, characterized in that it comprises the oligonucleotide of claim 10; and a pharmaceutically acceptable carrier. A method for inhibiting the expression of a nucleic acid sequence encoding brain derived neurotrophic factor or a portion thereof in a cell, comprising providing the cell with an effective amount of the oligonucleotide of claim 1. A method for inhibiting the expression of a nucleic acid sequence encoding brain derived neurotrophic factor or a portion thereof in a cell, comprising providing the cell with an effective amount of the oligonucleotide of claim 2. A method for diagnosing the presence of a tumor in a patient of a tumor type characterized by the expression of a brain derived neurotrophic factor gene which comprises detecting the expression of a brain derived neurotrophic factor gene in which is suspected to be tumor cells, the expression being detected by detection of the production of RNA or brain derived neurotrophic factor protein. 24. The method of claim 23, wherein the tumor is a neuroblastoma. A method according to claim 23, characterized in that the tumor is a small cell lung carcinoma. A method for identifying a cell expressing brain-derived neurotrophic factor re-ceptors, comprising: (a) exposing the cell in vitro to (i) a cytotoxic amount of the oligonucleotide of claim 1, and (ii) an amount of brain derived neurotrophic factor; and (b) detecting cell survival, indicating the survival of the cell in the presence of the oligonucleotide and the brain-derived neurotrophic factor, the expression of brain-derived neurotrophic factor receptors by the cell. A method for identifying a cell expressing neurotrophin-3 receptors, comprising: (a) exposing the cell in vitro to (i) a cytotoxic amount of the oligonucleotide of claim 1, and (ii) an appropriate amount of neurotrophin- 3; and (b) detecting cell survival, indicating the survival of the cell in the presence of the oligonucleotide and neurotrophin-3 the expression of neurotrophin-3 receptors by the cell. A method for identifying a cell expressing nerve growth factor receptors, comprising: (a) exposing the cell in vitro to (i) a cytotoxic amount of the oligonucleotide of claim 1, and (ii) an appropriate amount of nerve growth factor; and (b) detecting cell survival, indicating the survival of the cell in the presence of the oligonucleotide and nerve growth factor, the expression of nerve growth factor receptors by the cell. A pharmaceutically acceptable composition comprising as active ingredient a pharmaceutically effective amount of K252a with at least one substance selected from the group consisting of carriers, diluents, Excipients and pharmaceutical adjuvants, for the treatment of BDNF-expressing tumors. The composition of claim 29, further comprising at least one further chemotherapeutic agent selected from the group consisting of antimetabolites, alkylating agents, vinca alkaloids, antineoplastic antibiotics, platinum derivatives, substituted ureas, adrenocorticosteroids, cytokines, interleukins , interferons and antibodies. A method for identifying an agent capable of inhibiting the growth or killing of a cell, comprising: (a) exposing a first and second cells to an agent, said first cell expressing a recombinant receptor for a factor neurotrophic factor and the survival of the first cell depending on the presence of the neurotrophic factor, and the second cell is of the same type as the first cell and said second cell does not express the recombinant receptor and does not depend on the presence of the neurotrophic factor for survival; and (b) detecting a decrease in growth or survival of the first cell in the presence of the agent and the neurotrophic factor, relative to the growth or survival of the second cell in the presence of the agent, whereby the decrease indicates the ability of the agent to inhibit or cause death of the cell. A method according to claim 31, wherein said neurotrophic factor is BDNF. A method according to claim 31, wherein said first and second cell types are fibroblasts. A method according to claim 33, characterized in that said fibroblasts are NIH3T3 cells. 73 889 6526-089-118 -62- A method according to claim 31, wherein said neurotrophic factor is present by exogenous addition. A method according to claim 31, wherein said neurotrophic factor is present by endogenous production by the cell. A composition comprising the oligonucleotide of claim 1 for use in a method of reducing growth or to cause death of a BDNF-expressing tumor cell. Use of a composition comprising K252a or its derivatives, staurosporine or derivatives thereof, or a thiazolidinedione or derivatives thereof for the manufacture of a medicament for reducing growth or to cause the death of tumor cells expressing BDNF receptor. A cell line comprising a recombinant nucleic acid encoding a neurotrophic factor and a recombinant nucleic acid encoding a receptor for said factor, said cell being capable of surviving in serum-free medium. The cell line of claim 39, wherein said neurotrophic factor is selected from the group consisting of BDNF, ciliary neurotrophic factor, nerve growth factor, neurotrophin-3 and neurotrophin-4. The cell line of claim 39, wherein said neurotrophic factor is BDNF. The cell line of claim 41, wherein said receptor is trkB. 43 - Cell line, characterized in that the MBx cell line is deposited with the American Type Culture Collection with the access number 63- 73 889 6526-089-118 .... A method according to claim 31, wherein said first cell type is MBx. Lisbon, 3; JBL 1992 By REGENERON PHARMACEUTICALS, INC. = 0 FACTORY AGENT =